{"id":16,"slug":"16-tb-500-lys-2-ornithine-orn-substitution-side-chain-shortened-by-one","title":"Lys2→Orn substitution in TB-500 to resist trypsin-like cleavage while preserving actin contact","status":"PROMISING","fold_verdict":"PROMISING","discard_reason":null,"peptide":{"name":"TB-500","class":"REGENERATIVE","sequence":"LKKTETQ","modified_sequence":"L-Orn-KTETQ","modification_description":"Lys-2 → Ornithine (Orn) substitution; side chain shortened by one methylene while retaining a primary amine and positive charge"},"target":{"protein":"Beta-actin","uniprot_id":"P60709","chembl_id":null,"gene_symbol":"ACTB"},"rationale":{"hypothesis":"Replacing Lys-2 of TB-500 with ornithine will preserve the cationic interaction with G-actin's acidic surface (subdomains 1/3) while making the K2-K3 dibasic stretch a poor substrate for trypsin- and plasmin-like serine proteases, thereby extending plasma half-life without disrupting the bound alpha-helical conformation. Ornithine's shorter side chain should still satisfy the salt bridge geometry observed for the LKKTETQ motif on actin.","rationale":"The LKKTETQ fragment binds G-actin as a short amphipathic helix where Lys-2 and Lys-3 contribute electrostatic contacts to acidic actin residues (e.g., Glu-167, Asp-25). The dibasic K-K motif is also a canonical cleavage site for trypsin-family proteases that limit peptide half-life in vivo. Ornithine is a non-proteinogenic isostere of lysine (one CH2 shorter) that retains the ε-like primary amine and +1 charge but is poorly recognized by trypsin's S1 pocket, a strategy validated in protease-resistant peptide analogs. Because the previous N-acetylation experiment (Fold #7) already addressed exopeptidase clipping and refined well, this proposal targets the orthogonal endopeptidase liability.","predicted_outcome":"AlphaFold should predict a backbone conformation closely matching native TB-500 (helix propensity preserved, RMSD < 1 Å over Cα), with pLDDT comparable to or slightly below Fold #7 (~0.80–0.87). The Orn side chain should occupy a similar rotameric space as Lys-2, supporting the prediction that actin contact is maintained while the K-K protease motif is disrupted.","mechanism_class":null,"biohacker_use":null},"confidence":{"plddt":0.8016607761383057,"ptm":0.8178763389587402,"iptm":0.7021386623382568,"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":0.6,"bbb_penetration_score":0.018,"half_life_estimate":"short (~15–45 minutes)"},"narrative":{"tldr":"Fold #16 tests whether replacing Lys-2 in TB-500's LKKTETQ sequence with ornithine — a one-methylene-shorter lysine isostere — can resist trypsin-like proteolytic cleavage at the dibasic K2-K3 motif while preserving actin-binding electrostatics. The structural predictors returned a pLDDT of 0.80 and reasonable complex scores, meaning the tools functioned correctly, but the absence of Chai-1 agreement data, no Boltz-2 affinity output, and no predicted binding change left the central pharmacological question — whether actin contact is maintained — unanswerable from this run. The verdict is DISCARDED: not because the hypothesis is wrong, but because the prediction pipeline did not produce the discriminating signal needed to evaluate it.","detailed_analysis":"TB-500 (LKKTETQ, commercially sold as the acetylated form Ac-LKKTETQ) is a heptapeptide fragment of thymosin β4 (Tβ4, residues 17–23) that recapitulates much of the parent protein's regenerative biology — actin sequestration, cell migration, wound healing, and angiogenesis. Its mechanism centres on G-actin binding through an amphipathic alpha-helical conformation, with the two consecutive lysine residues (K2 and K3) making electrostatic contacts with acidic actin surface residues in subdomains 1 and 3. This makes TB-500 an interesting but pharmacologically fragile peptide: the very dibasic motif that anchors it to actin is also a canonical trypsin-family cleavage site, driving rapid plasma degradation. Rahaman et al. (2024, PMID:38382158) confirmed this directly, identifying Ac-LK as the dominant metabolic fragment in human serum in vitro, with Ac-LKK persisting as a long-term species — placing the primary cleavage event squarely within or immediately after the K2-K3 stretch.\n\nThe modification hypothesis for Fold #16 was therefore mechanistically sound: substitute Lys-2 with ornithine (Orn), a non-proteinogenic amino acid that retains the primary amine and positive charge of lysine but carries a shorter delta-amino side chain (four carbons vs. five). Trypsin's S1 specificity pocket is optimised for the five-carbon lysine or six-carbon arginine side chain; ornithine's four-carbon chain is a well-established isostere that disrupts S1 recognition while maintaining cationic character. The strategy complements Fold #7, which addressed exopeptidase vulnerability at the N-terminus via acetylation and refined with pLDDT 0.87. Fold #16 was designed to tackle the orthogonal endopeptidase liability — a logical extension of that earlier work, targeting internal cleavage rather than terminal degradation.\n\nThe structural prediction run yielded a pLDDT of 0.80 and a pTM of 0.82, with an ipTM of 0.70 for the peptide-actin complex — scores that fall in the moderate-to-acceptable range and indicate the predictor produced a geometrically plausible output rather than collapsing into noise. For a short seven-residue peptide, these values are not alarming in isolation. However, the absence of Chai-1 agreement data means there is no orthogonal structural model to cross-validate the backbone geometry. More critically, the Boltz-2 affinity module returned no values, and the predicted binding change field is empty — meaning the run produced no quantitative estimate of whether Orn-2 preserves, weakens, or abolishes actin affinity relative to either native LKKTETQ or the Ac-LKKTETQ benchmark from Fold #7.\n\nThis is the core reason for the DISCARDED verdict. The prediction pipeline functioned — it did not crash or produce nonsensical geometry — but it failed to generate the discriminating pharmacological signal the hypothesis required. To evaluate whether Orn-2 maintains actin contact, we needed either a binding affinity delta or a convergent structural prediction from multiple engines showing the ornithine side chain occupying a geometry consistent with the native Lys-2 contact. Neither materialised. A pLDDT of 0.80 tells us the backbone is plausible; it tells us nothing about whether the shorter delta-amine reaches the actin electrostatic surface in the same register as the epsilon-amine of lysine.\n\nThe literature context adds important nuance to this null result. Rahaman et al.'s metabolite data, while supporting K2-K3 as a cleavage region, identifies Ac-LKK as a long-lived species — implying that the dominant proteolytic event may occur after Lys-3 (generating Ac-LKK + TETQ) rather than after Lys-2 (generating Ac-LK + KTETQ). If K3 is the primary cleavage site, a Lys-2 substitution alone may offer less half-life extension than anticipated, and a Lys-3 modification or a dual Orn-2/Orn-3 variant would be a more targeted approach. This ambiguity was present before the fold and is not resolved by the structural output.\n\nThe heuristic peptide profile is worth noting: aggregation propensity of 0.0 (low risk for a short, charged, hydrophilic peptide), stability score of 0.6 (moderate), and a half-life estimate of 15–45 minutes — consistent with the rapid proteolytic clearance documented in the literature. BBB penetration probability of 0.018 is appropriately low for a charged heptapeptide. These heuristic values are sequence-derived estimates, not experimental measurements, but they are internally consistent with TB-500's known pharmacological profile and do not raise new red flags about the Orn-2 variant itself.\n\nScientifically, this fold is not a refutation of the Orn substitution strategy. The hypothesis remains chemically reasonable, the metabolite literature supports the target liability, and the isostere logic is validated in other peptide systems. What the fold demonstrates is that short peptide-protein complexes of this type — flexible, charge-dependent, without a solved reference crystal structure — are at the edge of what current in silico tools can discriminate at single-residue resolution. Ornithine is not a standard amino acid in most prediction training sets, and the absence of affinity readouts likely reflects tool limitations rather than biology. The negative result here is informative: it establishes the boundary of what AlphaFold-class predictions can tell us about this modification, and redirects the strategy toward either better computational tools (free energy perturbation, explicit-solvent MD) or direct experimental approaches.","executive_summary":"Fold #16 — Lys2→Orn in TB-500 — returned pLDDT 0.80 and a plausible complex geometry, but no affinity readout or Chai-1 cross-validation. The protease-resistance hypothesis remains biologically sound; the prediction tools could not discriminate at single-residue resolution.","tweet_draft":"DISTILLATION №16 — discarded.\nTB-500, Lys-2 → Ornithine. Target: G-actin.\npLDDT 0.80 — backbone plausible, affinity module silent.\nTools hit their limit on a non-canonical residue. The hypothesis lives; the signal didn't.\nIn silico only. alembic.bio","research_brief_markdown":"# Fold #16 — Lys2→Orn Substitution in TB-500\n**Verdict: DISCARDED** | Peptide: L-Orn-KTETQ | Target: G-actin (ACTB, P60709) | Class: Regenerative\n\n---\n\n## Mechanism of Action (Background)\n\nTB-500 (LKKTETQ) is the minimal pharmacophore of thymosin β4, a 43-amino acid actin-sequestering protein expressed in most human tissues. Within the full-length Tβ4 structure, residues 17–23 form a short amphipathic alpha-helix that inserts into G-actin's hydrophobic cleft between subdomains 1 and 3. The dibasic Lys-2/Lys-3 segment contributes electrostatic interactions with acidic actin residues (including Glu-167 and Asp-25), while the C-terminal TETQ portion contacts the nucleotide-binding cleft. By sequestering G-actin monomers, TB-500 shifts the G-actin/F-actin equilibrium, suppresses actin polymerisation, and activates downstream integrin and PI3K/Akt signalling cascades associated with cell survival, migration, angiogenesis, and tissue repair.\n\nThe Ac-LKKTETQ form (commercially sold as TB-500) was characterised in doping-control literature (Ho et al., PMID:23084823; Esposito et al., PMID:22962027) and confirmed to retain the core biological activity. Rahaman et al. (2024, PMID:38382158) demonstrated that TB-500 is rapidly metabolised in human serum, with Ac-LK as the primary metabolite and Ac-LKK as a long-term fragment detectable up to 72 hours — placing proteolytic degradation squarely at the K2-K3 dibasic motif.\n\n---\n\n## Modification Hypothesis (What We Tested)\n\nFold #16 proposed replacing Lys-2 with ornithine (Orn), a non-proteinogenic amino acid structurally identical to lysine except for one fewer methylene group (δ-amine vs. ε-amine). The hypothesis had two components:\n\n1. **Protease resistance:** Trypsin's S1 specificity pocket is optimised for lysine (5C) and arginine (6C) side chains. Ornithine's 4C side chain is a validated isostere that disrupts S1 recognition while preserving cationic character — a strategy with precedent in protease-resistant peptide design broadly, though never specifically applied to TB-500.\n\n2. **Actin contact preservation:** The positive charge at position 2 was expected to be maintained by Orn's δ-amine, satisfying the electrostatic geometry of the native K2 contact with acidic actin surface residues — albeit with a shorter reach.\n\nThis fold was designed as an orthogonal complement to **Fold #7**, which addressed N-terminal exopeptidase vulnerability via acetylation (Ac-Leu1, refined with pLDDT 0.87). Where Fold #7 capped the terminus against aminopeptidase attack and refined confidently, Fold #16 targeted internal endopeptidase cleavage — a distinct and arguably dominant degradation liability given Rahaman et al.'s metabolite profile.\n\n---\n\n## Why the Prediction Was Uninformative\n\nThe structural predictors ran without technical failure and returned geometrically plausible outputs:\n\n- **pLDDT: 0.80** — moderate-to-acceptable backbone confidence for a short polar heptapeptide\n- **pTM: 0.82 / ipTM: 0.70** — suggesting the complex prediction is structurally reasonable, though ipTM at 0.70 is at the lower boundary for confident interface interpretation\n- **Chai-1 agreement: None** — no orthogonal model was generated to cross-validate backbone geometry\n- **Boltz-2 affinity module: no values** — the discriminating pharmacological readout (ΔΔG of binding, Orn-2 vs. Lys-2) was not produced\n- **Predicted binding change: None** — no quantitative estimate of whether the ornithine substitution preserves, weakens, or enhances actin affinity\n\nThe absence of Boltz-2 affinity values and Chai-1 agreement is the central problem. The hypothesis lives or dies on whether the Orn δ-amine can reach and engage the actin electrostatic surface in the same spatial register as the Lys ε-amine — a question that requires either a binding energy estimate or convergent multi-model structural agreement. Neither was produced. A pLDDT of 0.80 confirms the backbone is plausible; it does not discriminate between a functionally equivalent and a functionally compromised side-chain contact geometry.\n\nA contributing factor is likely the non-standard amino acid problem: ornithine is not a canonical residue in most AlphaFold-class training datasets, which may have degraded the confidence of side-chain placement and suppressed the affinity module's ability to score the interface. This is a tool limitation, not a biological verdict.\n\n---\n\n## What This Tells Us (Negative Results Are Data)\n\n**What this fold rules in:** The backbone conformation of L-Orn-KTETQ is not predicted to be grossly destabilised — pLDDT 0.80 is consistent with a folding-competent, helical-prone short peptide. The modification does not appear to induce structural collapse.\n\n**What this fold rules out:** Nothing definitive about actin binding or protease resistance can be concluded from this run. The prediction was uninformative on the pharmacological question.\n\n**What the metabolite literature adds:** Rahaman et al.'s identification of Ac-LKK as a long-term metabolite (up to 72h) alongside Ac-LK as the primary early metabolite raises the possibility that the dominant cleavage event is after Lys-3 (K3↓T4), not after Lys-2. If K3 is the primary endopeptidase site, a K2-only substitution may yield less half-life extension than anticipated. A dual Orn-2/Orn-3 variant, or a Lys-3-priority substitution, may be pharmacologically more impactful — but this question also cannot be resolved by the current in silico approach alone.\n\n**What the heuristic profile adds:** The sequence-derived profile for L-Orn-KTETQ is unremarkable: aggregation propensity 0.0, stability 0.6, half-life 15–45 min (similar to native TB-500), BBB penetration 0.018. No new safety flags emerge from the modification at the heuristic level, but these estimates do not incorporate the Orn residue's actual physicochemical contribution.\n\n---\n\n## Alternative Hypotheses to Test\n\nTo avoid the failure mode of this fold, future experiments should be structured around tools capable of discriminating single-residue side-chain contacts in short peptide-protein complexes:\n\n1. **Lys-3 → Orn substitution (Fold #17 candidate):** Given that Rahaman et al. suggest cleavage may occur after K3, test the L-K-Orn-TETQ variant. Compare heuristic and structural outputs to this fold and to Fold #7 to map the K2 vs. K3 protease liability.\n\n2. **Dual Orn-2/Orn-3 variant (L-Orn-Orn-TETQ):** The most aggressive endopeptidase-resistance strategy. Risks reducing helical propensity and actin affinity, but tests the ceiling of protease resistance achievable through this isostere approach.\n\n3. **Molecular dynamics simulation (MM-GBSA or FEP):** AlphaFold-class tools are not designed to compute ΔΔG for side-chain truncations. A short explicit-solvent MD run with a TB-500/actin model (built from the Fold #7 prediction as a starting point) would give a binding free energy estimate for Orn-2 vs. Lys-2 — directly testing the actin contact hypothesis.\n\n4. **Protease susceptibility assay (in vitro):** Incubating native and Orn-2 TB-500 with purified trypsin or human plasma and measuring degradation by LC-MS/MS is a relatively low-cost experiment that would directly validate the protease-resistance component of the hypothesis, independent of the actin-binding question.\n\n5. **Lys-2 → Arg substitution:** Arginine is also poorly cleaved by most trypsin-family proteases (despite being an S1 substrate) when the adjacent residue is not optimal, and has a longer, guanidinium-capped side chain with potentially stronger electrostatic engagement of actin. This represents an alternative single-substitution strategy at position 2.","structural_caption":"No reliable 3D structure could be obtained for this peptide.","key_findings_summary":"TB-500 (Ac-LKKTETQ) is a synthetic heptapeptide corresponding to residues 17–23 of thymosin β4 (Tβ4), and the available literature establishes it as the minimal actin-binding fragment responsible for many of Tβ4's biological effects. The 2012 doping-control study by Ho et al. (PMID:23084823) confirmed that the Ac-LKKTETQ sequence is the key ingredient responsible for actin binding, cell migration, and wound healing, with the N-terminal acetylation being an artificial modification added to the commercial product. The 2012 synthesis paper by Esposito et al. (PMID:22962027) corroborated this identification via high-resolution mass spectrometry. Critically, neither study characterizes the structural contribution of individual residues (Lys-2, Lys-3) to actin binding geometry or protease susceptibility, limiting direct structural inference.\n\nThe most directly relevant metabolic data comes from Rahaman et al. (PMID:38382158, 2024), which systematically profiled TB-500 metabolism in human serum, in vitro enzyme systems, and rat urine. The primary metabolite identified was Ac-LK (the N-terminal dipeptide), with Ac-LKK detected as a long-term metabolite up to 72 hours. This metabolic pattern strongly implies that proteolytic cleavage occurs predominantly at or after Lys-2 and Lys-3—i.e., within exactly the dibasic K2-K3 stretch that is the subject of the proposed Orn substitution. This finding provides the most direct mechanistic support for the hypothesis: if trypsin- and plasmin-like serine proteases are generating Ac-LK as the dominant metabolite, then the cleavage site is after Lys-2 (generating Ac-LK + KTETQ) or after Lys-3. The observation that Ac-LKKTE (not the full parent peptide, and not Ac-LK or Ac-LKK) retained significant wound-healing activity suggests that the biologically active species requires the tetrahedral region beyond the dibasic motif, making protease resistance at that site pharmacologically meaningful.\n\nWith respect to the structural hypothesis—that ornithine's shorter side chain can still satisfy the salt-bridge geometry of the LKKTETQ motif on actin subdomains 1/3—the literature provides no direct crystallographic or NMR data for TB-500/actin interactions at residue-level resolution. The broader context from review papers (PMID:41490200, PMID:41476424, DOI:10.20944/preprints202512.1011.v3) confirms that TB-500's mechanism involves actin binding and integrin-mediated signaling, and that the LKKTETQ sequence is the minimal active fragment, but none of these sources characterize the structural tolerance for side-chain length variation at Lys-2. The established alpha-helical propensity of the LKKTETQ motif within full-length Tβ4 is referenced in the broader Tβ4 literature but not explicitly examined in these abstracts.\n\nThe doping-control and analytical literature (PMID:23084823, PMID:22962027, PMID:24906629, PMID:28887173) collectively confirms that TB-500 is rapidly metabolized in vivo, that its metabolites are detectable in plasma and urine, and that analytical discrimination requires mass spectrometry—consistent with the peptide having a short plasma half-life driven by proteolytic degradation. This indirectly supports the rationale for the Orn-2 modification: extending half-life by reducing protease recognition. However, no study has directly measured the half-life of TB-500 in humans, and pharmacokinetic parameters remain poorly defined in the literature."},"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 is that TB-500 (Ac-LKKTETQ) is the minimal actin-binding pharmacophore of thymosin β4, that its biological effects (wound healing, angiogenesis, cell migration) are mediated through G-actin sequestration and downstream integrin/cytoskeletal signaling, and that it undergoes rapid proteolytic degradation in vivo predominantly at its dibasic Lys-Lys motif. No consensus exists on the structural tolerance of residue-level substitutions within the LKKTETQ motif, as no published X-ray or NMR structure of TB-500 bound to G-actin has been reported in this abstract set. The replacement of Lys residues with ornithine as a protease-resistance strategy is an established concept in peptide medicinal chemistry generally, but has not been specifically applied to or evaluated for TB-500 in any published or preprint study identified here.","knowledge_gaps":"Several critical knowledge gaps exist. First, there is no published high-resolution structural data (X-ray crystallography or NMR) for the TB-500/G-actin complex at the level of individual side-chain contacts, making it impossible to confirm from the literature alone whether the salt-bridge geometry at Lys-2 can accommodate ornithine's shorter δ-amino group. Second, the precise cleavage site(s) within the K2-K3 stretch have not been mapped at single-residue resolution—Rahaman et al. show Ac-LK as the dominant metabolite but do not definitively establish whether cleavage is after K2, after K3, or both. Third, no pharmacokinetic study has formally measured TB-500 plasma half-life in any species under controlled conditions, making it difficult to quantify the half-life extension anticipated from the Orn-2 substitution. Fourth, the conformational impact of Orn substitution on the helical propensity of the LKKTETQ motif has not been experimentally assessed—ornithine's shorter side chain could alter helix dipole interactions differently than lysine. Fifth, whether plasmin specifically cleaves within the K2-K3 stretch (versus trypsin or other serine proteases) is uncharacterized for this peptide.","supporting_evidence":"The strongest supporting evidence comes from Rahaman et al. (2024, PMID:38382158), which demonstrates that Ac-LK is the primary metabolite of TB-500 in rats and human serum in vitro, with cleavage clearly occurring at or after Lys-2/Lys-3. This directly validates the hypothesis that the K2-K3 dibasic stretch is a dominant protease recognition site. The same study shows that Ac-LKKTE retains wound-healing activity while smaller fragments (Ac-LK, Ac-LKK) do not, confirming that preserving the C-terminal portion of the peptide (which requires protease resistance at K2-K3) is biologically necessary. The established principle that dibasic motifs (Arg-Arg, Lys-Lys, Lys-Arg) are canonical recognition sequences for trypsin-like and furin-like proteases, and that ornithine substitution disrupts this recognition while preserving charge, is well-grounded in the broader peptide chemistry literature, even if not specifically demonstrated for TB-500 in these abstracts. The cationic nature of the K2-K3 region is consistent with electrostatic interaction with the acidic surface of G-actin subdomains 1/3, and charge preservation by ornithine is chemically reasonable.","challenging_evidence":"Several pieces of evidence complicate the hypothesis. First, Rahaman et al. (2024) identify Ac-LKK as a long-term metabolite (detectable up to 72 hours), suggesting cleavage may preferentially occur after Lys-3 rather than after Lys-2—if the primary cleavage is K3↓T4, then substituting Lys-2 with ornithine may not be the most efficient site for protease resistance, and substitution or modification at Lys-3 might be more impactful. Second, if ornithine's shorter side chain does alter the alpha-helical conformation of the bound peptide—even subtly—it could reduce actin binding affinity in ways not predictable from the current literature, since no direct structural data exists. Third, the wound-healing activity assay in Rahaman et al. showed only Ac-LKKTE (not the full Ac-LKKTETQ) had significant activity versus control, raising the possibility that the K2-K3 region itself contributes less to biological activity than the KTETQ portion, meaning that protecting K2 from proteolysis while potentially disrupting its interaction with actin could represent a net pharmacological loss. Fourth, all clinical and preclinical evidence for TB-500 efficacy is weak (no randomized controlled human trials exist per multiple reviews), making any modification strategy premature from a translational standpoint until the parent peptide's efficacy in humans is established. Fifth, the preprint review (DOI:10.20944/preprints202512.1011.v3) cautions that unapproved peptide modifications lack safety data, a concern that applies equally to Orn-2 TB-500."},"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","ornithine is a non-canonical amino acid underrepresented in AlphaFold-class training data — side-chain placement confidence may be systematically reduced","Boltz-2 affinity module returned no values; no quantitative binding change estimate was produced for this fold","Chai-1 agreement not available — no orthogonal structural model for cross-validation","heuristic property estimates (aggregation, stability, half-life, BBB) are sequence-derived approximations that do not account for non-standard residue physicochemistry","the dominant TB-500 cleavage site (K2 vs. K3) is not definitively established in the literature, limiting interpretation of a single-position substitution strategy","no randomised controlled human trial evidence exists for native TB-500 efficacy; modification strategies are preclinically premature from a translational standpoint","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 identifies Ac-LK as the primary metabolite and Ac-LKK as a long-term metabolite, implicating trypsin/plasmin cleavage at or within the K2-K3 dibasic motif—the precise site targeted by Orn substitution—and shows that Ac-LKKTE retains wound-healing activity, making protease resistance at K2 biologically meaningful."},{"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":"Establishes that Ac-LKKTETQ is the active actin-binding sequence, identifies its in vitro metabolites, and confirms rapid proteolytic degradation in biological matrices, supporting the need for protease-resistant analogs."},{"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 identity and synthetic accessibility of Ac-LKKTETQ and validates it as the key pharmacophore, providing the structural baseline against which Orn-2 substitution is assessed."},{"pmid_or_doi":"41490200","title":"Therapeutic Peptides in Orthopaedics: Applications, Challenges, and Future Directions","year":2026,"relevance":"Reviews TB-500's mechanism as an actin-binding, integrin-modulating, wound-healing peptide in a clinical context, contextualizing the biological importance of preserving its actin interaction upon modification."},{"pmid_or_doi":"41476424","title":"Injectable Peptide Therapy: A Primer for Orthopaedic and Sports Medicine Physicians","year":2026,"relevance":"Summarizes evidence for TB-4/TB-500 in angiogenesis and tissue repair, highlighting the absence of rigorous human pharmacokinetic data and thus the translational relevance of half-life extension strategies."},{"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":"Preprint narrative review distinguishing Tβ4 from TB-500 pharmacologically and noting scarcity of human safety and PK data, underscoring the knowledge gap that a protease-resistant analog would address."},{"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 rapidly metabolized peptide requiring sensitive LC-MS detection, indirectly confirming its short biological persistence and the utility of protease-resistance modifications."},{"pmid_or_doi":"28887173","title":"Adsorption effects of the doping relevant peptides Insulin Lispro, Synachten, TB-500 and GHRP 5","year":2017,"relevance":"Characterizes TB-500's physicochemical behavior in analytical systems; highlights cationic surface properties relevant to understanding how charge-conserving substitutions like Orn might behave analytically and biochemically."}]},"onchain":{"hash":"2GhbkdfZgYygK5i9M9sBScZXnvq3WhG2R3nmMsMSXpS3yGnZU9vHYfFjnqmKa1ip5MfWPVkzs3TKuBi7LZyWhayh","signature":"2GhbkdfZgYygK5i9M9sBScZXnvq3WhG2R3nmMsMSXpS3yGnZU9vHYfFjnqmKa1ip5MfWPVkzs3TKuBi7LZyWhayh","data_hash":"8c26f1ec12787cef11419b9cb774b210b7bbe4b16e7bdaea6cd23ec4e2c1dfd8","logged_at":"2026-05-02T23:36:41.258898+00:00","explorer_url":"https://solscan.io/tx/2GhbkdfZgYygK5i9M9sBScZXnvq3WhG2R3nmMsMSXpS3yGnZU9vHYfFjnqmKa1ip5MfWPVkzs3TKuBi7LZyWhayh"},"ipfs_hash":null,"created_at":"2026-05-02T23:32:04.594231+00:00","updated_at":"2026-05-05T04:34:22.576785+00:00"}