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Kenneth B Chapman,1– 3 Dawood Sayed,4 Tim Lamer,5 Corey Hunter,6 Jacqueline Weisbein,7 Kiran V Patel,1– 3 David Dickerson,8,9 Jonathan M Hagedorn,10 David W Lee,11 Kasra Amirdelfan,12 Timothy Deer,13 Krishnan Chakravarthy14,15 1The Spine & Pain Institute of New York, New York, NY, USA; 2Department of Anesthesiology, Zucker School of Medicine at Hofstra Northwell, Manhasset, NY, USA; 3Department of Anesthesiology, NYU Langone Medical Center, New York, NY, USA; 4Department of Anesthesiology, The University of Kansas Medical Center (KUMC), Kansas City, KS, USA; 5Department of Anesthesiology and Perioperative Medicine, Division of Pain Medicine, Mayo Clinic, Rochester, MN, USA; 6Ainsworth Institute of Pain Management, New York, NY, USA; 7Napa Valley Orthopedic Medical Group, Napa, CA, USA; 8Department of Anesthesiology, Critical Care and Pain Medicine, NorthShore University Health System, Evanston, IL, USA; 9Department of Anesthesia & Critical Care, University of Chicago, Chicago, IL, USA; 10iSpine Pain Physicians, Maple Grove, MN, USA; 11Fullerton Orthopedic Surgery Medical Group, Fullerton, CA, USA; 12IPM Medical Group, Inc., Walnut Creek, CA, USA; 13The Spine and Nerve Center of the Virginias, Charleston, WV, USA; 14Department of Anesthesiology and Pain Medicine, University of California San Diego Health Sciences, San Diego, CA, USA; 15VA San Diego Healthcare System, San Diego, CA, USACorrespondence: Kenneth B Chapman, NYU Langone Medical Center, Zucker School of Medicine at Hofstra/Northwell, Pain Medicine at Staten Island University Hospital, 1360 Hylan Boulevard, Staten Island, NY, 10305, USA, Email chapmanken@spinepainny.comAbstract: With continued innovations in neuromodulation comes the need for evolving reviews of best practices. Dorsal root ganglion stimulation (DRG-S) has significantly improved the treatment of complex regional pain syndrome (CRPS), and it has broad applicability across a wide range of other conditions. Through funding and organizational leadership by the American Society for Pain and Neuroscience (ASPN), this best practices consensus document has been developed for the selection, implantation, and use of DRG stimulation for the treatment of chronic pain syndromes. This document is composed of a comprehensive narrative literature review that has been performed regarding the role of the DRG in chronic pain and the clinical evidence for DRG-S as a treatment for multiple pain etiologies. Best practice recommendations encompass safety management, implantation techniques, and mitigation of the potential complications reported in the literature. Looking to the future of neuromodulation, DRG-S holds promise as a robust intervention for otherwise intractable pain.Keywords: dorsal root ganglion, neurostimulation, chronic pain, best practice, guidelines

Timothy R Deer,1 Jay S Grider,2 Jason E Pope,3 Tim J Lamer,4 Sayed E Wahezi,5 Jonathan M Hagedorn,6 Steven Falowski,7 Reda Tolba,8 Jay M Shah,9 Natalie Strand,10 Alex Escobar,11 Mark Malinowski,12 Anjum Bux,13 Navdeep Jassal,14 Jennifer Hah,15 Jacqueline Weisbein,16 Nestor D Tomycz,17 Jessica Jameson,18 Erika A Petersen,19 Dawood Sayed20 1Centers for Pain Relief, Charleston, WV, USA; 2UK HealthCare Pain Services, Department of Anesthesiology, University of Kentucky College of Medicine, Lexington, KY, USA; 3Evolve Restorative Center, Santa Rosa, CA, USA; 4Division of Pain Medicine, Department of Anesthesiology, Mayo Clinic, Rochester, MN, USA; 5Montefiore Medical Center, SUNY-Buffalo, Buffalo, NY, USA; 6Department of Anesthesiology and Perioperative Medicine, Division of Pain Medicine, Mayo Clinic, Rochester, MN, USA; 7Director Functional Neurosurgery, Neurosurgical Associates of Lancaster, Lancaster, PA, USA; 8Pain Management Department, Anesthesiology Institute, Cleveland Clinic, Abu Dhabi, UAE; 9SamWell Institute for Pain Management, Colonia, NJ, USA; 10Department of Anesthesiology, Division of Pain Medicine, Mayo Clinic, Phoenix, AZ, USA; 11Department of Anesthesiology and Pain Medicine, University of Toledo Medical Center, Toledo, OH, USA; 12OhioHealth, Neurological Physicians, Columbus, OH, USA; 13Bux Pain Management, Lexington, KY, USA; 14Excel Pain and Spine, Lakeland, FL, USA; 15Department of Anesthesiology, Perioperative, and Pain Medicine, Stanford University, Palo Alto, CA, USA; 16Napa Valley Orthopaedic Medical Group, Inc., Napa, CA, USA; 17Department of Neurological Surgery, Allegheny General Hospital, Allegheny Health Network, Pittsburgh, PA, USA; 18Axis Spine Center, Coeur d’Alene, ID, USA; 19Department of Neurosurgery, University of Arkansas for Medical Sciences, Little Rock, AR, USA; 20Pain Medicine, Multidisciplinary Pain Fellowship, The University of Kansas Health System, Kansas City, KS, USACorrespondence: Timothy R Deer, The Spine and Nerve Centers of the Virginias, 400 Court Street, Suite 100, Charleston, WV, 25301, USA, Tel +1 304 347-6141, Email doctdeer@aol.comIntroduction: Lumbar spinal stenosis (LSS) is a common spinal disease of aging with a growing patient population, paralleling population growth. Minimally invasive treatments are evolving, and the use of these techniques needs guidance to provide the optimal patient safety and efficacy outcomes.Methods: The American Society of Pain and Neuroscience (ASPN) identified an educational need for guidance on the prudent use of the innovative minimally invasive surgical therapies for the treatment of symptomatic LSS. The executive board nominated experts spanning anesthesiology, physiatry, orthopedic surgery, and neurosurgery based on expertise, publications, research, diversity and field of practice. Evidence was reviewed, graded using the United States Preventive Services Task Force (USPSTF) criteria for evidence and recommendation strength and grade, and expert opinion was added to make consensus points for best practice.Results: The world literature in English was searched using Medline, EMBASE, Cochrane CENTRAL, BioMed Central, Web of Science, Google Scholar, PubMed, Current Contents Connect, Scopus, and meeting abstracts to identify and compile the evidence (per section) for LSS-related pain. Search words were selected based upon the section represented. Identified peer-reviewed literature was critiqued using USPSTF criteria and consensus points are presented.Discussion: The algorithm for patient selection in the management of symptomatic spinal stenosis is evolving. Careful consideration of patient selection and anatomic architecture variance is critical for improved outcomes and patient safety.Conclusion: ASPN created a guidance for best practice for minimally invasive surgical treatment of symptomatic spinal stenosis.Keywords: percutaneous image-guided lumbar decompression, interspinous spacers, intrathecal drug delivery, open decompression, neurostimulation, epidural steroid injections

Coronaviral infections are an important cause of enteric and respiratory diseases in humans and animals that are generally associated with a high level of morbidity and mortality. Similarly, picornavirus infections can lead to various illnesses that severely impact human and animal health. Despite belonging to different virus families, viral replication in all of these pathogens relies on the action of a central cysteine protease called 3C/3CL or main protease (M pro ). Due to the high functional and structural conservation of this enzyme among viral species and robustness against mutation it is considered a good target for antiviral inhibitor development. The evaluation of inhibitor potency, expressed as IC 50 , in many studies is achieved by measuring the inhibition of cleavage of a fluorogenic substrate in a Fluorescence Resonance Energy Transfer (FRET)-type assay. The FRET substrate is engineered after common recognition sequences of each viral M pro , resulting in different sequences and limited comparability of IC 50 between species. Our aim was to overcome this inconsistency by identifying common recognition motives of coronavirus and picornavirus M pro s to develop a unique FRET substrate that can be used universally for FRET assay tests of these enzymes. We synthesized a variety of FRET substrates with common recognition sequences and compared their cleavage kinetics towards main proteases from different species to determine the optimal sequence for universal application in FRET assays., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Fusobacterium nucleatum is an oral commensal bacterium that can act as an opportunistic pathogen and is implicated in diseases such as periodontitis, adverse pregnancy outcomes, colorectal cancer, and Alzheimer's disease. F. nucleatum synthesizes lanthionine for its peptidoglycan, rather than meso-2,6-diaminopimelic acid (DAP) used by most Gram-negative bacteria. Despite lacking the biosynthetic pathway for DAP, the genome of F. nucleatum ATCC 25586 encodes a predicted DAP epimerase. A recent study hypothesized that this enzyme may act as a lanthionine epimerase, but the authors found a very low turnover rate, suggesting that this enzyme likely has another more favored substrate. Here, we characterize this enzyme as a histidine racemase (HisR), and found that catalytic turnover is ∼10,000× faster with L-histidine than with L,L-lanthionine. Kinetic experiments suggest that HisR functions as a cofactor-independent racemase and that turnover is specific for histidine, while crystal structures of catalytic cysteine to serine mutants (C67S or C209S) reveal this enzyme in its substrate-unbound, open conformation. Currently, the only other reported cofactor-independent histidine racemase is CntK from Staphylococcus aureus, which is used in the biosynthesis of staphylopine, a broad-spectrum metallophore that increases virulence of S. aureus. However, CntK shares only 28% sequence identity with HisR, and their genes exist in different genomic contexts. Knockout of hisR in F. nucleatum results in a small but reproducible lag in growth compared to WT during exponential phase, suggesting that HisR may play a role in growth of this periodontal pathogen., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ibuzatrelvir (1) was recently disclosed and patented by Pfizer for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has received fast-track status from the USA Food and Drug Administration (FDA) and has entered phase III clinical trials as a possible replacement for Paxlovid. Like nirmatrelvir (2) in Paxlovid, this orally active drug candidate is designed to target viral main proteases (M pro ) through reversible covalent interaction of its nitrile warhead with the active site thiol of the chymotrypsin-like cysteine protease (3CL protease). Inhibition of M pro hinders the processing of the proteins essential for viral replication in vivo . However, ibuzatrelvir apparently does not require ritonavir (3), which is coadministered in Paxlovid to block human oxidative metabolism of nirmatrelvir. Here, we report the crystal structure of the complex of ibuzatrelvir with the active site of SARS-CoV-2 M pro at 2.0 Å resolution. In addition, we show that ibuzatrelvir also potently inhibits the M pro of Middle East respiratory syndrome-related coronavirus (MERS-CoV), which is fortunately not widespread but can be dangerously lethal (∼36% mortality). Co-crystal structures show that the binding mode of the drug to both active sites is similar and that the trifluoromethyl group of the inhibitor fits precisely into a critical S2 substrate binding pocket of the main proteases. However, our results also provide a rationale for the differences in potency of ibuzatrelvir for these two proteases due to minor differences in the substrate preferences leading to a weaker H-bond network in MERS-CoV M pro . In addition, we examined the reversibility of compound binding to both proteases, which is an important parameter in reducing off-target effects as well as the potential immunogenicity. The crystal structures of the ibuzatrelvir complexes with M pro of SARS-CoV-2 and of MERS-CoV will further assist drug design for coronaviral infections in humans and animals., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Importance: Many bacteriocins target the sugar transporter mannose phosphotransferase system (man-PTS) to exert their antibacterial activity. The elucidation in recent years of the structure of man-PTS has facilitated our understanding of how bacteriocins might interact with the receptor and which domains of the transporter are involved in bacteriocin resistance. Here, we show that missense mutations in the sugar-binding domain of the man-PTS not only impede the uptake of sugars but also prevent the antibacterial activity of the bacteriocins lactococcin A and garvicin Q., Competing Interests: The authors declare no conflict of interest.

Objectives: Since its foundation in the 1960s, neuromodulation has become an increasingly used treatment option for chronic pain. This bibliometric analysis examines the most cited research in this field with the aim of uncovering existing trends and future directions., Materials and Methods: Clarivate's Web of Science data base was searched for the top 25 most cited studies focusing on neuromodulation for chronic pain. Various bibliometric parameters were then extracted and analyzed. Randomized controlled trials (RCTs) were compared with non-RCTs., Results: The top 25 articles had a mean of 347 citations and 22.2 citations per year, with more recent articles having a higher citation rate. Most were published in the last two decades and predominantly originated from the United States. There were 13 RCTs, which were significantly more recent (p = 0.004) and more cited per year (p = 0.001) than the 12 non-RCTs. Sources included 15 journals with a mean impact factor of 13.896. The most studied modality was spinal cord stimulation with 20 articles (76.9%), followed by intrathecal drug delivery (15.4%), dorsal root ganglion stimulation (3.8%), and peripheral nerve stimulation (3.8%)., Conclusions: Analysis of the most cited articles on neuromodulation reveals a focal shift from historical reports to innovative RCTs that have increasingly guided pain practice in the recent years. As novel techniques and technologies continue to develop, high-quality evidence coupled with broadening indications will likely direct further expansion of this field., (Copyright © 2022 International Neuromodulation Society. Published by Elsevier Inc. All rights reserved.)

This study explores the relationship between structural alterations of nirmatrelvir, such as homologation and deuteration, and metabolic stability of newly synthesized derivatives. We developed a reliable synthetic protocol toward dideutero-nirmatrelvir and its homologated analogues with high isotopic incorporation. Deuteration of the primary metabolic site of nirmatrelvir provides a 3-fold improvement of its human microsomal stability but is accompanied by an increased metabolism rate at secondary sites. Homologation of the lactam ring allows the capping group modification to decrease and delocalize the molecule's lipophilicity, reducing the metabolic rate at secondary sites. The effect of deuteration was less pronounced for the 6-membered lactam than for its 5-membered analogue in human microsomes, but the trend is reversed in the case of mouse microsomes. X-ray data revealed that the homologation of the lactam ring favors the orientation of the drug's nitrile warhead for interaction with the catalytic sulfur of the SARS-CoV-2 M pro , improving its binding. Comparable potency against SARS-CoV-2 M pro from several variants of concern and selectivity over human cysteine proteases cathepsin B, L, and S was observed for the novel deuterated/homologated derivative and nirmatrelvir. Synthesized compounds displayed a large interspecies variability in hamster, rat, and human hepatocyte stability assays. Overall, we aimed to apply a rational approach in changing the physicochemical properties of the drug to refine its biochemical and biological parameters., Competing Interests: The authors declare the following competing financial interest(s): The Governors of the University of Alberta has filed a patent application that also contains a few of the molecules in this manuscript and currently some authors are listed as inventors., (© 2023 The Authors. Published by American Chemical Society.)

Background: Some peptides are targets for degradation when heterologously expressed as fusion proteins in E. coli, which can limit yields after isolation and purification. We recently reported that peptide degradation may be prevented by production of a "sandwiched" SUMO-peptide-intein (SPI) fusion protein, which protects the target peptide sequence from truncation and improves yield. This initial system required cloning with two commercially available vectors. It used an N-terminal polyhistidine tagged small ubiquitin-like modifier (SUMO) protein and a C-terminal engineered Mycobacterium xenopii DNA Gyrase A intein with an inserted chitin binding domain (CBD) to create "sandwiched" fusion proteins of the form: His 6 -SUMO-peptide-intein-CBD. However, the major drawback of this previously reported fusion protein "sandwich" approach is the increased time and number of steps required to complete the cloning and isolation procedures, relative to the simple procedures to produce recombinant peptides in E. coli from a single (non-"sandwiched") fusion protein system., Results: In this work we generate the plasmid pSPIH6, which improves upon the previous system by encoding both the SUMO and intein proteins and allows facile construction of a SPI protein in a single cloning step. Additionally, the Mxe GyrA intein encoded in pSPIH6 contains a C-terminal polyhistidine tag, resulting in SPI fusion proteins of the form: His 6 -SUMO-peptide-intein-CBD-His 6 . The dual polyhistidine tags greatly simplify isolation procedures compared to the original SPI system, which we have here demonstrated with two linear bacteriocin peptides: leucocin A and lactococcin A. The yields obtained for both peptides after purification were also improved compared to the previous SPI system as a result of this streamlined protocol., Conclusions: This modified SPI system and its simplified cloning and purification procedures described here may be generally useful as a heterologous E. coli expression system to obtain pure peptides in high yield, especially when degradation of the target peptide is an issue., (© 2023. The Author(s).)

The main protease of SARS-CoV-2 (M pro ) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in M pro may alter the structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOCs) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and the rate of cleavage of a viral peptide. Crystal structures of 11 M pro mutants provided structural insight into their altered functionality. Additionally, we show M pro mutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and a subsequent significant decrease in cytokine secretion, providing evidence for alterations in the escape of host-antiviral mechanisms. Accordingly, mutations associated with the Gamma VOC and highly virulent Delta VOC resulted in a significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting M pro will remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Heterologous expression in Escherichia coli is a commonly used method to produce ribosomally synthesized peptides for further study. This generally requires expression of the target protein with an affinity fusion tag, followed by isolation of the fusion protein from a cellular lysate by affinity purification, and finally by removal of the fusion tag and purification of the desired peptide. Sometimes, however, fusion proteins may be degraded during recombinant expression in E. coli. We recently reported an expression system that sandwiches the target peptide between an N-terminal small ubiquitin-like modifier (SUMO) protein and a C-terminal intein. This SUMO-peptide-intein (SPI) fusion protein protects the central peptide from degradation and can lead to improved peptide yield after purification. In this report, we detail the cloning, expression, and isolation procedures for the SPI fusion system, with comments on conditions that can be optimized for different peptides to obtain maximal yield for each construct. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cloning to construct SPI gene Basic Protocol 2: Expression of SPI fusion proteins in E. coli BL21(DE3) Support Protocol: Optimization of expression and induction conditions Basic Protocol 3: Isolation and purification of SPI fusion proteins with a chitin column Alternate Protocol: Isolation and purification of SPI fusion proteins without chitin., (© 2022 Wiley Periodicals LLC.)

Recombinant peptide production in Escherichia coli is often accomplished through cloning and expression of a fusion protein. The fusion protein partner generally has two requirements: (a) it contains an affinity tag to assist with purification and (b) it can be cleaved off to leave only the desired peptide sequence behind. Common soluble fusion partners include small ubiquitin-like modifier protein (SUMO), maltose-binding protein (MBP), glutathione S-transferase (GST), or intein proteins. However, heterologously expressed peptides can suffer from proteolytic degradation or instability. This degradation can pose a major issue for applications requiring a large amount of purified peptide, such as NMR structural assignments or biochemical assays. Improving peptide yield by testing various expression and isolation conditions requires a significant amount of effort and may not lead to improved results. Here, we cloned and expressed four different peptides as SUMO fusion proteins. These peptides (lactococcin A, leucocin A, faerocin MK, neopetrosiamide A) were truncated during expression and isolation as SUMO fusions, resulting in low yields of purified peptide. To prevent this degradation and improve yield, we designed a new expression system to create a "sandwiched" fusion protein of the form: His 6 -SUMO-peptide-intein (SPI). These sandwiched peptides were more stable and protected against degradation, resulting in improved yields (up to 17-fold) under a set of standard expression and isolation procedures. This SPI expression system uses only two commercially available vectors and standard protein purification techniques, and therefore may offer an economical and facile route to improve yields for peptides that undergo degradation., (© 2022 The Protein Society.)

Coronaviruses infect a variety of hosts in the animal kingdom, and while each virus is taxonomically different, they all infect their host via the same mechanism. The coronavirus main protease (M pro , also called 3CL pro ), is an attractive target for drug development due to its essential role in mediating viral replication and transcription. An M pro inhibitor, GC376, has been shown to treat feline infectious peritonitis (FIP), a fatal infection in cats caused by internal mutations in the feline enteric coronavirus (FECV). Recently, our lab demonstrated that the feline drug, GC373, and prodrug, GC376, are potent inhibitors of SARS-CoV-2 M pro and solved the structures in complex with the drugs; however, no crystal structures of the FIP virus (FIPV) M pro with the feline drugs have been published so far. Here, we present crystal structures of FIPV M pro -GC373/GC376 complexes, revealing the inhibitors covalently bound to Cys144 in the active site, similar to SARS-CoV-2 M pro . Additionally, GC376 has a higher affinity for FIPV M pro with lower nanomolar K i values compared to SARS-CoV and SARS-CoV-2 M pro . We also show that improved derivatives of GC376 have higher potency for FIPV M pro . Since GC373 and GC376 represent strong starting points for structure-guided drug design, determining the crystal structures of FIPV M pro with these inhibitors are important steps in drug optimization and structure-based broad-spectrum antiviral drug discovery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lu, Chen, Khan, Brassard, Arutyunova, Lamer, Vuong, Fischer, Young, Vederas and Lemieux.)

Recurring coronavirus outbreaks, such as the current COVID-19 pandemic, establish a necessity to develop direct-acting antivirals that can be readily administered and are active against a broad spectrum of coronaviruses. Described in this Article are novel α-acyloxymethylketone warhead peptidomimetic compounds with a six-membered lactam glutamine mimic in P1. Compounds with potent SARS-CoV-2 3CL protease and in vitro viral replication inhibition were identified with low cytotoxicity and good plasma and glutathione stability. Compounds 15e , 15h , and 15l displayed selectivity for SARS-CoV-2 3CL protease over CatB and CatS and superior in vitro SARS-CoV-2 antiviral replication inhibition compared with the reported peptidomimetic inhibitors with other warheads. The cocrystallization of 15l with SARS-CoV-2 3CL protease confirmed the formation of a covalent adduct. α-Acyloxymethylketone compounds also exhibited antiviral activity against an alphacoronavirus and non-SARS betacoronavirus strains with similar potency and a better selectivity index than remdesivir. These findings demonstrate the potential of the substituted heteroaromatic and aliphatic α-acyloxymethylketone warheads as coronavirus inhibitors, and the described results provide a basis for further optimization.