Increasing Proteome Coverage Through a Reduction in Analyte Complexity in Single-Cell Equivalent Samples.

The advancement of sophisticated instrumentation in mass spectrometry has catalyzed an in-depth exploration of complex proteomes. This exploration necessitates a nuanced balance in experimental design, particularly between quantitative precision and the enumeration of analytes detected. In bottom-up proteomics, a key challenge is that oversampling of abundant proteins can adversely affect the identification of a diverse array of unique proteins. This issue is especially pronounced in samples with limited analytes, such as small tissue biopsies or single-cell samples. Methods such as depletion and fractionation are suboptimal to reduce oversampling in single cell samples, and other improvements on LC and mass spectrometry technologies and methods have been developed to address the trade-off between precision and enumeration. We demonstrate that by using a monosubstrate protease for proteomic analysis of single-cell equivalent digest samples, an improvement in quantitative accuracy can be achieved, while maintaining high proteome coverage established by trypsin. This improvement is particularly vital for the field of single-cell proteomics, where single-cell samples with limited number of protein copies, especially in the context of low-abundance proteins, can benefit from considering analyte complexity. Considerations about analyte complexity, alongside chromatographic complexity, integration with data acquisition methods, and other factors such as those involving enzyme kinetics, will be crucial in the design of future single-cell workflows.

Integrated Neurophotonics: Toward Dense Volumetric Interrogation of Brain Circuit Activity-at Depth and in Real Time.

We propose a new paradigm for dense functional imaging of brain activity to surmount the limitations of present methodologies. We term this approach "integrated neurophotonics"; it combines recent advances in microchip-based integrated photonic and electronic circuitry with those from optogenetics. This approach has the potential to enable lens-less functional imaging from within the brain itself to achieve dense, large-scale stimulation and recording of brain activity with cellular resolution at arbitrary depths. We perform a computational study of several prototype 3D architectures for implantable probe-array modules that are designed to provide fast and dense single-cell resolution (e.g., within a 1-mm3 volume of mouse cortex comprising ∼100,000 neurons). We describe progress toward realizing integrated neurophotonic imaging modules, which can be produced en masse with current semiconductor foundry protocols for chip manufacturing. Implantation of multiple modules can cover extended brain regions.

Effect of pulsed electromagnetic field treatment on programmed resolution of inflammation pathway markers in human cells in culture.

Inflammation is a complex process involving distinct but overlapping biochemical and molecular events that are highly regulated. Pulsed electromagnetic field (PEMF) therapy is increasingly used to treat pain and edema associated with inflammation following surgery involving soft tissue. However, the molecular and cellular effects of PEMF therapy on pathways involved in the resolution of inflammation are poorly understood. Using cell culture lines relevant to trauma-induced inflammation of the skin (human dermal fibroblasts, human epidermal keratinocytes, and human mononuclear cells), we investigated the effect of PEMF on gene expression involved in the acute and resolution phases of inflammation. We found that PEMF treatment was followed by changes in the relative amount of messenger (m)RNAs encoding enzymes involved in heme catabolism and removal of reactive oxygen species, including an increase in heme oxygenase 1 and superoxide dismutase 3 mRNAs, in all cell types examined 2 hours after PEMF treatment. A relative increase in mRNAs encoding enzymes involved in lipid mediator biosynthesis was also observed, including an increase in arachidonate 12- and 15-lipoxygenase mRNAs in dermal fibroblasts and epidermal keratinocytes, respectively. The relative amount of both of these lipoxygenase mRNAs was elevated in mononuclear cells following PEMF treatment relative to nontreated cells. PEMF treatment was also followed by changes in the mRNA levels of several cytokines. A decrease in the relative amount of interleukin 1 beta mRNA was observed in mononuclear cells, similar to that previously reported for epidermal keratinocytes and dermal fibroblasts. Based on our results, we propose a model in which PEMF therapy may promote chronic inflammation resolution by mediating gene expression changes important for inhibiting and resolving inflammation.

An open-label pilot study of pulsed electromagnetic field therapy in the treatment of failed back surgery syndrome pain.

Persistent pain following back surgery remains a major treatment challenge. The primary objective of this open-label exploratory study was to investigate the analgesic effectiveness of pulsed electromagnetic field therapy administered twice daily over a 45-day period in 34 subjects (68% female) with persistent or recurrent pain following back surgery. A secondary goal was to guide the design of future randomized controlled trials that could target responsive subpopulations. All predefined primary and secondary outcomes, including change in pain intensity (PI), physical function (Oswestry Disability Index), analgesic consumption, and overall well-being (Patient Global Impression of Change), are reported. A responder analysis (≥30% reduction in PI versus baseline) was added as a post hoc evaluation. Safety outcomes, as well as results of a cost-avoidance survey, are also summarized. Of the 30 per-protocol subjects who completed the study, 33% reported a clinically meaningful (≥30%) reduction in PI. A higher response rate (60%) was reported for subjects who had undergone discectomy prior to the trial compared to subjects who had undergone other types of surgical interventions (decompression or fusion) without discectomy. Improvements in PI were paralleled by improvements in secondary outcomes. Relative to baseline, responders reported an average 44% and 55% reduction in back PI and leg PI (respectively), and an average 13% improvement in Oswestry Disability Index scores. In the per-protocol population, 50% of responders and 12% of nonresponders reported less analgesia consumption at the end of treatment versus baseline. Sixty-seven percent of per-protocol responders and 0% of nonresponders reported clinically meaningful improvement in overall well-being on the Patient Global Impression of Change scale.

The management of intractable pain with adjuvant pulsed electromagnetic field therapy.

This case describes a 51-year-old woman who reported experiencing severe, constant pain, diffusely located in the region of her right mandible neck (primarily involving the mandible, lower right molars, the neck, the upper back, and the shoulder) during the course of several years. Surgical interventions (root canal, spinal fusion) were performed to address potential sources of pain. Despite these interventions, the patient reported severe pain after both surgeries, which persisted beyond the acute postoperative period. Additional pharmacological interventions and physical therapy were also attempted; nonetheless, the patient reported that pain remained severe and constant for approximately 2 years. On the basis of the patient's poor response to conventional treatments, a novel approach of botulinum toxin (BTX) injections was initiated. When pulsed electromagnetic field therapy was added, the need for BTX injections decreased, with the patient reporting a noticeable decrease in pain intensity and an improvement in quality of life measures. Currently, the patient continues to use pulsed electromagnetic field therapy regularly for pain management, which has allowed her to reduce the use of other interventions and avoid continued use of narcotic medications. Considering the need for multifaceted pain management approaches in the treatment of chronic pain, this case is relevant for wound care practitioners attending to patients with chronic postincisional wound pain because the outcome highlights the utility of a nonpharmacological, complementary pain management intervention for closed, yet persistently painful, postoperative wounds.

Pulsed radio frequency energy therapy use for pain relief following surgery for tendinopathy-associated chronic pain: two case reports.

Chronic tendon pain from overuse is a common condition, with limited options for ongoing pain management. Two cases are presented in which pulsed radio frequency energy (PRFE) therapy was used for pain relief following surgical intervention for chronic tendinopathy-associated pain, unresponsive to conventional therapies. Both patients showed a dramatic reduction in pain following PRFE therapy after 2 to 3 weeks of treatment, and at the 7-month (case 1) and 6-month (case 2) follow-up visits, both patients reported that pain had not returned. Recent molecular evidence suggests a possible mechanism underlying PRFE-mediated pain relief. Further study into this promising technology is warranted.

Activation of endogenous opioid gene expression in human keratinocytes and fibroblasts by pulsed radiofrequency energy fields.

BACKGROUND: Pulsed radiofrequency energy (PRFE) fields are being used increasingly for the treatment of pain arising from dermal trauma. However, despite their increased use, little is known about the biological and molecular mechanism(s) responsible for PRFE-mediated analgesia. In general, current therapeutics used for analgesia target either endogenous factors involved in inflammation, or act on endogenous opioid pathways. METHODS AND RESULTS: Using cultured human dermal fibroblasts (HDF) and human epidermal keratinocytes (HEK), we investigated the effect of PRFE treatment on factors, which are involved in modulating peripheral analgesia in vivo. We found that PRFE treatment did not inhibit cyclooxygenase enzyme activity, but instead had a positive effect on levels of endogenous opioid precursor mRNA (proenkephalin, pro-opiomelanocortin, prodynorphin) and corresponding opioid peptide. In HEK cells, increases in opioid mRNA were dependent, at least in part, on endothelin-1. In HDF cells, additional pathways also appear to be involved. PRFE treatment was also followed by changes in endogenous expression of several cytokines, including increased levels of interleukin-10 mRNA and decreased levels of interleukin-1ß mRNA in both cell types. CONCLUSION: These findings provide a new insight into the molecular mechanism underlying PRFE-mediated analgesia reported in the clinical setting.

Meta-analysis of clinical efficacy of pulsed radio frequency energy treatment.

OBJECTIVE: To statistically evaluate published clinical efficacy data related to the use of pulsed radio frequency energy (PRFE) therapy in 3 clinical applications. BACKGROUND: Numerous clinical studies have reported efficacy outcomes for PRFE therapy use in the palliative treatment of both postoperative and nonpostoperative pain and edema, and for its use as an adjunctive wound-healing (WH) therapeutic. Although diverse in design and endpoint, these studies are amenable to systematic review using both a vote-counting and P-value combination meta-analytic technique. METHODS: A meta-analysis of efficacy outcomes reported in clinical trials was performed using a vote-counting procedure. In addition, when possible, the sum of logs method of P-value combination was used to determine a significance level for the combined evidence within each endpoint and clinical area. RESULTS: Of the 186 clinical articles identified after application of selection criteria, there were 25 controlled trials that met criteria for inclusion in vote-counting and P-value combination methods and were used for formal statistical analysis. In total, 1332 patients receiving PRFE treatment were studied. Vote-counting procedure applied to clinical outcomes from controlled studies resulted in a greater number of positive outcomes than neutral outcomes, and zero negative outcomes, for each of the clinical application groups evaluated. The sum of logs P-value method found statistically significant improvement in pain, reduction in edema, and improvement in WH outcomes. CONCLUSIONS: On the basis of statistical evaluation of published clinical efficacy data, there is strong statistical evidence that PRFE therapy is effective in the treatment of postoperative and nonpostoperative pain and edema and in WH applications.

Pulsed radio frequency energy (PRFE) use in human medical applications.

A number of electromagnetic field-based technologies are available for therapeutic medical applications. These therapies can be broken down into different categories based on technical parameters employed and type of clinical application. Pulsed radio frequency energy (PRFE) therapy is a non invasive, electromagnetic field-based therapeutic that is based on delivery of pulsed, shortwave radio frequency energy in the 13-27.12 MHz carrier frequency range, and designed for local application to a target tissue without the intended generation of deep heat. It has been studied for use in a number of clinical applications, including as a palliative treatment for both postoperative and non postoperative pain and edema, as well as in wound healing applications. This review provides an introduction to the therapy, a summary of clinical efficacy studies using the therapy in specific applications, and an overview of treatment-related safety.

Pro-hepcidin is unable to degrade the iron exporter ferroportin unless maturated by a furin-dependent process.

BACKGROUND/AIMS: The iron-regulatory peptide hepcidin is synthesized in the liver as an 84-aa pre-pro-hormone maturated by proteolysis through a consensus furin cleavage site to generate the bioactive 25-aa peptide secreted in the circulation. This peptide regulates iron export from enterocytes and macrophages by binding the membrane iron exporter, ferroportin, leading to its degradation. Whether pro-hepcidin could be secreted and reflect hepcidin levels remains an open question. However, the activity of the pro-peptide on ferroportin degradation has never been addressed. METHODS: To answer this question, we produced recombinant pro-hepcidin, both the wild-type form and a furin cleavage site mutant, and tested their activity on ferroportin levels in macrophagic J774 cells. Furin activity was also modulated using furin inhibitor or siRNA-mediated furin mRNA knockdown. RESULTS: We found that pro-hepcidin could fully induce ferroportin degradation, but only when processed by furin to generate the mature hepcidin-25 form. Pro-hepcidin activity was abolished in the presence of furin inhibitor and diminished after siRNA-mediated knockdown of furin mRNA. Furthermore, the mutated version of pro-hepcidin was completely inefficient at degrading ferroportin in macrophages. CONCLUSIONS: Our results demonstrate that pro-hepcidin lacks biological activity, unless fully maturated by a furin-dependent process to yield the bioactive 25-aa peptide.

von Hippel Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step.

HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin-proteasome pathway. Here, we identify von Hippel-Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel-Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin-VHL-proteasome pathway in the integration-transcription transition of the viral replication cycle.

Luman, a new partner of HIV-1 TMgp41, interferes with Tat-mediated transcription of the HIV-1 LTR.

In our search for new partners of the HIV-1 envelope glycoprotein (Env), we found that the cytoplasmic domain of the TMgp41 (TMgp41 CD) subunit of HIV-1 Env interacted with Luman, a transcription factor of the CREB/ATF family. Luman is anchored in the endoplasmic reticulum membrane and subjected to activation by regulated intramembrane proteolysis (RIP). The RIP process permits the release of the activated amino-terminal fragment of Luman into the cytoplasm, and its import into the nucleus. Here, we demonstrate that interaction between the TMgp41 CD and Luman requires a region encompassing the b-Zip and TM domains of Luman and decreases the stability of this factor. Moreover, we found that overexpression of a constitutively active form of Luman in cells transfected with HXB2R HIV-1 provirus decreased the intracellular expression of Gag and Env and led to a decrease in virion release. This negative effect of activated Luman on HIV-1 production was correlated to the inhibition of Tat transactivation of the HIV-1 LTR, which might be related to an interaction of activated Luman with Tat. Altogether, these results show that Luman acts as a partner of two major HIV-1 proteins: the TMgp41 Env subunit and Tat. The interaction between the TMgp41 subunit of Env and Luman affects the stability of the full-length Luman protein, the precursor of the activated, nuclear form of Luman, which acts negatively on Tat-mediated HIV-1 transactivation.

Deacetylation of the herpes simplex virus type 1 latency-associated transcript (LAT) enhancer and a decrease in LAT abundance precede an increase in ICP0 transcriptional permissiveness at early times postexplant.

Only the latency-associated transcript (LAT) of the herpes simplex virus type 1 (HSV-1) genome is transcribed during latency, while the lytic genes are suppressed, possibly by LAT antisense mechanisms and/or chromatin modifications. In the present study, latently infected dorsal root ganglia were explanted to assess both relative levels of LAT and histone H3 (K9, K14) acetylation of the LAT locus and ICP0 promoter at early times postexplant. We observed that a decrease in both LAT enhancer histone H3 (K9, K14) acetylation and LAT RNA abundance occurs prior to an increase in acetylation, or transcriptional permissiveness, at the ICP0 promoter.

The herpes simplex virus type 1 latency-associated transcript (LAT) enhancer/rcr is hyperacetylated during latency independently of LAT transcription.

During herpes simplex virus type 1 (HSV-1) latency, only one region of the viral genome is actively transcribed: the region encoding the latency-associated transcript (LAT). A previous study demonstrated that during latency the LAT promoter is hyperacetylated at histone H3 (K9, K14) relative to lytic genes examined. In the present study, we examine the acetylation profile of regions downstream of the LAT promoter during a latent infection of murine dorsal root ganglia. These analyses revealed the following: (i) the region of the genome containing the 5' exon of the LAT primary transcript was at least as enriched in acetylated H3 as the LAT promoter, and (ii) the region of hyperacetylation does not extend to the ICP0 promoter. In order to assess the contribution of LAT transcription to the acetylation of the 5' exon region, the acetylation profile of KOS/29, a recombinant with a deletion of the LAT promoter, was examined. The region containing the 5' exon of KOS/29 was hyperacetylated relative to lytic gene regions in the absence of detectable LAT transcription. These results indicate that the region containing the 5' exon of LAT, known to contain enhancer activities and to be critical for induced reactivation (rcr), exists in a chromatin structure during latency that is distinct from other lytic gene regions. This result suggests a role for the 5' exon LAT enhancer region as a cis-acting regulator of transcription that maintains a transcriptionally permissive chromatin domain in the HSV-1 latent episome.

Specific histone tail modification and not DNA methylation is a determinant of herpes simplex virus type 1 latent gene expression.

During herpes simplex virus type 1 (HSV-1) latency, gene expression is tightly repressed except for the latency-associated transcript (LAT). The mechanistic basis for this repression is unknown, but its global nature suggests regulation by an epigenetic mechanism such as DNA methylation. Previous work demonstrated that latent HSV-1 genomes are not extensively methylated, but these studies lacked the resolution to examine methylation of individual CpGs that could repress transcription from individual promoters during latency. To address this point, we employed established models to predict genomic regions with the highest probability of being methylated and, using bisulfite sequencing, analyzed the methylation profiles of these regions. We found no significant methylation of latent DNA isolated from mouse dorsal root ganglia in any of the regions examined, including the ICP4 and LAT promoters. This analysis indicates that methylation is unlikely to play a major role in regulating HSV-1 latent gene expression. Subsequently we focused on differential histone modification as another epigenetic mechanism that could regulate latent transcription. Chromatin immunoprecipitation analysis of the latent HSV-1 DNA repeat regions demonstrated that a portion of the LAT region is associated with histone H3 acetylated at lysines 9 and 14, consistent with a euchromatic and nonrepressed structure. In contrast, the chromatin associated with the HSV-1 DNA polymerase gene located in the unique long segment was not enriched in H3 acetylated at lysines 9 and 14, suggesting a transcriptionally inactive structure. These data suggest that histone composition may be a major regulatory determinant of HSV latency.