Total Chemical Synthesis of Aggregation-Prone Disulfide-Rich Starfish Peptides.

A relaxin-like gonad-stimulating peptide (RGP), Aso-RGP, featuring six cysteine residues, was identified in the Crown-of-Thorns Starfish (COTS, Acanthaster cf. solaris) and initially produced through recombinant yeast expression. This method yielded a single-chain peptide with an uncleaved C-peptide (His Tag) and suboptimal purity. Our objective was to chemically synthesize Aso-RGP in its mature form, comprising two chains (A and B) and three disulfide bridges, omitting the C-peptide. Furthermore, we aimed to synthesize a newly identified relaxin-like peptide, Aso-RLP2, from COTS, which had not been previously synthesized. This paper reports the first total chemical synthesis of Aso-RGP and Aso-RLP2. Aso-RGP synthesis proceeded without major issues, whereas the A-chain of Aso-RLP2, in its reduced and unfolded state with two free thiols, presented considerable challenges. These were initially marked by "messy" RP-HPLC profiles, typically indicative of synthesis failure. Surprisingly, oxidizing the A-chain significantly improved the RP-HPLC profile, revealing the main issue was not synthesis failure but the peptide's aggregation tendency, which initially obscured analysis. This discovery highlights the critical need to account for aggregation in peptide synthesis and analysis. Ultimately, our efforts led to the successful synthesis of both peptides with purities exceeding 95 %.

Development of a synthetic relaxin-3/INSL5 chimeric peptide ligand for NanoBiT complementation binding assays.

INSL5 and relaxin-3 are relaxin family peptides with important roles in gut and brain function, respectively. They mediate their actions through the class A GPCRs RXFP4 and RXFP3. RXFP4 has been proposed to be a therapeutic target for colon motility disorders whereas RXFP3 targeting could be effective for neurological conditions such as anxiety. Validation of these targets has been limited by the lack of specific ligands and the availability of robust ligand-binding assays for their development. In this study, we have utilized NanoBiT complementation to develop a SmBiT-conjugated tracer for use with LgBiT-fused RXFP3 and RXFP4. The low affinity between LgBiT:SmBiT should result in a low non-specific luminescence signal and enable the quantification of binding without the tedious separation of non-bound ligands. We used solid-phase peptide synthesis to produce a SmBiT-labelled RXFP3/4 agonist, R3/I5, where SmBiT was conjugated to the B-chain N-terminus via a PEG12 linker. Both SmBiT-R3/I5 and R3/I5 were synthesized and purified in high purity and yield. Stable HEK293T cell lines expressing LgBiT-RXFP3 and LgBiT-RXFP4 were produced and demonstrated normal signaling in response to the synthetic R3/I5 peptide. Binding was first characterized in whole-cell binding kinetic assays validating that the SmBiT-R3/I5 bound to both cell lines with nanomolar affinity with minimal non-specific binding without bound and free SmBiT-R3/I5 separation. We then optimized membrane binding assays, demonstrating easy and robust analysis of both saturation and competition binding from frozen membranes. These assays therefore provide an appropriate rigorous binding assay for the high-throughput analysis of RXFP3 and RXFP4 ligands.

Developing insulin-like peptide 5-based antagonists for the G protein-coupled receptor, RXFP4.

Human insulin-like peptide 5 (INSL5) is a gut hormone produced by colonic L-cells, and its biological functions are mediated by Relaxin Family Peptide Receptor 4 (RXFP4). Our preliminary data indicated that RXFP4 agonists are potential drug leads for the treatment of constipation. More recently, we designed and developed a novel RXFP4 antagonist, A13-nR that was shown to block agonist-induced activity in cells and animal models. We showed that A13-nR was able to block agonist-induced increases in colon motility in mice of both genders that express the receptor, RXFP4. Our data also showed that colorectal propulsion induced by intracolonic administration of short-chain fatty acids was antagonized by A13-nR. Therefore, A13-nR is an important research tool and potential drug lead for the treatment of colon motility disorders, such as bacterial diarrhea. However, A13-nR acted as a partial agonist at high concentrations in vitro and demonstrated modest antagonist potency (∼35 nM). Consequently, the primary objective of this study is to pinpoint novel modifications to A13-nR that eliminate partial agonist effects while preserving or augmenting antagonist potency. In this work, we detail the creation of a series of A13-nR-modified analogues, among which analogues 3, 4, and 6 demonstrated significantly improved RXFP4 affinity (∼3 nM) with reduced partial agonist activity, enhanced antagonist potency (∼10 nM) and maximum agonist inhibition (∼80 %) when compared with A13-nR. These compounds have potential as candidates for further preclinical evaluations, marking a significant stride toward innovative therapeutics for colon motility disorders.

A Qualitative Study of Emergency Medicaid Programs From the Perspective of Hospital Stakeholders.

INTRODUCTION: Uninsured patients often have poor clinical outcomes associated with lower access to care. Hospital Presumptive Eligibility (HPE) provides up to 60-d emergency Medicaid coverage for uninsured, low-income patients. After obtaining 60-d HPE, patients must file for ongoing Medicaid to sustain coverage; however, navigating HPE approval is complex. We conducted a qualitative study to understand (1) stakeholder perspectives on the application process and workflow and (2) facilitators and barriers to HPE approval to understand process improvement opportunities. MATERIAL AND METHODS: We conducted semi-structured interviews between September-December 2021 with key stakeholders (social workers, financial counselors, case managers, and private third-party vendor representatives) involved in HPE coverage determination, screening, approval, and Medicaid sustainment at our institution. We performed a team-based thematic analysis to elicit factors influencing HPE screening and approval, and recommendations for process improvement. RESULTS: Study participants described the HPE application and Medicaid approval processes. Patient-level barriers included information disclosure and immigration status, inability to contact patients or next-of-kin, and knowledge gaps about insurance acquisition and sustainment. System-level barriers included technical challenges with the state HPE application portal, inadequate staffing for patient screening, and short emergency department stays that limited opportunities to initiate HPE. Stakeholders proposed improvements in education, patient outreach, and logistics. CONCLUSIONS: This qualitative study reveals the process of HPE approval and outlines barriers within HPE and Medicaid processing from the perspective of direct hospital stakeholders. We identified opportunities at the patient, hospital, and policy levels that could improve successful HPE application and approval rates.

Further Developments towards a Minimal Potent Derivative of Human Relaxin-2.

Human relaxin-2 (H2 relaxin) is a peptide hormone with potent vasodilatory and anti-fibrotic effects, which is of interest for the treatment of heart failure and fibrosis. H2 relaxin binds to the Relaxin Family Peptide Receptor 1 (RXFP1). Native H2 relaxin is a two-chain, three-disulfide-bond-containing peptide, which is unstable in human serum and difficult to synthesize efficiently. In 2016, our group developed B7-33, a single-chain peptide derived from the B-chain of H2 relaxin. B7-33 demonstrated poor affinity and potency in HEK cells overexpressing RXFP1; however, it displayed equivalent potency to H2 relaxin in fibroblasts natively expressing RXFP1, where it also demonstrated the anti-fibrotic effects of the native hormone. B7-33 reversed organ fibrosis in numerous pre-clinical animal studies. Here, we detail our efforts towards a minimal H2 relaxin scaffold and attempts to improve scaffold activity through Aib substitution and hydrocarbon stapling to re-create the peptide helicity present in the native H2 relaxin.

For-Profit Status and Geographic Distribution of Trauma Centers in the US.

This cohort study assesses geographic distribution of for-profit and not-for-profit trauma centers in the US designated by their states between 2014 and 2018.

Development of Novel High-Affinity Antagonists for the Relaxin Family Peptide Receptor 1.

H2 relaxin is a peptide hormone that exerts its biological actions through the G protein-coupled receptor, RXFP1. The numerous important biological functions of H2 relaxin, including potent renal, vasodilatory, cardioprotective, and anti-fibrotic actions, have resulted in considerable interest in its use as a therapeutic for various cardiovascular diseases and other fibrotic indications. Interestingly though, H2 relaxin and RXFP1 have been shown to be overexpressed in prostate cancer, allowing for the downregulation or blocking of relaxin/RXFP1 to decrease prostate tumor growth. These findings suggest the application of an RXFP1 antagonist for the treatment of prostate cancer. However, these therapeutically relevant actions are still poorly understood and have been hindered by the lack of a high-affinity antagonist. In this study, we chemically synthesized three novel H2 relaxin analogues that have complex insulin-like structures with two chains (A and B) and three disulfide bridges. We report here the structure-activity relationship studies on H2 relaxin that resulted in the development of a novel high-affinity RXFP1 antagonist, H2 B-R13HR (∼40 nM), that has only one extra methylene group in the side chain of arginine 13 in the B-chain (ArgB13) of H2 relaxin. Most notably, the synthetic peptide was shown to be active in a mouse model of prostate tumor growth in vivo where it inhibited relaxin-mediated tumor growth. Our compound H2 B-R13HR will be an important research tool to understand relaxin actions through RXFP1 and may be a potential lead compound for the treatment of prostate cancer.

A Lipidated Single-B-Chain Derivative of Relaxin Exhibits Improved In Vitro Serum Stability without Altering Activity.

Human relaxin-2 (H2 relaxin) is therapeutically very important due to its strong anti-fibrotic, vasodilatory, and cardioprotective effects. Therefore, relaxin's receptor, relaxin family peptide receptor 1 (RXFP1), is a potential target for the treatment of fibrosis and related disorders, including heart failure. H2 relaxin has a complex two-chain structure (A and B) and three disulfide bridges. Our laboratory has recently developed B7-33 peptide, a single-chain agonist based on the B-chain of H2 relaxin. However, the peptide B7-33 has a short circulation time in vitro in serum (t1/2 = ~6 min). In this study, we report structure-activity relationship studies on B7-33 utilizing different fatty-acid conjugations at different positions. We have shown that by fatty-acid conjugation with an appropriate spacer length, the in vitro half-life of B7-33 can be increased from 6 min to 60 min. In the future, the lead lipidated molecule will be studied in animal models to measure its PK/PD properties, which will lead to their pre-clinical applications.

Evaluating Emergency Medicaid Program Policy Changes During the COVID-19 Pandemic.

INTRODUCTION: Trauma patients are twice as likely to be uninsured as the general population, which can lead to limited access to postinjury resources and higher mortality. The Hospital Presumptive Eligibility (HPE) program offers emergency Medicaid for eligible patients at presentation. The HPE program underwent several changes during the COVID-19 pandemic; we quantify the program's success during this time and seek to understand features associated with HPE approval. METHODS: A mixed methods study at a Level I trauma center using explanatory sequential design, including: 1) a retrospective cohort analysis (2015-2021) comparing HPE approval before and after COVID-19 policy changes; and 2) semistructured interviews with key stakeholders. RESULTS: 589 patients listed as self-pay or Medicaid presented after March 16, 2020, when COVID-19 policies were first implemented. Of these, 409 (69%) patients were already enrolled in Medicaid at hospitalization. Among those uninsured at arrival, 160 (89%) were screened and 98 (61%) were approved for HPE. This marks a significant improvement in the prepandemic HPE approval rate (48%). In adjusted logistic regression analyses, the COVID-19 period was associated with an increased likelihood of HPE approval (versus prepandemic: aOR, 1.64; P = 0.005). Qualitative interviews suggest that mechanisms include state-based expansion in HPE eligibility and improvements in remote approval such as telephone/video conferencing. CONCLUSIONS: The HPE program experienced an overall increased approval rate and adapted to policy changes during the pandemic, enabling more patients' access to health insurance. Ensuring that these beneficial changes remain a part of our health policy is an important aspect of improving access to health insurance for our patients.

Despite Increasing Costs, Perfusion Machines Expand the Donor Pool of Livers and Could Save Lives.

INTRODUCTION: Liver transplantation is a highly successful treatment for liver failure and disease. However, demand continues to outstrip our ability to provide transplantation as a treatment. Many livers initially considered for transplantation are not used because of concerns about their viability or logistical issues. Recent clinical trials have shown discarded livers may be viable if they undergo machine perfusion, which allows a more objective assessment of liver quality. METHODS: Using the Scientific Registry of Transplant Recipients dataset, we examined discarded and unretrieved organs to determine their eligibility for perfusion. We then used a Markov decision-analytic model to perform a cost-effectiveness analysis of two competing transplant strategies: Static Cold Storage (SCS) alone versus Static Cold Storage and Normothermic Machine Perfusion (NMP) of discarded organs. RESULTS: The average predicted successful transplants after perfusion was 385, representing a 5.8% increase in the annual yield of liver transplants. Our cost-effectiveness analysis found that the SCS strategy generated 4.64 quality-adjusted life years (QALYs) and cost $479,226. The combined SCS + NMP strategy generated 4.72 QALYs and cost $481,885. The combined SCS + NMP strategy had an incremental cost-effectiveness ratio of $33,575 per additional QALY over the 10-year study horizon. CONCLUSIONS: Machine perfusion of livers currently not considered viable for transplant could increase the number of transplantable grafts by approximately 5% per year and is cost-effective compared to Static Cold Storage alone.

Reevaluating Liver Donor Risk in the Era of Improved Hepatitis C Virus Treatment.

This cohort study examines the risk of graft failure associated with donors with hepatitis C virus (HCV) infection before and after the introduction of direct-acting antiviral medications.

Evaluation of Potential DnaK Modulating Proline-Rich Antimicrobial Peptides Identified by Computational Screening.

The day is rapidly approaching where current antibiotic therapies will no longer be effective due to the development of multi-drug resistant bacteria. Antimicrobial peptides (AMPs) are a promising class of therapeutic agents which have the potential to help address this burgeoning problem. Proline-rich AMPs (PrAMPs) are a sub-class of AMPs, that have multiple modes of action including modulation of the bacterial protein folding chaperone, DnaK. They are highly effective against Gram-negative bacteria and have low toxicity to mammalian cells. Previously we used an in silico approach to identify new potential PrAMPs from the DRAMP database. Four of these peptides, antibacterial napin, attacin-C, P9, and PP30, were each chemically assembled and characterized. Together with synthetic oncocin as a reference, each peptide was then assessed for antibacterial activity against Gram-negative/Gram-positive bacteria and for in vitro DnaK modulation activity. We observed that these peptides directly modulate DnaK activity independently of eliciting or otherwise an antibiotic effect. Based on our findings, we propose a change to our previously established PrAMP definition to remove the requirement for antimicrobial activity in isolation, leaving the following classifiers: >25% proline, modulation of DnaK AND/OR the 70S ribosome, net charge of +1 or more, produced in response to bacterial infection AND/OR with pronounced antimicrobial activity.

Cost-Effectiveness of Dapagliflozin for Non-diabetic Chronic Kidney Disease.

BACKGROUND: In the USA, chronic kidney disease (CKD) affects 1 in 7 adults and costs $100 billion annually. The DAPA-CKD trial found dapagliflozin, a sodium glucose co-transporter 2 (SGLT2) inhibitor, to be effective in reducing CKD progression and mortality in patients with diabetic and non-diabetic CKD. Currently, SGLT2 inhibitors are not considered standard of care for patients with non-diabetic CKD. OBJECTIVE: Determine the cost-effectiveness of adding dapagliflozin to standard management of patients with non-diabetic CKD. DESIGN: Markov model with lifetime time horizon and US healthcare sector perspective. PATIENTS: Patients with non-diabetic CKD INTERVENTION: Dapagliflozin plus standard care versus standard care only. MAIN MEASURES: Quality-adjusted life years (QALYs), costs, and incremental cost-effectiveness ratios (ICERs), all discounted at 3% annually; total incidence of kidney failure on kidney replacement therapy; average years on kidney replacement therapy. KEY RESULTS: Adding dapagliflozin to standard care improved life expectancy by 2 years, increased discounted QALYS (from 6.75 to 8.06), and reduced the total incidence of kidney failure on kidney replacement therapy (KRT) (from 17.4 to 11.0%) and average years on KRT (from 0.77 to 0.43) over the lifetime of the cohort. Dapagliflozin plus standard care was more effective than standard care alone while increasing lifetime costs (from $245,900 to $324,8900, or $60,000 per QALY gained). Results were robust to variations in assumptions about dapagliflozin's efficacy over time and by CKD stage, added costs of kidney replacement therapy, and expected population annual CKD progression rates and sensitive to the cost of dapagliflozin. The net 1-year budgetary implication of treating all US patients with non-diabetic CKD could be up to $21 billion. CONCLUSIONS: Dapagliflozin improved life expectancy and reduced progression of CKD, the proportion of patients requiring kidney replacement therapy, and time on kidney replacement therapy in patients with non-diabetic CKD. Use of dapagliflozin meets conventional criteria for cost-effectiveness.

Scalable and Efficient In Planta Biosynthesis of Sunflower Trypsin Inhibitor-1 (SFTI) Peptide Therapeutics.

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14 amino acid cyclic peptide which has been effectively employed as a scaffold for engineering a range of peptide therapeutic candidates. Typically, synthesis of SFTI-1-based therapeutics is performed via solid-phase peptide synthesis and native chemical ligation, with significant financial and environmental costs associated. In planta synthesis of SFTI-1 based therapeutics serves as a greener approach for environmentally sustainable production. Here, we detail the methods for the transient expression, production, and purification of SFTI-1-based therapeutic peptides in Nicotiana benthamiana using a scalable and high-throughput approach. We demonstrate that a prerequisite for this is the co-expression of specialized asparaginyl endopeptidases (AEPs) that perform the backbone cyclization of SFTI-1. In our founding study, we were able to achieve in planta yields of a plasmin inhibitor SFTI-1 peptide at yields of ~60 µg/g of dried plant material.

Cationic Antimicrobial Peptides Are Leading the Way to Combat Oropathogenic Infections.

Oral dental infections are one of the most common diseases affecting humans, with caries and periodontal disease having the highest incidence. Caries and periodontal disease arise from infections caused by oral bacterial pathogens. Current misuse and overuse of antibiotic treatments have led to the development of antimicrobial resistance. However, recent studies have shown that cationic antimicrobial peptides are a promising family of antibacterial agents that are active against oral pathogenic bacteria and also possess less propensity for development of antimicrobial resistance. This timely Review has a focus on two primary subjects: (i) the oral bacterial pathogens associated with dental infections and (ii) the current development of antimicrobial peptides targeting oral pathogens.

Sublingual Gland and Mylohyoid Muscle Advancement: A Novel Combined Local Soft Tissue Reconstruction.

ABSTRACT: Here the authors present the surgical management of a 78-year-old female with a superficial squamous cell carcinoma of the mucosa overlying her edentulous mandibular alveolus with no evidence of bony invasion.Combining the superficial nature of the lesion, patient age and substantial medical comorbidities, a limited intra-oral resection was planned, to avoid the need for free flap reconstruction. The operation required a wide local excision with clear clinical margins and a mandibular rim resection.All of the currently available reconstructions would result in prolonged surgical time, donor site morbidity, and possible secondary procedures. To overcome these pitfalls, a sublingual gland and mylohyoid muscle advancement flap was designed and executed. By combining the mylohyoid muscle and sublingual gland tissue as an advancement flap in a tension-free manner, secured to the remaining circumferential mucosa, a watertight closure was achieved.After an uneventful recovery without complication the patient was discharged the following day. Complete epithelialization was observed on day 25. Adjuvant radiotherapy was offered to reduce the risk of recurrence and progression. To date, no bony exposure and no pathological fractures have occurred.In conclusion, the entire procedure is simple and innovative. There is minimal donor site morbidity, with an immediate return to oral diet and tolerable surgical risks. It requires a small amount of surgical time compared to other reconstructive options and an overall reduced inpatient stay.

Cyclotide Structures Revealed by NMR, with a Little Help from X-ray Crystallography.

This review highlights the predominant role that NMR has had in determining the structures of cyclotides, a fascinating class of macrocyclic peptides found in plants. Cyclotides contain a cystine knot, a compact structural motif that is constrained by three disulfide bonds and able to resist chemical and biological degradation. Their resistance to proteolytic degradation has made cyclotides appealing as drug leads. Herein, we examine the developments that led to the identification and conclusive determination of the disulfide connectivity of cyclotides and describe in detail the structural features of exemplar cyclotides. We also review the role that X-ray crystallography has played in resolving cyclotide structures and describe how racemic crystallography opened up the possibility of obtaining previously inaccessible X-ray structures of cyclotides.

Novel technique for the direct closure of the radial forearm flap donor site defect with a local bilobed flap.

BACKGROUND: The donor site defect remains the major disadvantage of the radial forearm free flap (RFFF). The purpose of this case series was to evaluate the effectiveness and safety of a local bilobed flap in direct closure of the RFFF donor site defect. METHODS: Between February 2017 and July 2017, a prospective study was designed with 13 patients who had undergone RFFF reconstruction of the oral cavity. The primary purpose was to assess the effectiveness and safety of a bilobed flap closure of the RFFF donor site with primary healing. The late endpoint was to evaluate functional morbidity of the donor hand by comparing preoperative and postoperative outcomes. RESULTS: The donor site defect healed primarily in 12 of the 13 patients. A small area of skin necrosis (4%) developed in one patient, which was managed conservatively and healed by secondary intention. A significant reduction of wrist extension (mean difference [MD] = 2.64°, P = .01) and grip strength (MD = 3.68 kg, P = .04) was observed between preoperative and postoperative measurements. No statistically significant difference was observed regarding flexion, radial deviation, ulnar deviation, supination, pronation and pinch strength (P > .05). CONCLUSIONS: The bilobed flap is a reliable and effective method for closure of a RFFF donor site defect, ≤5 cm in greatest length. The advantages are excellent regional skin color match and avoidance of a second donor site and its inherent complications.

Metastasising Pleomorphic Salivary Adenoma: A Rare Case Report of a Massive Untreated Minor Salivary Gland Pleomorphic Adenoma with Concurrent Ipsilateral Cervical Node Metastases.

Salivary gland tumours constitute approximately 1-5% of all human neoplasms. Pleomorphic adenoma (PA) is the commonest benign neoplasm affecting the parotid gland most often (> 75%), followed by the submandibular gland (13%), then the palate (9%). Metastasising pleomorphic adenoma (MPA) is extremely rare. The effects can be severe and a reported 40% of MPA patients die with disease. This case represents the first known case in English literature of an untreated minor salivary gland PSA of the palate metastasising to an ipsilateral cervical node. We report a 61 year old female who presented with a large tumour occupying the palatal vault, and cervical neck mass. The oral tumour was believed to have been growing over four decades. The patient died eight months following surgical resection. Of known cases, male: female ratio is 35:51 and the mean age at diagnosis is 49.2. Most commonly, MPA is detected in bone 33.3% (n = 29), lung 31% (n = 27) and cervical lymph nodes 20.7% (n = 18). Thorough reporting is deemed essential to further understand the biological differences of non metastasising and metastasising PAs, treatment outcomes, prognosis and survival rates.