Analysis of sympathetic responses to cognitive stress and pain through skin sympathetic nerve activity and electrodermal activity.

We explored the non-invasive evaluation of the sympathetic nervous system (SNS) by employing two distinct physiological signals: skin sympathetic nerve activity (SKNA), extracted from electrocardiogram (ECG) signals, and electrodermal activity (EDA), a well-studied marker in the context of the SNS assessment. Our investigation focused on cognitive stress and pain; two conditions closely associated with the SNS. We sought to determine if the information and dynamics of EDA could be derived from the novel SKNA signal. To this end, ECG and EDA signals were recorded simultaneously during three experiments aimed at sympathetic stimulation, Valsalva maneuver (VM), Stroop test, and thermal-grill pain test. We calculated the integral area under the rectified SKNA signal (iSKNA) and decomposed the EDA signal to its phasic component (EDAphasic). An average delay of more than 4.6 s was observed in the onset of EDAphasic bursts compared to their corresponding iSKNA bursts. After shifting the EDAphasic segments by the extent of this delay and smoothing the corresponding iSKNA bursts, our results revealed a strong average correlation coefficient of 0.85±0.14 between the iSKNA and EDAphasic bursts, indicating a noteworthy similarity between the two signals. We also reconstructed the EDA signals with time-varying sympathetic (TVSymp) and modified TVSymp (MTVSymp) methods. Then we extracted the following features from iSKNA, EDAphasic, TVSymp, and MTVSymp signals: peak amplitude, average amplitude (aSKNA), standard deviation (vSKNA), and the cumulative duration during which the signals had higher amplitudes than a specified threshold (HaSKNA). A strong average correlation of 0.89±0.18 was found between vSKNA and subjects' self-rated pain levels during the pain test. Our statistical analysis also included applying Linear Mixed-Effects Models to check if there were significant differences in features across baseline and different levels of SNS stimulation. We then assessed the discriminating power of the features using Area Under the Receiver Operating Characteristic Curve (AUROC) and Fisher's Ratio. Finally, using all the four EDA features, a multi-layer perceptron (MLP) classifier reached the classification accuracies 95.56%, 89.29%, and 67.88% for the VM, Stroop, and thermal-grill pain control and stimulation classes. On the other hand, the highest classification accuracies based on SKNA features were achieved using K-nearest neighbors (KNN) (98.89%), KNN (89.29%), and MLP (95.11%) classifiers for the same experiments. Our comparative analysis showed the feasibility of SKNA as a novel tool for assessing the SNS with accurate classification capability, with a faster onset of amplitude increase in response to SNS activity, compared to EDA.

Human Bronchial Epithelial Cell Growth on Homologous Versus Heterologous Tissue Extracellular Matrix.

BACKGROUND: Extracellular matrix (ECM) bioscaffolds produced by decellularization of source tissue have been effectively used for numerous clinical applications. However, decellularized tracheal constructs have been unsuccessful due to the immediate requirement of a functional airway epithelium on surgical implantation. ECM can be solubilized to form hydrogels that have been shown to support growth of many different cell types. The purpose of the present study is to compare the ability of airway epithelial cells to attach, form a confluent monolayer, and differentiate on homologous (trachea) and heterologous (urinary bladder) ECM substrates for potential application in full tracheal replacement. MATERIALS AND METHODS: Porcine tracheas and urinary bladders were decellularized. Human bronchial epithelial cells (HBECs) were cultured under differentiation conditions on acellular tracheal ECM and urinary bladder matrix (UBM) bioscaffolds and hydrogels and were assessed by histology and immunolabeling for markers of ciliation, goblet cell formation, and basement membrane deposition. RESULTS: Both trachea and urinary bladder tissues were successfully decellularized. HBEC formed a confluent layer on both trachea and UBM scaffolds and on hydrogels created from these bioscaffolds. Cells grown on tracheal and UBM hydrogels, but not on bioscaffolds, showed positive-acetylated tubulin staining and the presence of mucus-producing goblet cells. Collagen IV immunolabeling showed basement membrane deposition by these cells on the surface of the hydrogels. CONCLUSIONS: ECM hydrogels supported growth and differentiation of HBEC better than decellularized ECM bioscaffolds and show potential utility as substrates for promotion of a mature respiratory epithelium for regenerative medicine applications in the trachea.

Role of 4-hydroxybutyrate in increased resistance to surgical site infections associated with surgical meshes.

An increased resistance to surgical site infections has been associated with surgical meshes composed of naturally occurring materials, including poly-4-hydroxybutrate (4HB). 4HB is a naturally occurring short-chain fatty acid that has been shown to promote endogenous expression of the Cramp gene coding for the antimicrobial peptide (AMP) cathelicidin LL-37 in murine bone marrow-derived macrophages. The molecular pathways involved in the 4HB-induced cathelicidin LL-37 expression have not yet been identified. The present study showed that transcriptional activation of the Cramp gene by 4HB is independent of inhibition of histone deacetylase (HDAC) activity, and that upregulation of Cramp is modulated by the G-protein coupled receptor GPR109A. Furthermore, an intracellular signaling cascade that promotes the activation of the MAP kinases, p38 and JNK, and a subsequent NF-κB phosphorylation downstream from p38 is essential for the AMP transcriptional response in 4HB-stimulated macrophages. The findings provide a solid scientific basis and rationale for the decreased incidence of surgical site infections with the use of this type of surgical meshes. Further clinical significance is found in the fact that the 4HB activated molecular pathway includes common targets of frequently used nonsteroidal anti-inflammatory drugs (NSAIDs) and other FDA approved drugs recognizing G-protein coupled receptors.

Ultrasonic cavitation to prepare ECM hydrogels.

Hydrogels composed of extracellular matrix (ECM) have been used as a substrate for 3D organoid culture, and in numerous preclinical and clinical applications to facilitate repair and reconstruction of a variety of tissues. However, these ECM hydrogel materials are fabricated using lengthy methods that have focused on enzymatic digestion of the ECM with an acid protease in an acidic solution; or the use of chaotropic extraction buffers and dialysis procedures which can affect native protein structure and function. Herein we report a method to prepare hydrogels from ECM bioscaffolds using ultrasonic cavitation. The solubilized ECM can be induced to rapidly self-assemble into a gel by adjusting temperature, and the material properties of the gel can be tailored by adjusting ECM concentration and sonication parameters. The present study shows that ECM bioscaffolds can be successfully solubilized without enzymatic digestion and induced to repolymerize into a gel form capable of supporting cell growth. STATEMENT OF SIGNIFICANCE: ECM hydrogels have been used in numerous preclinical studies to facilitate repair of tissue following injury. However, there has been relatively little advancement in manufacturing techniques, thereby impeding progress in advancing this technology toward the clinic. Laboratory techniques for producing ECM hydrogels have focused on protease digestion methods, which require lengthy incubation times. The significance of this work lies in the development of a fundamentally different approach whereby an ECM hydrogel is rapidly formed without the need for acidic solutions or protease digestion. The ultrasonic cavitation method described herein represents a marked improvement in rheological properties and processing time over traditional enzymatic methods, and may lend itself as a platform for large-scale manufacturing of ECM hydrogels.

Discovery and Characterization of Bacteriophage LuckyBarnes.

Here, we report the genome sequence of LuckyBarnes, a newly isolated singleton siphovirus that infects Brevibacterium iodinum ATCC 15728 and has a 50,774-bp genome with 67 predicted genes.

The challenge of stress incontinence and pelvic organ prolapse: revisiting biologic mesh materials.

PURPOSE OF REVIEW: The present article reviews the history of mesh-related complications and regulations in SUI and POP repair settings, clinical outcomes associated with the use of biologic and synthetic mesh materials, and novel approaches using modified mesh materials. RECENT FINDINGS: Treatment of pelvic floor disorders, such as stress urinary incontinence (SUI) and pelvic organ prolapse (POP) commonly involves implantation of synthetic surgical mesh materials like polypropylene. Many synthetic mesh materials, however, are associated with a foreign body response upon implantation, which is characterized by fibrotic encapsulation. Complications, including erosion, infections, bleeding, and chronic pain, have led to warnings by regulatory agencies and the recall of several mesh products. To mitigate such complications, biologic mesh materials have been proposed as alternatives for SUI and POP repair. SUMMARY: Clinical outcomes of surgical repair of POP/SUI are similar between biologic and synthetic meshes, but biologic meshes have a lower incidence of adverse effects. Several strategies for modifying or functionalizing biological and synthetic meshes have shown promising results in preclinical studies.

Characterization of embryonic stem cell-differentiated fibroblasts as mesenchymal stem cells with robust expansion capacity and attenuated innate immunity.

BACKGROUND: Mesenchymal stem cells (MSCs) isolated from adult tissues (Ad-MSCs) have shown great promise for use in regenerative medicine. However, their poor in vitro expansion capacity and tissue scarcity have been major limitations. In this study, we demonstrate that mouse embryonic stem cells (mESCs) can differentiate into cells with MSC properties. METHODS: Using previously established methods that characterize Ad-MSCs, we analyzed mESC-differentiated fibroblasts (mESC-FBs), including plastic adherence, clonogenic growth, MSC marker expression, tri-lineage differentiation potential, and the capacity to express immunomodulators. RESULTS: Although previously characterized as mESC-differentiated fibroblasts (mESC-FBs), these cells exhibit major properties of Ad-MSCs. However, mESC-FBs also display unique features inherited from ESCs, including robust expansion capacity, senescence resistance, and attenuated innate immunity. In particular, mESC-FBs are insensitive to bacterial endotoxin (lipopolysaccharide, LPS) and do not express LPS-induced inflammatory molecules, in contrast to bone marrow (BM)-MSCs. We further demonstrate that mESC-FBs are resistant to the cytotoxicity associated with inflammatory cytokines, bacterial endotoxins (LPS and heat-killed bacteria), and macrophage-mediated inflammation. CONCLUSIONS: While it remains to be determined how the unique properties of mESC-FBs will affect their immunoregulatory activity under an in vivo condition, our findings demonstrate that ESCs could be used as an alternative source to generate a new class of ESC-MSCs with unique features potentially useful in regenerative medicine.

Genome Sequences of Mycobacteriophages Amgine, Amohnition, Bella96, Cain, DarthP, Hammy, Krueger, LastHope, Peanam, PhelpsODU, Phrank, SirPhilip, Slimphazie, and Unicorn.

We report the genome sequences of 14 cluster K mycobacteriophages isolated using Mycobacterium smegmatis mc²155 as host. Four are closely related to subcluster K1 phages, and 10 are members of subcluster K6. The phage genomes span considerable sequence diversity, including multiple types of integrases and integration sites.

The Molecular Basis for the Lack of Inflammatory Responses in Mouse Embryonic Stem Cells and Their Differentiated Cells.

We reported previously that mouse embryonic stem cells do not have a functional IFN-based antiviral mechanism. The current study extends our investigation to the inflammatory response in mouse embryonic stem cells and mouse embryonic stem cell-differentiated cells. We demonstrate that LPS, TNF-α, and viral infection, all of which induce robust inflammatory responses in naturally differentiated cells, failed to activate NF-κB, the key transcription factor that mediates inflammatory responses, and were unable to induce the expression of inflammatory genes in mouse embryonic stem cells. Similar results were obtained in human embryonic stem cells. In addition to the inactive state of NF-κB, the deficiency in the inflammatory response in mouse embryonic stem cells is also attributed to the lack of functional receptors for LPS and TNF-α. In vitro differentiation can trigger the development of the inflammatory response mechanism, as indicated by the transition of NF-κB from its inactive to active state. However, a limited response to TNF-α and viral infection, but not to LPS, was observed in mouse embryonic stem cell-differentiated fibroblasts. We conclude that the inflammatory response mechanism is not active in mouse embryonic stem cells, and in vitro differentiation promotes only partial development of this mechanism. Together with our previous studies, the findings described in this article demonstrate that embryonic stem cells are fundamentally different from differentiated somatic cells in their innate immunity, which may have important implications in developmental biology, immunology, and embryonic stem cell-based regenerative medicine.

Genome Sequence of Mycobacterium Phage Waterfoul.

Waterfoul is a newly isolated temperate siphovirus of Mycobacterium smegmatis mc2155. It was identified as a member of the K5 cluster of Mycobacterium phages and has a 61,248-bp genome with 95 predicted genes.

Development of Antiviral Innate Immunity During In Vitro Differentiation of Mouse Embryonic Stem Cells.

The innate immunity of embryonic stem cells (ESCs) has recently emerged as an important issue in ESC biology and in ESC-based regenerative medicine. We have recently reported that mouse ESCs (mESCs) do not have a functional type I interferon (IFN)-based antiviral innate immunity. They are deficient in expressing IFN in response to viral infection and have limited ability to respond to IFN. Using fibroblasts (FBs) as a cell model, the current study investigated the development of antiviral mechanisms during in vitro differentiation of mESCs. We demonstrate that mESC-differentiated FBs (mESC-FBs) share extensive similarities with naturally differentiated FBs in morphology, marker expression, and growth pattern, but their development of antiviral mechanisms lags behind. Nonetheless, the antiviral mechanisms are inducible during mESC differentiation as demonstrated by the transition of nuclear factor kappa B (NFκB), a key transcription factor for IFN expression, from its inactive state in mESCs to its active state in mESC-FBs and by increased responses of mESC-FBs to viral stimuli and IFN during their continued in vitro propagation. Together with our previously published study, the current data provide important insights into molecular basis for the deficiency of IFN expression in mESCs and the development of antiviral innate immunity during mESC differentiation.

Neural latencies across auditory cortex of macaque support a dorsal stream supramodal timing advantage in primates.

Sensory systems across the brain are specialized for their input, yet some principles of neural organization are conserved across modalities. The pattern of anatomical connections from the primate auditory cortex to the temporal, parietal, and prefrontal lobes suggests a possible division into dorsal and ventral auditory processing streams, with the dorsal stream originating from more caudal areas of the auditory cortex, and the ventral stream originating from more rostral areas. These streams are hypothesized to be analogous to the well-established dorsal and ventral streams of visual processing. In the visual system, the dorsal processing stream shows substantially faster neural response latencies than does the ventral stream. However, the relative timing of putative dorsal and ventral stream processing has yet to be explored in other sensory modalities. Here, we compare distributions of neural response latencies from 10 different areas of macaque auditory cortex, confirmed by individual anatomical reconstructions, to determine whether a similar timing advantage is found for the hypothesized dorsal auditory stream. Across three varieties of auditory stimuli (clicks, noise, and pure tones), we find that latencies increase with hierarchical level, as predicted by anatomical connectivity. Critically, we also find a pronounced timing differential along the caudal-to-rostral axis within the same hierarchical level, with caudal (dorsal stream) latencies being faster than rostral (ventral stream) latencies. This observed timing differential mirrors that found for the dorsal stream of the visual system, suggestive of a common timing advantage for the dorsal stream across sensory modalities.

Auditory cortical tuning to band-pass noise in primate A1 and CM: a comparison to pure tones.

We examined multiunit responses to tones and to 1/3 and 2/3 octave band-pass noise (BPN) in the marmoset primary auditory cortex (A1) and the caudomedial belt (CM). In both areas, BPN was more effective than tones, evoking multiunit responses at lower intensity and across a wider frequency range. Typically, the best responses to BPN remained at the characteristic frequency. Additionally, in both areas responses to BPN tended to be of greater magnitude and shorter latency than responses to tones. These effects are consistent with the integration of more excitatory inputs driven by BPN than by tones. While it is generally thought that single units in A1 prefer narrow band sounds such as tones, we found that best responses for multi units in both A1 and CM were obtained with noises of narrow spectral bandwidths.

Coding of FM sweep trains and twitter calls in area CM of marmoset auditory cortex.

The primate auditory cortex contains three interconnected regions (core, belt, parabelt), which are further subdivided into discrete areas. The caudomedial area (CM) is one of about seven areas in the belt region that has been the subject of recent anatomical and physiological studies conducted to define the functional organization of auditory cortex. The main goal of the present study was to examine temporal coding in area CM of marmoset monkeys using two related classes of acoustic stimuli: (1) marmoset twitter calls; and (2) frequency-modulated (FM) sweep trains modeled after the twitter call. The FM sweep trains were presented at repetition rates between 1 and 24 Hz, overlapping the natural phrase frequency of the twitter call (6-8 Hz). Multiunit recordings in CM revealed robust phase-locked responses to twitter calls and FM sweep trains. For the latter, phase-locking quantified by vector strength (VS) was best at repetition rates between 2 and 8 Hz, with a mean of about 5 Hz. Temporal response patterns were not strictly phase-locked, but exhibited dynamic features that varied with the repetition rate. To examine these properties, classification of the repetition rate from the temporal response pattern evoked by twitter calls and FM sweep trains was examined by Fisher's linear discrimination analysis (LDA). Response classification by LDA revealed that information was encoded not only by phase-locking, but also other components of the temporal response pattern. For FM sweep trains, classification was best for repetition rates from 2 to 8 Hz. Thus, the majority of neurons in CM can accurately encode the envelopes of temporally complex stimuli over the behaviorally-relevant range of the twitter call. This suggests that CM could be engaged in processing that requires relatively precise temporal envelope discrimination, and supports the hypothesis that CM is positioned at an early stage of processing in the auditory cortex of primates.

Neurosurgical access to cortical areas in the lateral fissure of primates.

In this report, a method is presented for gaining direct access to cortical areas within the lateral fissure of primates for neuroanatomical tracer injections and electrode array implantation. Compared to areas on the surface of the brain, the anatomical and physiological properties of areas within the fissure are poorly understood. Typically, access to these areas is indirectly achieved by ablating or passing through intervening areas. To enable direct experimental access, a neurosurgical technique was developed in primates whereby the banks of the lateral fissure were retracted with sparing of the vascular network and intervening areas. In some animals, anatomical tracers were directly injected into target fields without contamination of other areas. In others, multichannel electrode arrays were implanted into target areas for chronic recording of neural activity. Since, these techniques could be adapted for exploration of areas within other sulci, the approach represents an important advance in efforts to elucidate the functional organization of the primate cerebral cortex.

GABA( A) synapses shape neuronal responses to sound intensity in the inferior colliculus.

Neurons in the inferior colliculus (IC) change their firing rates with sound pressure level. Some neurons maintain monotonic increases in firing rate over a wide range of sound intensities, whereas other neurons are monotonic over limited intensity ranges. We examined the conditions necessary for monotonicity in this nucleus in vitro in rat brain slices and in vivo in the unanesthetized rabbit. Our in vitro recordings indicate that concurrent activation of GABA(A) synapses with excitatory inputs facilitates monotonic increases in firing rate with increases in stimulus strength. In the absence of synaptic inhibition, excitatory input to IC neurons causes large depolarizations that result in firing block and nonmonotonicity. In vivo, although GABA(A) synapses decrease the firing rate in all IC neurons, they can have opposing effects on rate-level functions. GABAergic inputs activated by all sound intensities maintain monotonicity by keeping the postsynaptic potential below the level at which depolarization block occurs. When these inputs are blocked, firing block can occur and rate-level functions become nonmonotonic. High-threshold GABAergic inputs, in contrast, cause nonmonotonic responses by decreasing the firing rate at high intensities. Our results suggest that a dynamic regulation of the postsynaptic membrane potential by synaptic inhibition is necessary to allow neurons to respond monotonically to a wide range of sound intensities.

Sources of the scalp-recorded amplitude-modulation following response.

The scalp-recorded amplitude-modulation following response (AMFR) is gaining recognition as an objective audiometric tool, but little is known about the neural sources that underlie this potential. We hypothesized, based on our human studies and single-unit recordings in animals, that the scalp-recorded AMFR reflects the interaction of multiple sources. We tested this hypothesis using an animal model, the unanesthetized rabbit. We compared AMFRs recorded from the surface of the brain at different locations and before and after the administration of agents likely to enhance or suppress neural generators. We also recorded AMFRs locally at several stations along the auditory neuraxis. We conclude that the surface-recorded AMFR is indeed a composite response from multiple brain generators. Although the response at any modulation frequency can reflect the activity of more than one generator, the AMFRs to low and high modulation frequencies appear to reflect a strong contribution from cortical and subcortical sources, respectively.