{"id":77,"slug":"77-tb-500-n-terminal-myristoylation-covalent-attachment-of-a-myristoyl","title":"TB-500 N-terminal myristoylation for membrane targeting and depot half-life extension","status":"PROMISING","fold_verdict":"PROMISING","discard_reason":null,"peptide":{"name":"TB-500","class":"REGENERATIVE","sequence":"LKKTETQ","modified_sequence":"Myr-LKKTETQ","modification_description":"N-terminal myristoylation: covalent attachment of a myristoyl (C14) fatty acid to the α-amine of Leu-1 via a stable amide bond, yielding Myr-LKKTETQ"},"target":{"protein":"Beta-actin","uniprot_id":"P60709","chembl_id":null,"gene_symbol":"ACTB"},"rationale":{"hypothesis":"We hypothesize that N-terminal myristoylation of TB-500 will improve cellular uptake and create a membrane-anchored depot effect, allowing the peptide to partition into plasma membranes and gradually release into the cytosol where G-actin sequestration occurs. Unlike the previously refined C-terminal palmitoyl-γGlu (Fold #38, albumin-binding strategy), a shorter C14 chain directly on the N-terminus is expected to favor membrane insertion over albumin binding, providing a complementary PK mechanism while also capping the aminopeptidase-vulnerable Leu-1 (building on the Fold #7 acetylation success).","rationale":"Myristoylation is a well-established intracellular targeting motif (e.g., Src-family kinases, MARCKS) where the C14 chain provides reversible, weak membrane affinity (~10⁻⁴ M partition) suitable for dynamic localization — distinct from the strong albumin sequestration of C16/C18 diacids. Since TB-500 must reach intracellular G-actin pools, membrane partitioning followed by flip-flop release could improve cytosolic delivery, a route untested for this peptide. This diverges from the last 3 folds (truncation/DISCARDED, Asp→Asp substitution/REFINED, lipid-extension on Tirzepatide/PROMISING) by combining DELIVERY focus with N-terminal mono-acyl lipidation rather than ε-amine fatty diacid albumin binders. The Leu-1 amide cap also addresses the aminopeptidase liability identified as a key TB-500 weakness.","predicted_outcome":"Boltz/Chai should predict the Myr-LKKTETQ peptide retaining the canonical LKKT motif geometry against actin subdomain 1/2, with the myristoyl chain projecting away from the binding interface (no steric clash) since the N-terminus of TB-500 in the actin-bound state is solvent-exposed. Expect pLDDT ≥0.80 on the LKKTETQ core and a flexible/disordered lipid tail. Interface contacts (Lys-2/Lys-3 with actin acidic patch, Thr-4 H-bonds) should be preserved relative to unmodified TB-500.","mechanism_class":null,"biohacker_use":null},"confidence":{"plddt":0.8731597661972046,"ptm":0.9156717658042908,"iptm":0.6029306650161743,"chai_agreement":null,"chai1_gated_decision":"SKIPPED_HIGH_CONFIDENCE","binding_probability":null,"binding_pic50":null,"predicted_binding_change":null},"profile":{"aggregation_propensity":0.0,"stability_score":0.5,"bbb_penetration_score":0.067,"half_life_estimate":"short (~15–45 minutes)"},"narrative":{"tldr":"DISTILLATION №77 tests N-terminal myristoylation of TB-500 (Myr-LKKTETQ), hypothesizing that a C14 fatty acid chain on Leu-1 would create a membrane-anchored depot for improved intracellular delivery to G-actin pools. Despite a surprisingly strong pLDDT of 0.87 on the peptide core, the fold was DISCARDED due to a tool-limit failure: AlphaFold/Boltz-2 cannot model the non-canonical myristoyl lipid moiety, meaning the predicted structure reflects the bare LKKTETQ backbone only and not the actual modified molecule. The binder probability module returned no values, and the ipTM of 0.60 reflects insufficient complex-level confidence to call binding. This is a tool resolution failure, not a biological invalidation — the membrane depot hypothesis remains untested.","detailed_analysis":"TB-500 (Ac-LKKTETQ) is the heptapeptide core of thymosin β4, responsible for G-actin sequestration via its LKKT pharmacophore. The canonical commercial formulation already incorporates N-terminal acetylation — a small, flat C2 cap that protects Leu-1 from aminopeptidase attack while remaining sterically unobtrusive at the actin-binding interface. This fold escalates that concept dramatically by substituting the acetyl for a myristoyl (C14 saturated fatty acid) chain, a modification borrowed from endogenous membrane-targeting proteins like Src-family kinases and HIV-1 Gag p17. The hypothesis is that the C14 chain would partition into plasma membranes, creating a reversible depot from which the peptide gradually releases into the cytosol to engage G-actin — a delivery route not previously explored for this peptide class.\n\nThe cross-fold context is rich and directly relevant. Fold #38 established the first lipidation precedent for TB-500, using a C-terminal palmitoyl-γGlu-Lys-7 construct targeting albumin-mediated half-life extension; it was REFINED with pLDDT 0.84, validating lipidation as a productive direction. Fold #7 (acetylation, referenced in the researcher's rationale) confirmed that N-terminal caps are well-tolerated. Fold #77 therefore represents a logical escalation: combining the aminopeptidase-protection benefit of Fold #7 with a lipid-delivery mechanism complementary to (but mechanistically distinct from) Fold #38's albumin strategy. The progression makes scientific sense on paper — the lab has now probed both ends of the molecule with lipid handles.\n\nThe structural prediction returned a paradoxically high pLDDT of 0.87 for the peptide backbone, which superficially looks encouraging. However, this score is an artifact of the tool's behavior: AF2/Boltz-2 strip non-standard chemical modifications and predict the unmodified or minimally modified backbone. The myristoyl chain — a 14-carbon lipid tail covalently bonded to the Leu-1 α-amine — is invisible to the model. What was predicted is essentially LKKTETQ geometry, not Myr-LKKTETQ behavior. The pTM of 0.92 likewise reflects intra-peptide confidence on a short sequence. The ipTM of 0.60 falls below the threshold for confident complex-level binding prediction, and critically, the Boltz-2 affinity module returned no values — the binding probability module simply could not adjudicate. Chai-1 agreement data were unavailable.\n\nFrom a biological standpoint, the literature raises legitimate concerns that compound the tool limitations. Leu-1 and Lys-2 are the N-terminal residues that anchor LKKTETQ to actin subdomain 1/2; N-terminal acetylation is tolerated, but a C14 chain represents a qualitatively different steric perturbation. The Rahaman et al. (2024) metabolic data confirm Leu-1's aminopeptidase vulnerability, supporting the protection rationale, but also show that only fragments ≥5 residues (Ac-LKKTE) retain wound healing activity — suggesting the N-terminal region contributes meaningfully to efficacy and cannot be freely decorated. Aqueous solubility and aggregation are practical concerns: C14 chains on heptapeptides may drive micelle formation below the critical micellar concentration (CMC) at therapeutically relevant concentrations, complicating dosing. The Judák et al. (2017) adsorption data show unmodified TB-500 already suffers surface binding problems; lipidation would worsen this.\n\nThe heuristic peptide profile (sequence-based estimates, not wet-lab measurements) paints a modest picture: aggregation propensity of 0.0 (likely underestimated for the lipidated form, as the tool sees the bare sequence), stability score of 0.5, and a short half-life estimate of 15–45 minutes — consistent with the known rapid catabolism of TB-500 in the Rahaman et al. data, though the myristoyl cap is expected to extend this somewhat by blocking aminopeptidase entry at Leu-1.\n\nThis discard sits in a pattern for this peptide. Fold #65 (D-Lys-3 chirality inversion) and Fold #51 (Thr-4 → 4F-Phe) were both discarded, while the structurally conservative modifications — Fold #38 (C-terminal palmitoyl), Fold #28 (lactam bridge) — achieved REFINED verdicts. The pattern suggests TB-500's actin-binding heptapeptide tolerates backbone conformational stabilization and C-terminal extensions better than N-terminal bulk or non-canonical substitutions at pharmacophore-adjacent positions. Fold #77's myristoylation sits squarely in the 'N-terminal bulk' category that has consistently challenged the predictors on this sequence.\n\nThe failure here is unambiguously a tool-limit failure, not a biological one. Standard structure-prediction pipelines are designed for proteinogenic amino acids in aqueous environments; lipid moieties require molecular dynamics with explicit membrane models, enhanced sampling, or coarse-grained force fields (CHARMM-GUI, GROMACS with Slipids/CHARMM36, or coarse-grained MARTINI simulations). The membrane depot hypothesis is pharmacologically coherent and literature-supported in principle — it simply cannot be evaluated by the current in silico stack. Wet-lab synthesis of Myr-LKKTETQ followed by SPR against immobilized G-actin, combined with liposome partitioning assays and CMC measurements, would provide the definitive answer the predictors cannot.","executive_summary":"Fold #77 myristoylates TB-500's N-terminus (Myr-LKKTETQ) for a membrane depot strategy complementing Fold #38's albumin-binding palmitoyl. DISCARDED: structure predictors are blind to the C14 lipid — pLDDT 0.87 reflects the bare peptide backbone, affinity module returned no values. Hypothesis intact; needs wet-lab or MD.","tweet_draft":"DISTILLATION №77 — DISCARDED (tool-limit).\nTB-500 N-terminal myristoylation (Myr-LKKTETQ).\nMembrane depot hypothesis — C14 chain invisible to AF2/Boltz.\npLDDT 0.87 (bare backbone). Affinity module: no output.\nNot disproved. Needs MD sim or wet lab.\nalembic.bio","research_brief_markdown":"# FOLD №77 — TB-500 N-terminal Myristoylation\n### Verdict: DISCARDED (Tool-Limit Failure)\n\n---\n\n## TLDR\n\nFold #77 was **DISCARDED** due to a tool-limit failure: current structure predictors (AlphaFold/Boltz-2/Chai-1) cannot model the N-terminal myristoyl (C14 fatty acid) modification, meaning the structural output reflects the bare LKKTETQ backbone only — not the actual Myr-LKKTETQ molecule under investigation. The Boltz-2 affinity module returned no binding probability values, and the ipTM of 0.60 is insufficient for a confident complex-level verdict. This is **not a biological invalidation** of the membrane depot hypothesis.\n\n---\n\n## What We Tried\n\nThis fold tested N-terminal myristoylation of TB-500 — specifically, covalent attachment of a myristoyl (C14 saturated fatty acid) chain to the α-amine of Leu-1 via a stable amide bond, yielding **Myr-LKKTETQ**. The hypothesis was that the C14 chain would partition into plasma membranes, creating a reversible lipid depot from which the active heptapeptide would gradually release into the cytosol to engage its target: G-actin sequestration via the canonical LKKT pharmacophore.\n\nThis is the lab's **second lipidation strategy** for TB-500 and the first to target the N-terminus. Fold #38 established that C-terminal palmitoyl-γGlu lipidation (LKKTETK(γGlu-Palm)-NH₂) was REFINED with pLDDT 0.84, validating lipidation as a productive direction for this peptide. Fold #77 was designed as a complementary, mechanistically distinct approach: where Fold #38 targeted albumin-mediated systemic half-life extension via a C16 chain at the C-terminus, this fold targeted **membrane insertion and intracellular depot formation** via a shorter C14 chain at the N-terminus — a strategy known from endogenous myristoylated proteins (Src, HIV-1 Gag, MARCKS).\n\nThe N-terminal position was also chosen because aminopeptidase cleavage at Leu-1 is a documented TB-500 liability (Rahaman et al., 2024, showing Ac-LK as the primary short-interval metabolite). The existing commercial formulation uses acetylation (Ac-LKKTETQ) to partially address this; a myristoyl amide bond was hypothesized to provide more robust protection. This builds directly on the rationale established in Fold #7 (N-terminal acetylation) and addresses a gap that the acetyl cap only partially closes.\n\n---\n\n## Why It Was Discarded\n\nThe discard is a **tool-limit failure**, not a structural or biological red flag:\n\n- **AlphaFold2/Boltz-2 blind to lipid chemistry**: Standard structure-prediction pipelines process proteinogenic amino acid sequences. The myristoyl chain — a non-canonical chemical modification attached to the Leu-1 α-amine — is stripped or ignored by the model. The resulting prediction describes the LKKTETQ backbone geometry, not Myr-LKKTETQ behavior in a membrane environment.\n- **No affinity module output**: The Boltz-2 binder probability module returned **no values** — the most direct signal that the modified molecule was outside the model's evaluable chemical space.\n- **ipTM 0.60**: Complex-level confidence fell below the threshold for a reliable binding call. On a heptapeptide without reliable interface atom placement for the modified terminus, this is expected rather than informative.\n- **High pLDDT (0.87) is an artifact**: The strong backbone confidence score reflects the model's familiarity with LKKTETQ as a short, well-behaved polar sequence — not confidence in the myristoylated molecule. A high pLDDT on a stripped sequence should not be interpreted as structural validation of the modification.\n- **Chai-1 agreement unavailable**: No ensemble cross-check was possible to assess prediction stability.\n\nBiological concerns (not the primary discard reason, but relevant context):\n- Leu-1 and Lys-2 are directly adjacent to the actin-binding pharmacophore; a C14 chain at this position may impose steric costs on LKKT–actin engagement that acetylation does not.\n- C14 chains on heptapeptides may drive **micelle formation** at physiological concentrations (CMC considerations), rather than monomeric membrane insertion.\n- No published data exist on any lipidated LKKTETQ analog; the membrane depot mechanism is entirely de novo.\n\n---\n\n## What This Doesn't Mean\n\n**DISCARDED does not mean disproved.** This verdict reflects the boundaries of the current in silico toolchain, not a judgment on the biological validity of N-terminal myristoylation for TB-500. The membrane depot hypothesis is pharmacologically coherent, literature-supported in analogous systems (Src-family kinases, lipidated cell-penetrating peptides), and mechanistically distinct from anything the lab has tested before for this peptide. The failure to obtain a binding probability value means the question was never answered — not that the answer is negative. It is entirely possible that Myr-LKKTETQ inserts into membranes with appropriate kinetics, releases active peptide into the cytosol, and engages G-actin with preserved or enhanced potency. That question requires wet-lab or specialized computational tools to resolve.\n\n---\n\n## What Would Answer the Question\n\n- **Solid-phase peptide synthesis + HPLC purification** of Myr-LKKTETQ, followed by **SPR (surface plasmon resonance)** against immobilized G-actin (rabbit muscle, Cytoskeleton Inc.) to directly measure K_D relative to Ac-LKKTETQ. This is the gold-standard binding assay for this target class and is directly analogous to the approach used in Tβ4 fragment SAR studies.\n- **CMC measurement and liposome partitioning assay** (pyrene fluorescence probe or DLS) to determine whether Myr-LKKTETQ forms micelles at physiologically relevant concentrations (1–100 µM range) or partitions as monomer into model POPC/POPE membranes — directly testing the depot hypothesis.\n- **Coarse-grained MD simulation** (MARTINI force field, GROMACS/CHARMM-GUI) of Myr-LKKTETQ in an explicit POPC bilayer to model membrane insertion depth, orientation, and flip-flop kinetics — the computational tool class appropriate for lipid-modified peptides that AF2 cannot handle.\n- **Aminopeptidase protection assay** (leucine aminopeptidase, human serum stability) comparing Myr-LKKTETQ vs. Ac-LKKTETQ degradation kinetics, directly testing the N-terminal cap hypothesis from the Rahaman et al. (2024) metabolic vulnerability data.\n\n---\n\n## Raw Metrics\n\n| Metric | Value | Interpretation |\n|---|---|---|\n| pLDDT | 0.873 | High — but reflects bare LKKTETQ backbone, not the lipidated molecule |\n| pTM | 0.916 | Strong intra-peptide fold confidence (short sequence artifact) |\n| ipTM | 0.603 | Below threshold for confident complex-level binding call |\n| Boltz-2 binder probability | — (no output) | Tool could not evaluate myristoylated molecule |\n| Chai-1 agreement | N/A | Not available for this fold |\n| Aggregation propensity (heuristic) | 0.0 | Likely underestimated; tool sees bare sequence, not C14 chain |\n| Stability score (heuristic) | 0.5 | Moderate; myristoyl cap expected to improve but unquantified |\n| Half-life estimate (heuristic) | 15–45 min | Consistent with known TB-500 rapid catabolism; actual improvement from myristoyl cap is untested |\n| BBB penetration (heuristic) | 0.067 | Not a relevant endpoint for this regenerative peptide |\n\n---\n\n*All values are in silico predictions. No wet-lab validation has been performed. This report is research context only, not medical advice.*","structural_caption":"No reliable 3D structure could be obtained for this peptide.","key_findings_summary":"TB-500 (Ac-LKKTETQ) is the N-terminal acetylated heptapeptide fragment (residues 17-23) of thymosin β4 (Tβ4), and the literature consistently identifies the LKKTETQ sequence as the actin-binding active site of Tβ4 responsible for G-actin sequestration, cell migration, wound healing, and angiogenesis. Multiple doping control and analytical chemistry papers (Ho et al. 2012, Esposito et al. 2012, Rahaman et al. 2024) confirm the commercial TB-500 formulation as Ac-LKKTETQ, with the N-terminal acetyl group being an intentional modification that both caps the free amine and provides some protection against aminopeptidase degradation at Leu-1. Importantly, the Rahaman et al. (2024) metabolic study demonstrates that TB-500 is rapidly degraded in human serum and rat urine, with Ac-LK identified as the primary short-interval metabolite and Ac-LKK as a long-lived metabolite detectable up to 72 hours—only the longer fragment Ac-LKKTE showed significant wound healing activity, underscoring the biological importance of preserving the full heptapeptide sequence for efficacy.\n\nThe mechanistic basis for the hypothesis centers on the well-established actin-binding function of the LKKTETQ motif. The crystal and NMR data in the broader Tβ4 literature (not captured in these abstracts but referenced by multiple review papers included here) confirms that residues 17-23 engage G-actin via the Leu-17/Lys-18 interface, meaning that any N-terminal modification must be compatible with this binding interaction. N-terminal acetylation (as in the existing TB-500 formulation) is already tolerated, providing a precedent that N-terminal modifications do not inherently abolish actin-binding activity. However, the jump from a small acetyl (C2) to a bulky myristoyl (C14) chain is a qualitatively different modification, and no literature directly evaluates myristoylated LKKTETQ or myristoylated Tβ4 fragments.\n\nMyristoylation as a general membrane-anchoring and cellular uptake strategy is well-established for proteins and peptides (e.g., Src-family kinases, HIV-1 Gag), with C14 fatty acid chains providing sufficient hydrophobicity for stable but reversible plasma membrane association. The proposed 'depot effect'—membrane partitioning followed by gradual cytosolic release—is pharmacologically plausible and has precedent in lipidated peptide drug design (e.g., liraglutide's albumin-binding palmitate, fatty-acid-modified cell-penetrating peptides). However, none of the TB-500 literature retrieved here specifically investigates lipidated variants. The review and sports medicine literature (Mendias & Awan 2026, Mayfield et al. 2026, Rahman et al. 2026) addresses TB-500 broadly as a regenerative peptide with promising preclinical data but absent human clinical trials, not lipidated analogs.\n\nFrom a pharmacokinetic standpoint, the Rahaman et al. (2024) metabolic data provide the most directly relevant challenge: TB-500 is degraded rapidly, and the metabolite profile suggests that N-terminal modifications (acetyl in the current form) are cleaved or bypass-metabolized relatively quickly in vivo. Myristoylation would more robustly protect the N-terminus against aminopeptidase cleavage compared to acetylation, which is consistent with the hypothesis. However, a C14 chain may also dramatically reduce aqueous solubility and could promote peptide aggregation or micelle formation rather than clean membrane insertion, potentially complicating dosing and delivery. No literature was retrieved on myristoyl-TB-500 specifically, leaving the membrane depot hypothesis empirically untested."},"structured":{"known_activity":null,"known_binders":null,"candidate_variants":null,"domain_annotations":null,"literature_context":{"pubmed":[{"pmid":"41490200","title":"Therapeutic Peptides in Orthopaedics: Applications, Challenges, and Future Directions.","abstract":"Therapeutic peptides are emerging as promising adjuncts in the management of orthopaedic injuries, grounded in their ability to modulate molecular signaling networks central to cellular medicine. By acting on key pathways such as PI3K/Akt, mTOR, MAPK, TGF-β, and AMPK, peptides exert influence over tissue regeneration, inflammation resolution, and neuromuscular recovery. Wound-healing peptides such as BPC-157, TB-500, and GHK-Cu promote angiogenesis, integrin-mediated extracellular matrix remodeling, and fibroblast activation, whereas growth hormone secretagogues like ipamorelin, CJC-1295, tesamorelin, sermorelin, and AOD-9604 activate IGF-1 signaling and satellite cell repair. Recovery-enhancing agents such as epithalon, delta sleep-inducing peptide, and pinealon target circadian and mitochondrial regulators, and neuroactive peptides like selank, semax, and dihexa enhance brain-derived neurotrophic factor and HGF/c-Met pathways critical to neuroplasticity. Although preclinical studies are promising, there is a current lack of clinical trials. This review integrates current mechanistic insights with orthopaedic relevance, emphasizing safety, efficacy, and future directions for responsible integration into musculoskeletal care.","authors":["Rahman Omar F","Lee Steven J","Seeds William A"],"year":2026,"journal":"Journal of the American Academy of Orthopaedic Surgeons. Global research & reviews"},{"pmid":"38382158","title":"Simultaneous quantification of TB-500 and its metabolites in in-vitro experiments and rats by UHPLC-Q-Exactive orbitrap MS/MS and their screening by wound healing activities in-vitro.","abstract":"BACKGROUND: TB-500 (Ac-LKKTETQ), derived from the active site of thymosin β4 (Tβ4), has various biological functions in its unacetylated form, LKKTETQ. These functions include actin binding, dermal wound healing, angiogenesis, and skin repair. The biological effects of TB-500, however, have not been documented. And the analysis of TB-500 and its metabolites have been neither simultaneously quantified nor structurally identified using synthesized authentic standards.\n\nMETHODS: This study was aimed to investigating simultaneous analytical methods of TB-500 and its metabolites in in-vitro and urine samples by using UHPLC-Q-Exactive orbitrap MS, and to comparing the biological activity of its metabolites with the parent TB-500. The metabolism of TB-500 was investigated in human serum, various in-vitro enzyme systems, and urine samples from rats treated with TB-500, and their biological activities measured by cytotoxicity and wound healing experiments were also evaluated in fibroblasts.\n\nRESULTS: The simultaneous analytical method for TB-500 and its metabolites was developed and validated. The study found that Ac-LK was the primary metabolite with the highest concentration in rats at 0-6 h intervals. Also, the metabolite Ac-LKK was a long-term metabolite of TB-500 detected up to 72 hr. No cytotoxicity of the parent and its metabolites was found. Ac-LKKTE only showed a significant wound healing activity compared to the control.\n\nCONCLUSION: The study provides a valuable tool for quantifying TB-500 and its metabolites, contributing to the understanding of metabolism and potential therapeutic applications. Our results also suggest that the previously reported wound-healing activity of TB-500 in literature may be due to its metabolite Ac-LKKTE rather than the parent form.","authors":["Rahaman Khandoker Asiqur","Muresan Anca Raluca","Min Hophil","Son Junghyun","Han Hyung-Seop","Kang Min-Jung","Kwon Oh-Seung"],"year":2024,"journal":"Journal of chromatography. B, Analytical technologies in the biomedical and life sciences"},{"pmid":"41476424","title":"Injectable Peptide Therapy: A Primer for Orthopaedic and Sports Medicine Physicians.","abstract":"BACKGROUND: Therapeutic peptides are short-chain amino acids that regulate cellular functions and facilitate biochemical processes. In recent years, there has been significant growth in the global market for therapeutic peptides and thus its popularity among patients. Given the increase in the development of peptides and increased marketing to patients for orthopaedic injuries, it is critical for orthopaedic surgeons to understand the current evidence behind these therapeutic peptides.\n\nPURPOSE: To evaluate the current evidence and applications of injectable peptide therapy, focusing on its potential in regenerative medicine and sports performance, to help orthopaedic providers better understand the current state of different therapeutic peptide approaches.\n\nSTUDY DESIGN: Narrative review.\n\nMETHODS: A comprehensive literature search was conducted using PubMed to identify biochemical and clinical studies on the most popular types of injectable peptide therapy. Key peptides evaluated included BPC-157, TB-4, TB-500, CJC-1295 + ipamorelin, tesamorelin, and GHK-Cu.\n\nRESULTS: BPC-157 demonstrated potential benefits in tendon and muscle repair, but these findings are largely unvalidated in human trials. A single human case series reported improvements in pain after intra-articular knee injections of BPC-157, although significant methodological flaws and a lack of controls limit its applicability and reliability. TB-4 and its derivative TB-500 promoted angiogenesis and tissue repair in preclinical models, but human orthopaedic data are lacking, and both remain banned substances in sports. CJC-1295 combined with ipamorelin showed significantly improved maximum tetanic tension in murine models with glucocorticoid-induced muscle loss, but these findings are limited to animal studies. Tesamorelin, approved for treating HIV-associated lipodystrophy, has no supporting orthopaedic evidence. GHK-Cu showed promise in wound healing and anti-inflammatory effects, but no clinical data support its use for musculoskeletal conditions.\n\nCONCLUSION: While peptide therapy may possess significant therapeutic and regenerative potential, it is critical that orthopaedic and sports medicine providers understand the current lack of evidence to support the clinical use of these peptides. Importantly, information regarding the indications, dosing, frequency, and duration of treatment remains unknown. Despite the popularity of these peptides in mainstream media and among patients, significant research regarding the safety and efficacy of these therapeutic methods is required before definitive recommendations can be made to patients.","authors":["Mayfield Cory K","Bolia Ioanna K","Feingold Cailan L","Lin Eric H","Liu Joseph N","Rick Hatch George F","Gamradt Seth C","Weber Alexander E"],"year":2026,"journal":"The American journal of sports medicine"},{"pmid":"23084823","title":"Doping control analysis of TB-500, a synthetic version of an active region of thymosin β₄, in equine urine and plasma by liquid chromatography-mass spectrometry.","abstract":"A veterinary preparation known as TB-500 and containing a synthetic version of the naturally occurring peptide LKKTETQ has emerged. The peptide segment (17)LKKTETQ(23) is the active site within the protein thymosin β(4) responsible for actin binding, cell migration and wound healing. The key ingredient of TB-500 is the peptide LKKTETQ with artificial acetylation of the N-terminus. TB-500 is claimed to promote endothelial cell differentiation, angiogenesis in dermal tissues, keratinocyte migration, collagen deposition and decrease inflammation. In order to control the misuse of TB-500 in equine sports, a method to definitely identify its prior use in horses is required. This study describes a method for the simultaneous detection of N-acetylated LKKTETQ and its metabolites in equine urine and plasma samples. The possible metabolites of N-acetylated LKKTETQ were first identified from in vitro studies. The parent peptide and its metabolites were isolated from equine urine or plasma by solid-phase extraction using ion-exchange cartridges, and analysed by liquid chromatography-mass spectrometry (LC/MS). These analytes were identified according to their LC retention times and relative abundances of the major product ions. The peptide N-acetylated LKKTETQ could be detected and confirmed at 0.02 ng/mL in equine plasma and 0.01 ng/mL in equine urine. This method was successful in confirming the presence of N-acetylated LKKTETQ and its metabolites in equine urine and plasma collected from horses administered with a single dose of TB-500 (containing 10mg of N-acetylated LKKTETQ). To our knowledge, this is the first identification of TB-500 and its metabolites in post-administration samples from horses.","authors":["Ho Emmie N M","Kwok W H","Lau M Y","Wong April S Y","Wan Terence S M","Lam Kenneth K H","Schiff Peter J","Stewart Brian D"],"year":2012,"journal":"Journal of chromatography. A"},{"pmid":"28887173","title":"Adsorption effects of the doping relevant peptides Insulin Lispro, Synachten, TB-500 and GHRP 5.","abstract":"The tendency of peptides to adsorb to surfaces can raise a concern in variety of analytical fields where the qualitative/quantitative measurement of low concentration analytes (ng/mL-pg/mL) is required. To demonstrate the importance of using the optimal glassware/plasticware, four doping relevant model peptides (GHRP 5, TB-500, Insulin Lispro, Synachten) were chosen and their recovery from various surfaces were evaluated. Our experiments showed that choosing expensive consumables with low-bind characteristics is not beneficial in all cases. A careful selection of the consumables based on the evaluation of the physico/chemical features of the peptide is recommended.","authors":["Judák Péter","Van Eenoo Peter","Deventer Koen"],"year":2017,"journal":"Analytical biochemistry"},{"pmid":"22962027","title":"Synthesis and characterization of the N-terminal acetylated 17-23 fragment of thymosin beta 4 identified in TB-500, a product suspected to possess doping potential.","abstract":"The formulation TB-500 is suspected to be used as doping agent in sport. This work describes the detection and the identification of the N-terminal acetylated 17-23 fragment of human thymosin beta 4 (Ac-LKKTETQ) in TB-500 by means of high-performance liquid chromatography/high resolution mass spectrometry using an Orbitrap Exactive benchtop mass spectrometer. Ac-LKKTETQ was also synthesized by solid-phase peptide synthesis, and an analytical strategy for detection in plasma and urine by high-performance liquid chromatography/low resolution triple-quadrupole mass spectrometry was suggested.","authors":["Esposito Simone","Deventer Koen","Goeman Jan","Van der Eycken Johan","Van Eenoo Peter"],"year":2012,"journal":"Drug testing and analysis"},{"pmid":"24906629","title":"Analytical approaches for the detection of emerging therapeutics and non-approved drugs in human doping controls.","abstract":"The number and diversity of potentially performance-enhancing substances is continuously growing, fueled by new pharmaceutical developments but also by the inventiveness and, at the same time, unscrupulousness of black-market (designer) drug producers and providers. In terms of sports drug testing, this situation necessitates reactive as well as proactive research and expansion of the analytical armamentarium to ensure timely, adequate, and comprehensive doping controls. This review summarizes literature published over the past 5 years on new drug entities, discontinued therapeutics, and 'tailored' compounds classified as doping agents according to the regulations of the World Anti-Doping Agency, with particular attention to analytical strategies enabling their detection in human blood or urine. Among these compounds, low- and high-molecular mass substances of peptidic (e.g. modified insulin-like growth factor-1, TB-500, hematide/peginesatide, growth hormone releasing peptides, AOD-9604, etc.) and non-peptidic (selective androgen receptor modulators, hypoxia-inducible factor stabilizers, siRNA, S-107 and ARM036/aladorian, etc.) as well as inorganic (cobalt) nature are considered and discussed in terms of specific requirements originating from physicochemical properties, concentration levels, metabolism, and their amenability for chromatographic-mass spectrometric or alternative detection methods.","authors":["Thevis Mario","Schänzer Wilhelm"],"year":2014,"journal":"Journal of pharmaceutical and biomedical analysis"},{"pmid":"40681595","title":"Comparative effects of dietary sodium butyrate and tributyrin on broiler chickens' performance, gene expression, intestinal histomorphometry, blood indices, and litter.","abstract":"Sodium butyrate and tributyrin are known to enhance broiler chicken performance. In this study, 1,000 Arbor Acres broiler chicks were assigned to four dietary treatments (250 birds each; six replicates of 40-42 birds): a control basal diet (CON), or the same diet supplemented with either 500 g/ton tributyrin (40%) + copper + essential oils (TB-500), 300 g/ton di- and tri-butyrin (60%) (TB-300), or 500 g/ton coated sodium butyrate (40%) (SB-500). Weekly growth parameters were recorded, and on Day 35, carcass traits, serum biochemistry, immunity, gene expression (mTOR, TLR4, NBN), intestinal morphology, caecal microbiota, and litter hygiene were assessed. TB-300 improved body weight (+ 4.6%, P = 0.014), FCR (- 5.2%, P = 0.032), and European Production Efficiency Factor (EPEF) (+ 14.9%, P = 0.006). SB-500 significantly reduced litter Clostridia (P < 0.0001) and aerobic bacteria (P = 0.026) counts, while all butyrate treatments lowered caecal aerobic bacterial levels (P = 0.041). TB-300 and SB-500 enhanced duodenal villi height (P < 0.0001) and crypt-villus ratio (P < 0.001); TB-500 had the deepest duodenal crypts (P = 0.003). Jejunal and ileal morphology improved with most of the supplements, particularly TB-500 (P < 0.0001; P = 0.050). All butyrate treatments increased serum total proteins (P = 0.015) and digestive enzymes (lipase, P < 0.0001; protease, P = 0.001). TB-300 and SB-500 significantly lowered serum lipids (P = 0.024), urea (P = 0.018), and aspartate aminotransferase (AST) (P = 0.027), while enhancing mTOR and NBN gene expression (P < 0.0001). TLR4 expression was upregulated in all butyrate-treated groups (P < 0.0001). Each form of butyrate supplementation exerts distinct beneficial effects on growth, gut health, and physiological performance in broiler chickens.","authors":["Ismael Elshaimaa","Kamel Shaimaa","Elleithy Ebtihal M M","Bekeer Manal R","Fahmy Khaled Nasr El-Din"],"year":2025,"journal":"Scientific reports"}],"biorxiv":[{"pmid":"","doi":"10.21203/rs.3.rs-8237978/v1","title":"Head-to-head comparison of [177Lu]Lu-FAP-2286 and [161Tb]Tb-FAP-2286 efficacy in a PDAC mouse model: Is there an added benefit of internal conversion and Auger electrons for FAP-TRT?","abstract":"<title>Abstract</title>  <p>  Background  Terbium-161 (Tb-161) emits internal conversion and Auger electrons, in addition to beta-minus radiation, which might be of added benefit for targeted radionuclide therapy (TRT) compared to Lutetium-177 (Lu-177). We extensively compared Lu-177 and Tb-161 for fibroblast activation protein (FAP)-targeted TRT in a preclinical setting. To study this, FAP-2286 was labeled with Lu-177 and Tb-161 and characterized in vitro on FAP-expressing cells and ex vivo using patient tumor samples. Moreover, in vivo studies (i.e. biodistribution and efficacy) were performed using a clinically representative pancreatic ductal adenocarcinoma (PDAC) mouse model. Biodistribution was performed 1, 4, 24, and 48 h post injection of 5 MBq/500 pmol [  <sup>177</sup>  Lu]Lu-FAP-2286 or [  <sup>161</sup>  Tb]Tb-FAP-2286. Subsequently, animals were treated with 4×40 MBq/500 pmol [  <sup>177</sup>  Lu]Lu-FAP-2286 or [  <sup>161</sup>  Tb]Tb-FAP-2286 and with alternating doses of 2×40 MBq/500 pmol of each radiopharmaceutical. Results  No difference in [  <sup>177</sup>  Lu]Lu-FAP-2286 and [  <sup>161</sup>  Tb]Tb-FAP-2286 uptake was observed in the cell models. In vivo studies did not show a survival benefit after 4×40 MBq/500 pmol [  <sup>177</sup>  Lu]Lu-FAP-2286 or [  <sup>161</sup>  Tb]Tb-FAP-2286, while Kaplan-Meier analyses demonstrated modestly prolonged survival after tandem therapy, in mice that first received [  <sup>177</sup>  Lu]Lu-FAP-2286 followed by [  <sup>161</sup>  Tb]Tb-FAP-2286. Dosimetry calculations based on autoradiography on patient tumor samples showed that even with lower binding, a higher absorbed dose to the tumor can be accomplished with [  <sup>161</sup>  Tb]Tb-FAP-2286. Conclusions  In our vitro and in vivo studies, [  <sup>177</sup>  Lu]Lu-FAP-2286 and [  <sup>161</sup>  Tb]Tb-FAP-2286 demonstrated similar behavior. In the applied PDAC mouse model, FAP-TRT showed limited therapeutic efficacy, with a modest response observed in the tandem therapy group that first received [  <sup>177</sup>  Lu]Lu-FAP-2286, followed by [  <sup>161</sup>  Tb]Tb-FAP-2286.  </p>","authors":["Heide CDvd","Ntihabose CM","Konijnenberg M","Ma H","Stuurman D","de Ridder C","Seimbille Y","Doukas MC","de Blois E","Dalm SU."],"year":2025,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.21203/rs.3.rs-8427494/v1","title":"Excess Tuberculosis Incidence in the United States During COVID-19: A State, Age, and Race/Ethnicity Analysis and Structural Drivers of Variation (2020–2023)","abstract":"<title>Abstract</title>  <p>  <bold>Background:</bold>  The COVID-19 pandemic disrupted healthcare systems and disease surveillance worldwide, potentially affecting tuberculosis (TB) detection and control. While global analyses have documented major TB setbacks, the extent to which pandemic-related disruptions altered TB incidence patterns across U.S. demographic and geographic groups remains unclear. This study aimed to quantify excess TB incidence (newly reported TB cases) across U.S. jurisdictions, age groups, and racial/ethnic populations during 2020--2023, and to assess structural factors associated with geographic disparities in excess TB burden.  <bold>Methods:</bold>  We used a sub-epidemic ensemble modeling framework applied to annual U.S. TB incidence data, defined as newly reported TB cases. Models were calibrated to pre-pandemic trends (2010--2019) and used to generate counterfactual forecasts for 2020--2023. Because publicly available TB surveillance data are one-way stratified, we calibrated separate models for each jurisdiction, age group, and racial/ethnic category. Excess TB cases were defined as the difference between observed and expected counts, with 95% prediction intervals estimated via bootstrap simulation. Analyses were classified by jurisdiction (50 states, along with D.C. and Puerto Rico), age (11 groups from younger than 1 to greater than 85 years), and race/ethnicity (8 groups). A Poisson error structure was applied consistently across all models. To investigate predictors of state-level excess TB burden, we performed backward stepwise ordinary least squares (OLS) regression using seven candidate predictors: population density, percentage foreign-born, poverty rate, HIV prevalence, incarceration rate, homelessness rate, and percentage American Indian/Alaska Native (AI/AN) population.  <bold>Results:</bold>  Excess TB burden varied widely across jurisdictions. Texas (410 cases [95%PI: 59--930]), New York (380 [200--680]), Florida (260 [61--600]), and California (200 [62--500]) had the highest excess case counts. Population-adjusted analyses revealed a markedly different pattern, with Alaska showing the largest excess rate (13 per 100,000 [0–35]), emphasizing disproportionate impacts in smaller but structurally vulnerable jurisdictions. Working-age adults carried the greatest excess burden, particularly those aged 35--44 (650 cases [300--1200]) and 25--34 (630 [330--1100]). Large racial and ethnic disparities were observed: the Hispanic population experienced the highest excess burden (1,700 cases [1,100--2,500]), with notable excess also among American Indian/Alaska Native populations (140 cases [61--210]) despite their small population share, while the Asian population showed no excess case counts. Several jurisdictions and the 55--64 age group had uncertainty intervals including zero, suggesting patterns consistent with pre-pandemic trends. Stepwise regression identified four predictors of state-level excess TB cases: percentage foreign-born (positive association), incarceration rate (positive association), homelessness rate (positive association), and population density (negative association), with an adjusted \\((R^2)\\) of 0.36.  <bold>Conclusions:</bold>  The COVID-19 pandemic had uneven effects on TB incidence across the United States. Estimated excess TB incidence likely reflects a combination of delayed diagnosis, disruptions to routine surveillance and care, and post-pandemic rebound in case detection, rather than increased transmission alone. Working-age Hispanic adults and residents of jurisdictions with high proportions of foreign-born individuals, elevated incarceration rates, and substantial homelessness experienced the greatest excess burden.  </p>","authors":["Karami H","Rajaram V","Lee S","Mamelund S","Chowell G."],"year":2026,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.20944/preprints202512.1011.v3","title":"Safety and Efficacy of Approved and Unapproved Peptide Therapies for Musculoskeletal Injuries and Athletic Performance","abstract":"Peptides are short chains of amino acids with a unique pharmacological niche between small-molecule drugs and large proteins. Their use in sports medicine is rapidly expanding, driven by patient demand for accelerated injury recovery and performance enhancement. While numerous peptide drugs have undergone a rigorous approval process that evaluates both safety and efficacy, a parallel \"gray market\" of unapproved compounds has emerged, operating largely outside regulatory oversight. Our objective is to present the pharmacological mechanisms, safety profiles, and regulatory status of prominent approved and unapproved peptides marketed direct to patients, including AOD-9604 (Anti-Obesity Drug 9604), BPC-157 (Body Protection Compound 157), CJC-1295, FS-344 (Follistatin-344), GHK-Cu (Glycyl-L-histidyl-L-lysine copper), ipamorelin, MOTS-C (Mitochondrial ORF of the 12S rRNA type-c), sermorelin, SS-31 (Elamipretide), tesamorelin (Egrifta), Tβ4 (thymosin beta-4), and TB-500 (thymosin beta-4 fragment). Many unapproved peptides demonstrate favorable tissue repair and metabolic outcomes in animal models, but rigorous human safety data is scarce, and there is potential for serious harm to patients. This narrative review focuses on the utilization of peptides in sports medicine, and alternative treatments that may be considered. We provide a framework to navigate patient discussions about peptides to better facilitate evidence-based practices for musculoskeletal healing and athletic performance. We also discuss the placebo effect as a mediator of peptide efficacy, and how social media amplifies this effect.","authors":["Mendias CL","Awan TM."],"year":2026,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.1101/2025.11.18.688276","title":"Reconstructing the emergence of the human chorion via HIPPO-mediated trophoblast induction","abstract":"The first lineage decision in the mammalian blastocyst commits outer cells to the trophectoderm and initiates the trajectory that gives rise to the placental chorion. The molecular sequence that unfolds downstream of HIPPO pathway inactivation, linking human trophectoderm specification to the early organization of the chorion, has remained unknown. Here, we establish a developmentally informed model that leverages HIPPO pathway modulation to induce the native trophectoderm trajectory in the absence of exogenous BMP or WNT signaling. We first transiently reset primed human pluripotent stem cells into a trophectoderm-competent ground state, followed by LATS kinase inhibition to set the trajectory in motion. To benchmark fidelity, we built an embryo-chorion single-cell reference integrating published early human and placental transcriptomes and applied a computational stage-matching tool to align our cultures to natural development. Stage matching revealed an ordered progression along the trophectoderm trajectory from early TE to post-implantation trophoblast. With extended culture, all major cell types of the nascent chorion emerged, encompassing both trophoblast and chorionic mesoderm lineages. Within the trophoblast, we identified proliferative and non-cycling villous cytotrophoblast, a columnar population connecting villous and extravillous domains, as well as syncytia and extravillous subtypes. When cultured in suspension, these lineages self-organized into three-dimensional organoids that recapitulated the stromal-epithelial architecture and proliferative-syncytial polarity of the emergent chorion. We identified CLDN6 as a defining surface marker of columnar trophoblast, the population that bridges villous and extravillous compartments. Prospective isolation of living CLDN6+ trophoblast revealed their capacity to reacquire a proliferative villous state and, under directed cues, generate both syncytial and extravillous fates, confirming their proposed dual developmental potential within the chorion. Together, these findings establish a developmentally informed framework that connects human trophectoderm specification to the emergent chorion and provides a dynamic platform for investigating the earliest steps of placental specification and the origins of implantation disorders.","authors":["Zhang M","Lim RL","Reis AH","Piszker W","Boyd WW","Pagon A","Mahajan A","Wu L","Zhao C","Petropoulos S","Ronda C","Simunovic M."],"year":2025,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.1101/2025.09.19.677076","title":"An immunocompetent murine model of virus-elicited liver fibrosis and hepatocellular carcinoma","abstract":"Hepatocellular carcinoma (HCC) is the third deadliest cancer worldwide. Over 75% of HCC cases are associated with chronic viral infections. Mechanistic studies and preclinical therapeutic development for virus-associated HCC have been limited by a paucity of small animal models of chronic hepatotropic virus infection that faithfully recapitulate human disease. Here we demonstrate the induction of chronic hepatitis, progressive liver fibrosis, and HCC in immunocompetent laboratory mice upon chronic viral infection with Norway rat hepacivirus (NrHV) - a virus closely related to hepatitis C virus (HCV). NrHV-elicited tumors resemble HCV-associated tumors and liver transcriptome analyses reveal numerous similarities between chronic NrHV and HCV. These findings establish an experimentally tractable, physiologically relevant, and immunocompetent mouse model of virus-elicited progressive liver fibrosis and oncogenesis.","authors":["Batista MN","Bordignon J","Mosimann ALP","Bobrowski T","Chen H","Tobin-Xet G","Barrall EA","Prokhnevska N","Vaidya AB","Lewy T","Dinnon KH","Seifert LL","Zeck B","Quirk C","Ho Y","Filiol A","Wolfisberg R","Jiang C","Cogliati B","Chiriboga L","Theise N","MacDonald MR","Kamphorst A","Scheel TKH","Sheahan TP","Billerbeck E","Lowe S","Rosenberg BR","Rice CM."],"year":2025,"journal":"PPR","source":"PPR","preprint":true}],"preprints":[{"pmid":"","doi":"10.21203/rs.3.rs-8237978/v1","title":"Head-to-head comparison of [177Lu]Lu-FAP-2286 and [161Tb]Tb-FAP-2286 efficacy in a PDAC mouse model: Is there an added benefit of internal conversion and Auger electrons for FAP-TRT?","abstract":"<title>Abstract</title>  <p>  Background  Terbium-161 (Tb-161) emits internal conversion and Auger electrons, in addition to beta-minus radiation, which might be of added benefit for targeted radionuclide therapy (TRT) compared to Lutetium-177 (Lu-177). We extensively compared Lu-177 and Tb-161 for fibroblast activation protein (FAP)-targeted TRT in a preclinical setting. To study this, FAP-2286 was labeled with Lu-177 and Tb-161 and characterized in vitro on FAP-expressing cells and ex vivo using patient tumor samples. Moreover, in vivo studies (i.e. biodistribution and efficacy) were performed using a clinically representative pancreatic ductal adenocarcinoma (PDAC) mouse model. Biodistribution was performed 1, 4, 24, and 48 h post injection of 5 MBq/500 pmol [  <sup>177</sup>  Lu]Lu-FAP-2286 or [  <sup>161</sup>  Tb]Tb-FAP-2286. Subsequently, animals were treated with 4×40 MBq/500 pmol [  <sup>177</sup>  Lu]Lu-FAP-2286 or [  <sup>161</sup>  Tb]Tb-FAP-2286 and with alternating doses of 2×40 MBq/500 pmol of each radiopharmaceutical. Results  No difference in [  <sup>177</sup>  Lu]Lu-FAP-2286 and [  <sup>161</sup>  Tb]Tb-FAP-2286 uptake was observed in the cell models. In vivo studies did not show a survival benefit after 4×40 MBq/500 pmol [  <sup>177</sup>  Lu]Lu-FAP-2286 or [  <sup>161</sup>  Tb]Tb-FAP-2286, while Kaplan-Meier analyses demonstrated modestly prolonged survival after tandem therapy, in mice that first received [  <sup>177</sup>  Lu]Lu-FAP-2286 followed by [  <sup>161</sup>  Tb]Tb-FAP-2286. Dosimetry calculations based on autoradiography on patient tumor samples showed that even with lower binding, a higher absorbed dose to the tumor can be accomplished with [  <sup>161</sup>  Tb]Tb-FAP-2286. Conclusions  In our vitro and in vivo studies, [  <sup>177</sup>  Lu]Lu-FAP-2286 and [  <sup>161</sup>  Tb]Tb-FAP-2286 demonstrated similar behavior. In the applied PDAC mouse model, FAP-TRT showed limited therapeutic efficacy, with a modest response observed in the tandem therapy group that first received [  <sup>177</sup>  Lu]Lu-FAP-2286, followed by [  <sup>161</sup>  Tb]Tb-FAP-2286.  </p>","authors":["Heide CDvd","Ntihabose CM","Konijnenberg M","Ma H","Stuurman D","de Ridder C","Seimbille Y","Doukas MC","de Blois E","Dalm SU."],"year":2025,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.21203/rs.3.rs-8427494/v1","title":"Excess Tuberculosis Incidence in the United States During COVID-19: A State, Age, and Race/Ethnicity Analysis and Structural Drivers of Variation (2020–2023)","abstract":"<title>Abstract</title>  <p>  <bold>Background:</bold>  The COVID-19 pandemic disrupted healthcare systems and disease surveillance worldwide, potentially affecting tuberculosis (TB) detection and control. While global analyses have documented major TB setbacks, the extent to which pandemic-related disruptions altered TB incidence patterns across U.S. demographic and geographic groups remains unclear. This study aimed to quantify excess TB incidence (newly reported TB cases) across U.S. jurisdictions, age groups, and racial/ethnic populations during 2020--2023, and to assess structural factors associated with geographic disparities in excess TB burden.  <bold>Methods:</bold>  We used a sub-epidemic ensemble modeling framework applied to annual U.S. TB incidence data, defined as newly reported TB cases. Models were calibrated to pre-pandemic trends (2010--2019) and used to generate counterfactual forecasts for 2020--2023. Because publicly available TB surveillance data are one-way stratified, we calibrated separate models for each jurisdiction, age group, and racial/ethnic category. Excess TB cases were defined as the difference between observed and expected counts, with 95% prediction intervals estimated via bootstrap simulation. Analyses were classified by jurisdiction (50 states, along with D.C. and Puerto Rico), age (11 groups from younger than 1 to greater than 85 years), and race/ethnicity (8 groups). A Poisson error structure was applied consistently across all models. To investigate predictors of state-level excess TB burden, we performed backward stepwise ordinary least squares (OLS) regression using seven candidate predictors: population density, percentage foreign-born, poverty rate, HIV prevalence, incarceration rate, homelessness rate, and percentage American Indian/Alaska Native (AI/AN) population.  <bold>Results:</bold>  Excess TB burden varied widely across jurisdictions. Texas (410 cases [95%PI: 59--930]), New York (380 [200--680]), Florida (260 [61--600]), and California (200 [62--500]) had the highest excess case counts. Population-adjusted analyses revealed a markedly different pattern, with Alaska showing the largest excess rate (13 per 100,000 [0–35]), emphasizing disproportionate impacts in smaller but structurally vulnerable jurisdictions. Working-age adults carried the greatest excess burden, particularly those aged 35--44 (650 cases [300--1200]) and 25--34 (630 [330--1100]). Large racial and ethnic disparities were observed: the Hispanic population experienced the highest excess burden (1,700 cases [1,100--2,500]), with notable excess also among American Indian/Alaska Native populations (140 cases [61--210]) despite their small population share, while the Asian population showed no excess case counts. Several jurisdictions and the 55--64 age group had uncertainty intervals including zero, suggesting patterns consistent with pre-pandemic trends. Stepwise regression identified four predictors of state-level excess TB cases: percentage foreign-born (positive association), incarceration rate (positive association), homelessness rate (positive association), and population density (negative association), with an adjusted \\((R^2)\\) of 0.36.  <bold>Conclusions:</bold>  The COVID-19 pandemic had uneven effects on TB incidence across the United States. Estimated excess TB incidence likely reflects a combination of delayed diagnosis, disruptions to routine surveillance and care, and post-pandemic rebound in case detection, rather than increased transmission alone. Working-age Hispanic adults and residents of jurisdictions with high proportions of foreign-born individuals, elevated incarceration rates, and substantial homelessness experienced the greatest excess burden.  </p>","authors":["Karami H","Rajaram V","Lee S","Mamelund S","Chowell G."],"year":2026,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.20944/preprints202512.1011.v3","title":"Safety and Efficacy of Approved and Unapproved Peptide Therapies for Musculoskeletal Injuries and Athletic Performance","abstract":"Peptides are short chains of amino acids with a unique pharmacological niche between small-molecule drugs and large proteins. Their use in sports medicine is rapidly expanding, driven by patient demand for accelerated injury recovery and performance enhancement. While numerous peptide drugs have undergone a rigorous approval process that evaluates both safety and efficacy, a parallel \"gray market\" of unapproved compounds has emerged, operating largely outside regulatory oversight. Our objective is to present the pharmacological mechanisms, safety profiles, and regulatory status of prominent approved and unapproved peptides marketed direct to patients, including AOD-9604 (Anti-Obesity Drug 9604), BPC-157 (Body Protection Compound 157), CJC-1295, FS-344 (Follistatin-344), GHK-Cu (Glycyl-L-histidyl-L-lysine copper), ipamorelin, MOTS-C (Mitochondrial ORF of the 12S rRNA type-c), sermorelin, SS-31 (Elamipretide), tesamorelin (Egrifta), Tβ4 (thymosin beta-4), and TB-500 (thymosin beta-4 fragment). Many unapproved peptides demonstrate favorable tissue repair and metabolic outcomes in animal models, but rigorous human safety data is scarce, and there is potential for serious harm to patients. This narrative review focuses on the utilization of peptides in sports medicine, and alternative treatments that may be considered. We provide a framework to navigate patient discussions about peptides to better facilitate evidence-based practices for musculoskeletal healing and athletic performance. We also discuss the placebo effect as a mediator of peptide efficacy, and how social media amplifies this effect.","authors":["Mendias CL","Awan TM."],"year":2026,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.1101/2025.11.18.688276","title":"Reconstructing the emergence of the human chorion via HIPPO-mediated trophoblast induction","abstract":"The first lineage decision in the mammalian blastocyst commits outer cells to the trophectoderm and initiates the trajectory that gives rise to the placental chorion. The molecular sequence that unfolds downstream of HIPPO pathway inactivation, linking human trophectoderm specification to the early organization of the chorion, has remained unknown. Here, we establish a developmentally informed model that leverages HIPPO pathway modulation to induce the native trophectoderm trajectory in the absence of exogenous BMP or WNT signaling. We first transiently reset primed human pluripotent stem cells into a trophectoderm-competent ground state, followed by LATS kinase inhibition to set the trajectory in motion. To benchmark fidelity, we built an embryo-chorion single-cell reference integrating published early human and placental transcriptomes and applied a computational stage-matching tool to align our cultures to natural development. Stage matching revealed an ordered progression along the trophectoderm trajectory from early TE to post-implantation trophoblast. With extended culture, all major cell types of the nascent chorion emerged, encompassing both trophoblast and chorionic mesoderm lineages. Within the trophoblast, we identified proliferative and non-cycling villous cytotrophoblast, a columnar population connecting villous and extravillous domains, as well as syncytia and extravillous subtypes. When cultured in suspension, these lineages self-organized into three-dimensional organoids that recapitulated the stromal-epithelial architecture and proliferative-syncytial polarity of the emergent chorion. We identified CLDN6 as a defining surface marker of columnar trophoblast, the population that bridges villous and extravillous compartments. Prospective isolation of living CLDN6+ trophoblast revealed their capacity to reacquire a proliferative villous state and, under directed cues, generate both syncytial and extravillous fates, confirming their proposed dual developmental potential within the chorion. Together, these findings establish a developmentally informed framework that connects human trophectoderm specification to the emergent chorion and provides a dynamic platform for investigating the earliest steps of placental specification and the origins of implantation disorders.","authors":["Zhang M","Lim RL","Reis AH","Piszker W","Boyd WW","Pagon A","Mahajan A","Wu L","Zhao C","Petropoulos S","Ronda C","Simunovic M."],"year":2025,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.1101/2025.09.19.677076","title":"An immunocompetent murine model of virus-elicited liver fibrosis and hepatocellular carcinoma","abstract":"Hepatocellular carcinoma (HCC) is the third deadliest cancer worldwide. Over 75% of HCC cases are associated with chronic viral infections. Mechanistic studies and preclinical therapeutic development for virus-associated HCC have been limited by a paucity of small animal models of chronic hepatotropic virus infection that faithfully recapitulate human disease. Here we demonstrate the induction of chronic hepatitis, progressive liver fibrosis, and HCC in immunocompetent laboratory mice upon chronic viral infection with Norway rat hepacivirus (NrHV) - a virus closely related to hepatitis C virus (HCV). NrHV-elicited tumors resemble HCV-associated tumors and liver transcriptome analyses reveal numerous similarities between chronic NrHV and HCV. These findings establish an experimentally tractable, physiologically relevant, and immunocompetent mouse model of virus-elicited progressive liver fibrosis and oncogenesis.","authors":["Batista MN","Bordignon J","Mosimann ALP","Bobrowski T","Chen H","Tobin-Xet G","Barrall EA","Prokhnevska N","Vaidya AB","Lewy T","Dinnon KH","Seifert LL","Zeck B","Quirk C","Ho Y","Filiol A","Wolfisberg R","Jiang C","Cogliati B","Chiriboga L","Theise N","MacDonald MR","Kamphorst A","Scheel TKH","Sheahan TP","Billerbeck E","Lowe S","Rosenberg BR","Rice CM."],"year":2025,"journal":"PPR","source":"PPR","preprint":true}],"consensus_view":"The literature consensus firmly establishes TB-500 as Ac-LKKTETQ, the N-terminally acetylated 17-23 fragment of Tβ4, with actin sequestration (G-actin binding), angiogenesis, and wound healing as its primary biological activities. N-terminal acetylation is the standard modification, providing modest aminopeptidase protection and is well-tolerated with respect to biological activity. TB-500 undergoes rapid in vivo catabolism, generating Ac-LK and Ac-LKK as major metabolites, with only longer fragments retaining wound healing activity. No consensus exists on lipidated or myristoylated analogs of TB-500, as this class of modification has not been studied in the published literature for this peptide. The broader peptide drug field recognizes N-myristoylation as a validated membrane-anchoring strategy for improving intracellular delivery and PK, but evidence for this approach is absent for short actin-binding heptapeptides specifically.","knowledge_gaps":"Several critical knowledge gaps exist: (1) No published data exist on myristoylated TB-500 (Myr-LKKTETQ) or any other lipidated variant of LKKTETQ, making the entire membrane depot hypothesis empirically untested for this peptide. (2) It is unknown whether a bulky C14 N-terminal chain is sterically compatible with LKKTETQ's actin-binding interface, as the Leu-1/Lys-2 residues that are modified are directly adjacent to the pharmacophore. (3) The aqueous solubility and aggregation behavior of Myr-LKKTETQ have not been characterized—at physiological conditions a C14 chain may drive micelle formation (critical micellar concentration considerations) rather than monomeric membrane insertion. (4) Whether cytosolic release from a plasma membrane depot would be kinetically sufficient to achieve therapeutic G-actin sequestration concentrations is unstudied. (5) The comparative PK between the proposed myristoyl N-terminal depot and the previously studied C-terminal palmitoyl-γGlu albumin-binding approach has no direct experimental precedent in any peptide of this size or sequence class.","supporting_evidence":"The existing N-terminal acetylation of TB-500 (Ac-LKKTETQ) establishes that N-terminal modifications are chemically feasible and biologically tolerated for this peptide—acetylation was intentionally employed to protect Leu-1 from aminopeptidase degradation, and this modified form is the canonical commercially available formulation. The Rahaman et al. (2024) metabolic data show that Ac-LK is the primary short-interval metabolite, confirming aminopeptidase vulnerability at the N-terminus and directly supporting the rationale for a more robust N-terminal cap (myristoyl amide bond is more stable than acetyl amide under aminopeptidase attack). N-myristoylation as a membrane-anchoring strategy is well-validated in the broader lipidated peptide and protein literature, and C14 chains are known to insert into plasma membranes with sufficient affinity for stable but reversible association. The concept of a fatty-acid-anchored peptide depot releasing active peptide into the cytosol is mechanistically sound in principle, consistent with the known behavior of myristoylated signaling peptides. Multiple reviews confirm TB-500's primary mechanism involves cytosolic G-actin sequestration, meaning enhanced intracellular delivery is mechanistically aligned with the therapeutic target.","challenging_evidence":"The most significant challenge is that Leu-1 and Lys-2 of LKKTETQ are the very N-terminal residues where myristoylation occurs, and these residues are likely involved in actin binding based on the known Tβ4 actin-binding domain structure. Adding a C14 chain to Leu-1 may sterically occlude the actin-binding interface, reducing or abolishing G-actin sequestration activity—this is a direct structural concern not addressed by any retrieved literature. The Rahaman et al. (2024) study shows that only Ac-LKKTE (not shorter fragments) retains wound healing activity, suggesting the N-terminal region contributes to overall efficacy; a bulky myristoyl modification could be functionally disruptive. Additionally, myristoylation will dramatically reduce aqueous solubility of an already short polar peptide, raising formulation and aggregation concerns at therapeutic concentrations. The adsorption study (Judák et al. 2017) demonstrates that even unmodified TB-500 has significant surface adsorption problems at low concentrations; a lipidated analog would exacerbate these handling challenges. Finally, there is no literature on membrane-to-cytosol release kinetics for myristoylated heptapeptides of this polarity class, making the 'gradual release depot' mechanism speculative without direct experimental support. The absence of any published myristoyl-TB-500 data means the entire approach is de novo hypothesis-driven with no confirmatory precedent."},"caveats":["in silico prediction only — requires wet lab validation","single-run prediction (not ensembled)","predicted properties may not reflect real-world biological behavior","this is research, not medical advice","AlphaFold/Boltz-2 cannot model non-canonical lipid modifications — the myristoyl chain was not represented in the structural prediction; pLDDT reflects the bare LKKTETQ backbone only","Boltz-2 affinity module returned no binder probability values — the modified molecule was outside evaluable chemical space","heuristic property estimates (aggregation, half-life, stability) are sequence-based and do not account for the C14 lipid chain; aggregation propensity is likely significantly underestimated for the myristoylated form","CMC and micelle formation behavior of Myr-LKKTETQ at therapeutic concentrations is unknown and could preclude monomeric membrane insertion","no published experimental data exist on any myristoylated or lipidated LKKTETQ analog — the entire membrane depot hypothesis is de novo and empirically untested","Verdict reclassified: DISCARDED → PROMISING. Raw metrics (pLDDT/pTM/ipTM) permit at least the higher tier; the original LLM discard reflected modification chemistry the predictor cannot represent (D-AA, lipid moiety, non-canonical residue). Per the metric-floor rule this is a caveat, not a verdict downgrade. Report text below pre-dates the rule and may still describe the fold as DISCARDED — the structural verdict shown is the authoritative one."],"works_cited":[{"pmid_or_doi":"38382158","title":"Simultaneous quantification of TB-500 and its metabolites in in-vitro experiments and rats by UHPLC-Q-Exactive orbitrap MS/MS and their screening by wound healing activities in-vitro","year":2024,"relevance":"Directly characterizes the metabolism of Ac-LKKTETQ in human serum, rat urine, and enzymatic systems, identifying Ac-LK as primary metabolite and Ac-LKK as long-term metabolite; demonstrates that only the longer Ac-LKKTE fragment retains wound healing activity, informing which portions of the sequence must be preserved after N-terminal myristoylation."},{"pmid_or_doi":"23084823","title":"Doping control analysis of TB-500, a synthetic version of an active region of thymosin β₄, in equine urine and plasma by liquid chromatography-mass spectrometry","year":2012,"relevance":"Confirms that the key ingredient of TB-500 is N-acetylated LKKTETQ and establishes metabolite profiles in vivo, providing baseline PK/metabolic data relevant to comparing N-terminal acetylation vs. proposed myristoylation."},{"pmid_or_doi":"22962027","title":"Synthesis and characterization of the N-terminal acetylated 17-23 fragment of thymosin beta 4 identified in TB-500, a product suspected to possess doping potential","year":2012,"relevance":"Confirms the chemical identity of TB-500 as Ac-LKKTETQ by HRMS and solid-phase synthesis, establishing the reference structure against which the N-terminal myristoylation modification (Myr-LKKTETQ) would be compared."},{"pmid_or_doi":"10.20944/preprints202512.1011.v3","title":"Safety and Efficacy of Approved and Unapproved Peptide Therapies for Musculoskeletal Injuries and Athletic Performance","year":2026,"relevance":"Provides a current regulatory and pharmacological framework for TB-500 and Tβ4 in sports medicine, noting favorable tissue repair outcomes in animal models but absence of rigorous human safety data; relevant context for any novel modification like myristoylation."},{"pmid_or_doi":"41490200","title":"Therapeutic Peptides in Orthopaedics: Applications, Challenges, and Future Directions","year":2026,"relevance":"Summarizes TB-500 mechanisms including angiogenesis, integrin-mediated ECM remodeling, and fibroblast activation, contextualizing the biological targets that a membrane-anchored depot form would need to engage."},{"pmid_or_doi":"41476424","title":"Injectable Peptide Therapy: A Primer for Orthopaedic and Sports Medicine Physicians","year":2026,"relevance":"Reviews clinical evidence for TB-4 and TB-500 in tissue repair, highlighting the lack of controlled human trials and the general landscape of peptide pharmacology relevant to evaluating modified variants."},{"pmid_or_doi":"24906629","title":"Analytical approaches for the detection of emerging therapeutics and non-approved drugs in human doping controls","year":2014,"relevance":"Discusses TB-500 as a modified peptidic doping agent and addresses analytical requirements arising from physicochemical properties and metabolism, providing context on how N-terminal modifications affect detection and stability."},{"pmid_or_doi":"28887173","title":"Adsorption effects of the doping relevant peptides Insulin Lispro, Synachten, TB-500 and GHRP 5","year":2017,"relevance":"Demonstrates that TB-500 has significant surface adsorption characteristics relevant to formulation and quantitative handling; a myristoylated analog with increased hydrophobicity would be expected to have even greater adsorption/aggregation challenges."}]},"onchain":{"hash":"27s7mpmhARN5999uGA9HwNhfbvvNLpuZsjfNTgddod3T4Umd9pjiZFyrYJWipB7pr12mSXpC2CbzhJZBAacg1XSL","signature":"27s7mpmhARN5999uGA9HwNhfbvvNLpuZsjfNTgddod3T4Umd9pjiZFyrYJWipB7pr12mSXpC2CbzhJZBAacg1XSL","data_hash":"b0b87dd8d9fa47d39a21e3fdeda7f358c42c2ace0fd0aa787a976e5c5fe1c8f1","logged_at":"2026-05-05T01:03:14.802507+00:00","explorer_url":"https://solscan.io/tx/27s7mpmhARN5999uGA9HwNhfbvvNLpuZsjfNTgddod3T4Umd9pjiZFyrYJWipB7pr12mSXpC2CbzhJZBAacg1XSL"},"ipfs_hash":null,"created_at":"2026-05-05T00:59:07.542765+00:00","updated_at":"2026-05-05T04:34:22.576795+00:00"}