Naturalistic acute pain states decoded from neural and facial dynamics.

Pain is a complex experience that remains largely unexplored in naturalistic contexts, hindering our understanding of its neurobehavioral representation in ecologically valid settings. To address this, we employed a multimodal, data-driven approach integrating intracranial electroencephalography, pain self-reports, and facial expression quantification to characterize the neural and behavioral correlates of naturalistic acute pain in twelve epilepsy patients undergoing continuous monitoring with neural and audiovisual recordings. High self-reported pain states were associated with elevated blood pressure, increased pain medication use, and distinct facial muscle activations. Using machine learning, we successfully decoded individual participants' high versus low self-reported pain states from distributed neural activity patterns (mean AUC = 0.70), involving mesolimbic regions, striatum, and temporoparietal cortex. High self-reported pain states exhibited increased low-frequency activity in temporoparietal areas and decreased high-frequency activity in mesolimbic regions (hippocampus, cingulate, and orbitofrontal cortex) compared to low pain states. This neural pain representation remained stable for hours and was modulated by pain onset and relief. Objective facial expression changes also classified self-reported pain states, with results concordant with electrophysiological predictions. Importantly, we identified transient periods of momentary pain as a distinct naturalistic acute pain measure, which could be reliably differentiated from affect-neutral periods using intracranial and facial features, albeit with neural and facial patterns distinct from self-reported pain. These findings reveal reliable neurobehavioral markers of naturalistic acute pain across contexts and timescales, underscoring the potential for developing personalized pain interventions in real-world settings.

Application of the novel Nalu™ Neurostimulation System for peripheral nerve stimulation.

Peripheral nerve stimulation is an established treatment modality for chronic neuropathic pain. Over the last decade, with the advent of innovative devices and delivery platforms, peripheral nerve stimulation has evolved from invasive open surgeries to image-guided, minimally invasive percutaneous procedures. The authors hereby present a novel device, the Nalu™ Neurostimulation System (Nalu Medical, CA, USA), which has established its advantages in providing predictable and reliable peripheral nerve stimulation therapy for chronic neuropathic pain management. This novel device is effective in treating chronic pain conditions such as post-herniorrhaphy pain syndrome, intercostal neuralgia, post-laminectomy syndrome, and complex regional pain syndrome and holds great promise for the treatment of peripheral neuropathic pain.

Chronic nerve pain is a debilitating condition that can affect quality of life and functioning. The Nalu™ Neurostimulation System (Nalu Medical, CA, USA) provides long-term pain relief without medications. There are numerous devices currently available that can be utilized to block pain signals using small wires. This system is unique because the wires placed over affected nerves are powered by an external battery that does not require permanent surgical implantation. Pain after hernia surgery, back surgery, hip surgery and knee surgery, as well as nerve pain can be effectively managed by this system.

Considerations in permanent implantation of peripheral nerve stimulation (PNS) for chronic neuropathic pain: An international cross-sectional survey of implanters.

AIM: Novel minimally invasive short-term and long-term peripheral nerve stimulation (PNS) systems have revolutionized targeted treatment of chronic neuropathic pain. We present an international survey of PNS-implanting pain physicians to assess what factors they consider when offering permanent PNS. METHODS: This cross-sectional study consisted of a survey (Qualtrics) that was distributed to PNS-implanting physicians in a device supplier's entire email database on November 13, 2020, with 3 weeks of response time. Physicians' contact information in the form of their email addresses had been previously collected by the supplier upon device distribution with permission to use survey responses for research. RESULTS: Of 2032 database physicians, 40 physicians representing 37 institutions responded to the survey. The most common application of PNS was mononeuropathic pain (57%). The most frequently targeted nerve was the suprascapular nerve (29%). 14% of physicians reported 81-100% of their implants were dual-lead. The representative physicians ranged broadly in their most frequently targeted nerves. Although mononeuropathic pain was the most common indication for PNS, there was still varied response regarding other indications such as CRPS and post-surgical chronic pain. CONCLUSION: In context of a low response rate, identifying such factors can help update the prevailing treatment algorithm for interventional therapies, assist pain physicians in better identifying which patients are the best candidates for PNS, and inform future clinical trial design on PNS efficacy.

Preclinical characterization of AB-506, an inhibitor of HBV replication targeting the viral core protein.

AB-506, a small-molecule inhibitor targeting the HBV core protein, inhibits viral replication in vitro (HepAD38 cells: EC50 of 0.077 µM, CC50 > 25 µM) and in vivo (HBV mouse model: ∼3.0 log10 reductions in serum HBV DNA compared to the vehicle control). Binding of AB-506 to HBV core protein accelerates capsid assembly and inhibits HBV pgRNA encapsidation. Furthermore, AB-506 blocks cccDNA establishment in HBV-infected HepG2-hNTCP-C4 cells and primary human hepatocytes, leading to inhibition of viral RNA, HBsAg, and HBeAg production (EC50 from 0.64 µM to 1.92 µM). AB-506 demonstrated activity across HBV genotypes A-H and maintains antiviral activity against nucleos(t)ide analog-resistant variants in vitro. Evaluation of AB-506 against a panel of core variants showed that T33N/Q substitutions results in >200-fold increase in EC50 values, while L30F, L37Q, and I105T substitutions showed an 8 to 20-fold increase in EC50 values in comparison to the wild-type. In vitro combinations of AB-506 with NAs or an RNAi agent were additive to moderately synergistic. AB-506 exhibits good oral bioavailability, systemic exposure, and higher liver to plasma ratios in rodents, a pharmacokinetic profile supporting clinical development for chronic hepatitis B.

Evidence for Continuing Buprenorphine in the Perioperative Period.

OBJECTIVE: Given there are conflicting recommendations for the perioperative management of buprenorphine, we conducted a retrospective cohort study of our surgery patients on buprenorphine whose baseline dose had been preoperatively continued, tapered, or discontinued. MATERIALS AND METHODS: We reviewed charts of patients on buprenorphine who had received elective surgery at Stanford Healthcare from January 1, 2013 to June 30, 2016. Our primary outcome of interest was the change in pain score, defined as mean postoperative pain score-preoperative pain score. We also collected data on patients' tapering procedure and any postoperative nonbuprenorphine opioid requirements. RESULTS: Out of ∼1200 patients on buprenorphine, 121 had surgery of which 50 were admitted and included in the study. Perioperative continuation of transdermal buprenorphine resulted in a significantly lower change in pain score postoperatively (0.606±0.878) than discontinuation (4.83±1.23, P=0.012). Among sublingual patients, there was no statistically significant difference in the change in pain score between those who were tapered to a nonzero dose versus discontinued (P=0.55). Continuation of sublingual buprenorphine resulted in fewer nonbuprenorphine scheduled opioid prescriptions than its taper or discontinuation (P=0.028). Finally, tapers were performed with great variability in the tapering team and rate of taper. DISCUSSION: On the basis of our findings, we implemented a policy at our institution for the continuation of perioperative buprenorphine whenever possible. Our work reveals crucial targets for the education of perioperative healthcare providers and the importance of coordination among all perioperative services and providers.

ARB-1740, a RNA Interference Therapeutic for Chronic Hepatitis B Infection.

Current approved nucleoside analogue treatments for chronic hepatitis B virus (HBV) infection are effective at controlling viral titer but are not curative and have minimal impact on the production of viral proteins such as surface antigen (HBsAg), the HBV envelope protein believed to play a role in maintaining the immune tolerant state required for viral persistence. Novel agents are needed to effect HBV cure, and reduction of HBV antigenemia may potentiate activation of effective and long-lasting host immune control. ARB-1740 is a clinical stage RNA interference agent composed of three siRNAs delivered using lipid nanoparticle technology. In a number of cell and animal models of HBV, ARB-1740 caused HBV RNA reduction, leading to inhibition of multiple elements of the viral life cycle including HBsAg, HBeAg, and HBcAg viral proteins as well as replication marker HBV DNA. ARB-1740 demonstrated pan-genotypic activity in vitro and in vivo, targeting three distinct highly conserved regions of the HBV genome, and effectively inhibited replication of nucleoside analogue-resistant HBV variants. Combination of ARB-1740 with a capsid inhibitor and pegylated interferon-alpha led to greater liver HBsAg reduction which correlated with more robust induction of innate immune responses in a human chimeric mouse model of HBV. The preclinical profile of ARB-1740 demonstrates the promise of RNA interference and HBV antigen reduction in treatment strategies driving toward a cure for HBV.

Preclinical Profile of AB-423, an Inhibitor of Hepatitis B Virus Pregenomic RNA Encapsidation.

AB-423 is a member of the sulfamoylbenzamide (SBA) class of hepatitis B virus (HBV) capsid inhibitors in phase 1 clinical trials. In cell culture models, AB-423 showed potent inhibition of HBV replication (50% effective concentration [EC50] = 0.08 to 0.27 µM; EC90 = 0.33 to 1.32 µM) with no significant cytotoxicity (50% cytotoxic concentration > 10 µM). Addition of 40% human serum resulted in a 5-fold increase in the EC50s. AB-423 inhibited HBV genotypes A through D and nucleos(t)ide-resistant variants in vitro Treatment of HepDES19 cells with AB-423 resulted in capsid particles devoid of encapsidated pregenomic RNA and relaxed circular DNA (rcDNA), indicating that it is a class II capsid inhibitor. In a de novo infection model, AB-423 prevented the conversion of encapsidated rcDNA to covalently closed circular DNA, presumably by interfering with the capsid uncoating process. Molecular docking of AB-423 into crystal structures of heteroaryldihydropyrimidines and an SBA and biochemical studies suggest that AB-423 likely also binds to the dimer-dimer interface of core protein. In vitro dual combination studies with AB-423 and anti-HBV agents, such as nucleos(t)ide analogs, RNA interference agents, or interferon alpha, resulted in additive to synergistic antiviral activity. Pharmacokinetic studies with AB-423 in CD-1 mice showed significant systemic exposures and higher levels of accumulation in the liver. A 7-day twice-daily administration of AB-423 in a hydrodynamic injection mouse model of HBV infection resulted in a dose-dependent reduction in serum HBV DNA levels, and combination with entecavir or ARB-1467 resulted in a trend toward antiviral activity greater than that of either agent alone, consistent with the results of the in vitro combination studies. The overall preclinical profile of AB-423 supports its further evaluation for safety, pharmacokinetics, and antiviral activity in patients with chronic hepatitis B.

Metabolic regulation of mycobacterial growth and antibiotic sensitivity.

Treatment of chronic bacterial infections, such as tuberculosis (TB), requires a remarkably long course of therapy, despite the availability of drugs that are rapidly bacteriocidal in vitro. This observation has long been attributed to the presence of bacterial populations in the host that are "drug-tolerant" because of their slow replication and low rate of metabolism. However, both the physiologic state of these hypothetical drug-tolerant populations and the bacterial pathways that regulate growth and metabolism in vivo remain obscure. Here we demonstrate that diverse growth-limiting stresses trigger a common signal transduction pathway in Mycobacterium tuberculosis that leads to the induction of triglyceride synthesis. This pathway plays a causal role in reducing growth and antibiotic efficacy by redirecting cellular carbon fluxes away from the tricarboxylic acid cycle. Mutants in which this metabolic switch is disrupted are unable to arrest their growth in response to stress and remain sensitive to antibiotics during infection. Thus, this regulatory pathway contributes to antibiotic tolerance in vivo, and its modulation may represent a novel strategy for accelerating TB treatment.

Contrasting transcriptional responses of a virulent and an attenuated strain of Mycobacterium tuberculosis infecting macrophages.

BACKGROUND: H37Rv and H37Ra are well-described laboratory strains of Mycobacterium tuberculosis derived from the same parental strain, H37, that show dramatically different pathogenic phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the transcriptomes of the two strains during axenic growth in broth and during intracellular growth within murine bone-marrow macrophages were compared by whole genome expression profiling. We identified and compared adaptations of either strain upon encountering an intracellular environment, and also contrasted the transcriptomes of the two strains while inside macrophages. In the former comparison, both strains induced genes that would facilitate intracellular survival including those involved in mycobactin synthesis and fatty acid metabolism. However, this response was stronger and more extensive for H37Rv than for H37Ra. This was manifested as the differential expression of a greater number of genes and an increased magnitude of expression for these genes in H37Rv. In comparing intracellular transcriptional signatures, fifty genes were found to be differentially expressed between the strains. Of these fifty, twelve were under control of the PhoPR regulon. Further differences between strains included genes whose products were members of the ESAT-6 family of proteins, or were associated with their secretion. CONCLUSIONS/SIGNIFICANCE: Along with the recent identification of single nucleotide polymorphisms in H37Ra when compared to H37Rv, our demonstration of differential expression of PhoP-regulated and ESX-1 region-related genes during macrophage infection further highlights the significance of these genes in the attenuation of H37Ra.

Characterization of genes differentially expressed within macrophages by virulent and attenuated Mycobacterium tuberculosis identifies candidate genes involved in intracellular growth.

To identify genes involved in the intracellular survival of Mycobacterium tuberculosis we compared the transcriptomes of virulent (H37Rv) and attenuated (H37Ra) strains during their interaction with murine bone-marrow-derived macrophages. Expression profiling was accomplished via the bacterial artificial chromosome fingerprint array (BACFA) technique. Genes identified with BACFA, and confirmed via qPCR to be upregulated in the attenuated H37Ra at 168 h post-infection, were frdB, frdC and frdD. Genes upregulated in the virulent H37Rv were pks2, aceE and Rv1571. Further qPCR analysis of these genes at 4 and 96 h post-infection revealed that the frd operon (encoding the fumarate reductase enzyme complex) is expressed at higher levels in the virulent H37Rv at earlier time points while the expression of aceE and pks2 is higher in the virulent strain throughout the course of infection. Assessment of frd transcripts in oxygen-limited cultures of M. tuberculosis H37Ra and H37Rv showed that the attenuated strain displayed a lag in frdA and frdB expression at the onset of microaerophilic culture, when compared to microaerophilic cultures of H37Rv and aerated cultures of H37Ra. Lastly, treatment of intracellular bacteria with a putative inhibitor of fumarate reductase resulted in a significant reduction of bacterial growth.

Bacterial artificial chromosome fingerprint arrays for the differentiation of transcriptomic differences in mycobacteria.

Although microarray technology has become more widespread as a discovery tool for bacterial pathogenesis, it remains a method available only to laboratories with access to expensive equipment and costly analysis software. Mycobacterium tuberculosis, the causative agent for tuberculosis (TB), afflicts one-third of the global population, and kills between 2 and 3 million people per year. While the majority of cases of TB occur in developing areas of the world, facilities in these regions may not be able to support microarray analysis. Additionally, a major limitation of microarrays is that only genes on the array are being assayed. With acquired virulence and drug resistance in microbes, a method less dependent on a predetermined list of gene targets is advantageous. We present a method of expression analysis based on bacterial artificial chromosomes (BACs) that can be applied with standard laboratory equipment and free analysis software. This technique, bacterial artificial chromosome fingerprint arrays (BACFA), was developed and utilised to identify expression differences between intracellular strains of M. tuberculosis, one virulent (H37Rv) and one attenuated (H37Ra). Southern blots of restriction-enzyme digested BAC fragments were sequentially hybridised with strain-specific cDNA probes to generate expression profiles that were used to isolate expression differences in broth grown and intracellular bacteria. Repeat comparisons of intracellular profiles via BACFA identified genomic regions differentially expressed by the two strains. Quantitative real-time PCR was used to assess the genes located in these fragments in order to confirm or deny the differential regulation of genes. In total, we identified six genes that were differentially regulated between strains inside the host cell (pks2, aceE, Rv1571, and frdBCD). We report that BACFA is an effective technique in the expression analysis of bacteria and can be considered complementary to the high-throughput analysis offered by microarrays.

Two-dimensional bacterial genome display: a method for the genomic analysis of mycobacteria.

Annually, Mycobacterium tuberculosis is the cause of approximately three million deaths worldwide. It would appear that currently available therapies for this disease are inadequate. The identification of genes involved in mycobacterial virulence will facilitate the design of new prophylactic and therapeutic interventions. A method for high-resolution comparison of bacterial genomes has been developed to facilitate the identification of genes possibly involved in the virulence of clinically relevant mycobacteria. This 'two-dimensional bacterial genome display' (2DBGD) method utilizes two-dimensional DNA electrophoresis to separate, on the basis of size and G+C content, genomic fragments generated with different restriction endonucleases. The use of this method to identify genomic differences between species, strains and, most importantly, isogenic mutants of mycobacteria is reported. That 2DBGD can be used to identify differences resulting from either insertional mutagenesis using a gentamicin-resistance gene or from a frameshift mutation is demonstrated.