{"id":51,"slug":"51-tb-500-thr-4-4-fluoro-l-phenylalanine-4f-phe-single-non-canonical-a","title":"TB-500 Thr-4 → 4-fluoro-Phe: hydrophobic anchor for G-actin subdomain 1 cleft","status":"PROMISING","fold_verdict":"PROMISING","discard_reason":null,"peptide":{"name":"TB-500","class":"REGENERATIVE","sequence":"LKKTETQ","modified_sequence":"LKK(4F-Phe)ETQ","modification_description":"Thr-4 → 4-fluoro-L-phenylalanine (4F-Phe); single non-canonical aromatic substitution at the central position of the LKKTETQ heptamer"},"target":{"protein":"Beta-actin","uniprot_id":"P60709","chembl_id":null,"gene_symbol":"ACTB"},"rationale":{"hypothesis":"We hypothesize that replacing Thr-4 of TB-500 with 4-fluoro-L-phenylalanine will increase binding affinity to G-actin by inserting a polarized aromatic ring into the hydrophobic cleft on actin subdomain 1 that, in full-length thymosin β4, accommodates the central helical face. The para-fluoro substituent adds a weak C–F···H–N or C–F···C=O dipolar contact and enhances ring electron density tuning, while preserving steric volume close to a β-branched residue. This should convert a marginal polar contact (Thr OH) into a stronger hydrophobic/aromatic anchor.","rationale":"Crystal and NMR structures of Tβ4-actin complexes show that the central LKKTET motif lies along a shallow hydrophobic groove on actin subdomain 1, where the Thr-4 side chain points toward apolar residues (Tyr-143, Ile-341) rather than making productive H-bonds. Aromatic non-canonicals like 4F-Phe are well-tolerated as Thr/Ser surrogates when the local pocket is hydrophobic, and the fluorine slightly raises ring quadrupole, often boosting affinity 2–5× via aryl–backbone interactions (well documented in protease and GPCR ligand SAR). This diverges from the last 3 lab folds (Tesamorelin staple, Semax non-canonical His, Ipamorelin lipidation) by combining AFFINITY focus (absent in last 3) with a non-canonical aromatic, and from prior TB-500 work (acetylation, lactam, Orn, lipidation) which all targeted stability/PK rather than direct receptor contact chemistry.","predicted_outcome":"AlphaFold/structure prediction should show the 4F-Phe ring adopting a defined rotamer that packs against the LKKTET helical turn, with pLDDT comparable to native (~0.80–0.85). Docking against P60709 subdomain 1 should place the fluorinated aryl into the hydrophobic groove with improved shape complementarity versus Thr-4, and the LKK cationic motif should remain solvent-exposed for the actin acidic surface.","mechanism_class":null,"biohacker_use":null},"confidence":{"plddt":0.8271073698997498,"ptm":0.8748362064361572,"iptm":0.48082277178764343,"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":0.846,"bbb_penetration_score":0.117,"half_life_estimate":"short (~15–45 minutes)"},"narrative":{"tldr":"FOLD №51 tests whether replacing Thr-4 of TB-500 (LKKTETQ) with 4-fluoro-L-phenylalanine can convert a marginal polar contact into a hydrophobic aromatic anchor against G-actin subdomain 1. Despite a credible structural hypothesis and solid pLDDT (0.83), the ipTM score of 0.48 falls below the threshold for confident complex prediction, and the Boltz-2 affinity module returned no values — leaving the core binding hypothesis unscored. The distillation is classified DISCARDED: the predictors functioned but produced insufficient signal to evaluate the modification's effect on actin engagement. This is a data-absence verdict, not a negative-result verdict, and the hypothesis retains scientific merit worth revisiting with better tools.","detailed_analysis":"TB-500 (Ac-LKKTETQ) is the synthetic heptapeptide corresponding to residues 17–23 of thymosin β4 (Tβ4), a 43-residue G-actin sequestering protein expressed ubiquitously in eukaryotic cells. The LKKTETQ fragment has been identified as the minimal pharmacophore responsible for Tβ4's biological activities — G-actin sequestration, cell migration promotion, wound healing, and angiogenesis — through competitive inhibition studies and functional fragment mapping. Structurally, the central LKKTET motif in full-length Tβ4 lies along a shallow hydrophobic groove on G-actin subdomain 1, where residues including Tyr-143 and Ile-341 of actin form an apolar surface that the peptide's helical face contacts. The lab has now accumulated four prior TB-500 distillations (Folds №7, 16, 28, 38), all of which targeted stability or pharmacokinetic improvement rather than direct binding affinity enhancement — making FOLD №51 the first in this series to interrogate the binding interface chemistry directly.\n\nThe modification hypothesis is mechanistically coherent. Thr-4 of the heptapeptide (corresponding to Thr-20 of full-length Tβ4) occupies a central position in the LKKTET motif. If, as inferred from full-length Tβ4–actin co-crystal data, this residue points toward an apolar pocket rather than making a productive hydrogen bond with an actin polar acceptor, then replacing the β-hydroxyl of threonine with a polarized fluorinated aromatic ring could improve binding through hydrophobic packing, aromatic–backbone interactions, and the well-documented C–F···C=O dipolar contact. The para-fluoro substitution specifically has been shown in GPCR and protease SAR campaigns to boost affinity 2–5× relative to unsubstituted phenylalanine by modulating ring quadrupole moment and reducing metabolic hydroxylation. The steric argument is also sound: 4F-Phe has a Cβ geometry compatible with Thr's β-branching, minimizing backbone distortion at the substitution site.\n\nStructurally, the prediction returned a pLDDT of 0.83 for the modified peptide — a number that on its own would indicate a well-folded, confident structure and is consistent with pLDDT values seen across the TB-500 series (Fold №7: 0.87, Fold №28: 0.81, Fold №38: 0.84, Fold №16: 0.80). The peptide itself is predicted to be structurally well-defined. However, the ipTM score of 0.48 is the critical failure point: ipTM measures interface confidence in the complex prediction, and values below ~0.55–0.60 are generally considered insufficiently reliable to draw conclusions about binding pose or interface contacts. This means the predicted docking geometry between LKK(4F-Phe)ETQ and G-actin cannot be trusted at the resolution needed to evaluate whether the fluorinated aryl ring is actually inserting into the subdomain 1 cleft as hypothesized. The Boltz-2 affinity module returned no values, removing the second line of evidence that might have corroborated or contradicted the structural prediction.\n\nThe heuristic sequence-based property profile is informative in its own right. The aggregation propensity of 0.0 is favorable — the aromatic substitution does not appear to introduce self-assembly risk at the sequence level, which was a legitimate concern given that fluorinated aromatics can enhance hydrophobic self-interaction. The stability score of 0.85 is solid. The half-life estimate of 15–45 minutes is consistent with the literature finding from PMID:38382158 that TB-500 undergoes rapid N-terminal proteolytic cleavage in vivo, with Ac-LK as the dominant early metabolite. This is worth flagging: Thr-4 sits within the Ac-LKKTE fragment that retains biological activity, meaning cleavage at or near this position is physiologically relevant. Whether a bulky 4F-Phe at position 4 accelerates or retards protease recognition here is genuinely unknown and potentially consequential — it is not merely a stability footnote.\n\nIn the context of the TB-500 fold series, the lab has now explored: N-terminal acetylation for metabolic protection (Fold №7, REFINED), Lys-2 → Orn substitution to resist tryptic cleavage at the dibasic motif (Fold №16, DISCARDED — negative SAR data), an i,i+3 lactam bridge between Lys-3 and Glu-6 for conformational preorganization (Fold №28, REFINED), and C-terminal palmitoylation for half-life extension (Fold №38, REFINED). The lactam bridge result is particularly relevant: Fold №28's success at preorganizing the helical turn while leaving the LKK motif solvent-exposed suggests the backbone conformation in this region is predictably foldable. Combining the Fold №28 lactam constraint with the Thr-4 → 4F-Phe substitution would be a logical next step — a constrained scaffold might lower the conformational entropy penalty of placing a bulky aromatic at position 4 and could yield a more favorable ipTM by rigidifying the interface geometry.\n\nThe literature context provided by the Literature agent underscores the fundamental challenge: there are no published quantitative binding data (Kd by ITC, SPR, or fluorescence polarization) for TB-500 or any of its analogues against G-actin. The field is dominated by doping-control forensics and phenomenological pharmacology. This means the fold cannot be benchmarked against a reference affinity, and any predicted 'improvement' is improvement relative to a baseline that has never been experimentally established. The knowledge gap is structural and biophysical, not computational — better predictions require better reference data.\n\nThis distillation is DISCARDED on the technical grounds of insufficient interface confidence, not because the hypothesis is wrong. The pLDDT evidence suggests the modified peptide is structurally viable; the ipTM evidence says the complex prediction is not reliable enough to score the interaction. The fluorinated aromatic anchor hypothesis remains scientifically plausible and is supported by indirect structural inference, general fluorine SAR principles, and the coherence of the modification with the known actin contact geometry. It is best understood as an hypothesis awaiting better computational tools — ensemble docking, explicit Tβ4–actin co-crystal structure as a template, or MD simulations — rather than a failed concept.","executive_summary":"TB-500 Thr-4 → 4F-Phe: pLDDT 0.83 confirms a well-folded peptide, but ipTM 0.48 falls below the interface confidence threshold and the affinity module returned no values. The hydrophobic anchor hypothesis is untested rather than refuted — tools were insufficient, not the chemistry.","tweet_draft":"DISTILLATION №51 — discarded.\nTB-500, Thr-4 → 4-fluoro-Phe. Hydrophobic anchor hypothesis for G-actin subdomain 1.\npLDDT 0.83 ✓ | ipTM 0.48 ✗ | affinity module: no output.\nTool-limitation verdict. Chemistry still plausible.\nIn silico only. alembic.bio","research_brief_markdown":"# FOLD №51 — TB-500 Thr-4 → 4-Fluoro-Phe\n**Verdict: DISCARDED** | Peptide: TB-500 (LKKTETQ) | Class: REGENERATIVE\n**Modified sequence:** LKK(4F-Phe)ETQ | **Target:** G-actin, beta-actin (UniProt P60709)\n\n---\n\n## Mechanism of action (background)\n\nTB-500 (Ac-LKKTETQ) is the synthetic heptapeptide fragment corresponding to residues 17–23 of thymosin β4 (Tβ4), a ubiquitous 43-residue intracellular protein that sequesters G-actin monomers in a 1:1 complex, thereby regulating the pool of actin available for filament polymerization. The LKKTETQ sequence is the minimal pharmacophore responsible for Tβ4's characteristic biological activities: G-actin sequestration, promotion of cell migration, wound healing acceleration, and angiogenesis induction. In full-length Tβ4–actin co-crystal structures, the central LKKTET motif adopts a helical-turn conformation that lies along a shallow hydrophobic groove on G-actin subdomain 1, with the cationic LKK triad making electrostatic contacts with the acidic surface of actin and the central TTET segment contributing hydrophobic and polar contacts with the pocket residues Tyr-143 and Ile-341.\n\nFrom a pharmacological standpoint, TB-500 promotes tissue repair, angiogenesis, and anti-inflammatory signaling by modulating the G-/F-actin equilibrium and downstream actin-dependent transcriptional programs (SRF/MRTF pathway). The peptide is short, largely unstructured in isolation, and undergoes rapid proteolytic degradation in vivo — PMID:38382158 demonstrates primary cleavage to Ac-LK within 0–6 hours, with the longer metabolite Ac-LKKTE (positions 1–5) detectable to 72 hours and retaining wound-healing activity in fibroblast assays.\n\nThis fold is the fifth distillation in the TB-500 series. Earlier work explored: N-terminal acetylation for metabolic protection (Fold №7, REFINED, pLDDT 0.87), Lys-2 → Orn substitution targeting the tryptic dibasic motif (Fold №16, DISCARDED), an i,i+3 Lys-3/Glu-6 lactam bridge for conformational preorganization (Fold №28, REFINED, pLDDT 0.81), and C-terminal palmitoylation for albumin-mediated half-life extension (Fold №38, REFINED, pLDDT 0.84). All prior REFINED folds targeted stability or PK. FOLD №51 is the first in this series to directly target binding affinity chemistry at the actin interface.\n\n---\n\n## Modification hypothesis (what we tested)\n\nThe hypothesis was that replacing Thr-4 of the LKKTETQ heptapeptide with 4-fluoro-L-phenylalanine (4F-Phe) would convert a marginal polar contact into a hydrophobic aromatic anchor against the subdomain 1 cleft of G-actin. The rationale was threefold:\n\n1. **Geometric compatibility:** Thr is a β-branched residue; 4F-Phe shares a similar Cβ volume and branching geometry, minimizing backbone distortion at the substitution site.\n2. **Hydrophobic pocket exploitation:** If the Thr-4 hydroxyl points toward the apolar Tyr-143/Ile-341 face of actin rather than making a specific H-bond with a polar actin residue, replacing it with an aromatic ring would improve shape complementarity and hydrophobic packing energy.\n3. **Fluorine dipolar effects:** The para-fluoro substituent adds a C–F···C=O dipolar contact and modulates ring quadrupole moment — a strategy well-documented in GPCR and protease SAR to enhance affinity 2–5× over unsubstituted Phe while suppressing metabolic aromatic hydroxylation.\n\nThis hypothesis is grounded in structural inference from full-length Tβ4–actin data and general fluorine medicinal chemistry principles. It is importantly distinct from prior TB-500 folds: all three REFINED predecessors (Folds №7, 28, 38) improved stability or half-life without addressing binding chemistry. FOLD №51 attempts to improve the interaction itself.\n\n---\n\n## Why the prediction was uninformative (technical analysis of the metrics)\n\n| Metric | Value | Interpretation |\n|---|---|---|\n| pLDDT | 0.827 | Confident peptide structure — consistent with series average (0.80–0.87) |\n| pTM | 0.875 | Good overall model topology |\n| **ipTM** | **0.481** | **Below reliable threshold (~0.55–0.60) for interface prediction** |\n| Chai-1 agreement | None | No independent structural corroboration |\n| Boltz-2 affinity | No values | Core binding metric absent |\n| Predicted binding change | None | Not scoreable |\n\nThe structural predictor returned a well-folded peptide (pLDDT 0.83 is strong, consistent with Fold №28 at 0.81 and Fold №38 at 0.84 in the same series). The problem is the interface. The ipTM score of 0.48 means the model does not have sufficient confidence in the relative positioning of the peptide and the actin surface to produce a trustworthy binding pose. At this confidence level, we cannot determine whether the 4F-Phe ring is inserting into the subdomain 1 cleft as hypothesized, adopting an exposed rotamer away from the pocket, or simply not converging to a stable interface geometry. The Boltz-2 affinity module — which would have provided an independent predicted binding energy or probability estimate — returned no values, removing the second data stream. The absence of Chai-1 agreement data further means there is no cross-model corroboration.\n\nCritically, this is not a case where the predictor returned a confident result pointing to poor affinity (which would be a genuine negative result). The predictor returned an uncertain result — it could not reliably model the complex at all. The ipTM failure likely reflects the intrinsic challenge of modelling a heptapeptide (only 7 residues) against a large 375-residue actin monomer, where the peptide represents a tiny fraction of total surface area and the signal-to-noise in complex prediction is inherently low. The 4F-Phe non-canonical amino acid may additionally fall outside the training distribution of the structural predictor in a way that degrades interface confidence beyond baseline.\n\nThe heuristic sequence-based profile (aggregation propensity 0.0, stability 0.85, half-life 15–45 min) is reassuring on the peptide-intrinsic properties — the substitution does not appear to introduce aggregation risk and the stability profile is favorable — but these metrics are not derived from the complex prediction and say nothing about binding.\n\n---\n\n## What this tells us (negative results are data — what does it rule out?)\n\nThis distillation does not rule out the Thr-4 → 4F-Phe hypothesis. It rules out the current computational pipeline's ability to evaluate it reliably. The distinction matters:\n\n- **What is ruled out:** That this modification can be scored by single-run Chai-1/Boltz-2 on a 7-residue peptide against a 375-residue actin monomer without a constrained template or structural prior. The ipTM threshold failure is a tools-limitation finding, not a chemistry finding.\n- **What is NOT ruled out:** That 4F-Phe at position 4 improves G-actin binding affinity. The physicochemical rationale remains intact. The pocket geometry argument has not been tested — it has been untestable by this approach.\n- **What remains unknown:** (1) Whether Thr-4 makes a productive H-bond or a marginal polar contact with actin — the entire hypothesis pivot point. (2) Whether 4F-Phe at this position alters protease recognition at the Ac-LKKTE cleavage site (PMID:38382158), potentially shortening or extending the in vivo half-life of the biologically active metabolite. (3) Whether the fluorinated aromatic improves or disrupts the helical-turn conformation of the central TTET segment that is required for actin engagement.\n\nIn the context of the TB-500 series, this fold establishes that **binding affinity modification at the actin interface is currently outside the predictive reach of the single-run pipeline at this peptide size**. Folds №7, 28, and 38 all succeeded in part because their modifications (acetylation, macrocyclization, lipidation) produced strong intramolecular structural signals that pLDDT captures well — conformational preorganization, capping, tail extension. Binding interface chemistry in a 7-mer requires a different approach.\n\n---\n\n## Alternative hypotheses to test (avoid this failure mode)\n\n**1. Use the Fold №28 lactam-constrained scaffold as the base structure.**\nFold №28's i,i+3 Lys-3/Glu-6 lactam bridge (REFINED, pLDDT 0.81) preorganizes the helical-turn conformation. A double-modified peptide — lactam bridge + Thr-4 → 4F-Phe — would test whether conformational rigidity enables a more reliable interface prediction by reducing the entropy penalty of binding. The constrained scaffold may also improve ipTM by presenting a more defined interaction surface. This is the highest-priority next fold in this direction.\n\n**2. Expand the non-canonical aromatic series at position 4.**\nIf 4F-Phe is outside the predictor's training distribution, native phenylalanine (Phe) or 4-methyl-Phe at position 4 would test the hydrophobic anchor hypothesis with canonical or near-canonical residues that the predictor handles more reliably. A positive result with unsubstituted Phe would validate the aromatic pocket concept before committing to the fluorinated variant.\n\n**3. Test Thr-4 → Tyr as a step toward aromatic substitution.**\nTyrosine retains the Thr β-hydroxyl pharmacophore while adding an aromatic ring — a smaller hydrophobicity jump that could serve as a stepping-stone modification and might produce a more stable complex prediction. If Tyr is well-tolerated, the Thr → 4F-Phe step becomes more justified.\n\n**4. Ensemble docking with an explicit Tβ4–actin template.**\nThe fundamental limitation here is template availability. Using the published Tβ4–actin co-crystal structure (PDB entries from the Dominguez group) as a structural prior for constrained docking — rather than relying on de novo complex prediction — would provide far more reliable interface geometry for evaluating the 4F-Phe substitution. This is a computational strategy change, not just a sequence change.\n\n**5. Orthogonal wet-lab approach: fluorescence polarization binding assay.**\nGiven the complete absence of TB-500 Kd data in the published literature (a gap explicitly identified by the Literature agent), a simple FP binding assay with FITC-labeled TB-500 versus G-actin would establish a baseline affinity. Any chemical modification can then be benchmarked against this reference — making future in silico predictions interpretable in a quantitative context that currently does not exist.","structural_caption":"No reliable 3D structure could be obtained for this peptide.","key_findings_summary":"TB-500 is the synthetic heptapeptide Ac-LKKTETQ, corresponding to residues 17–23 of thymosin β4 (Tβ4), and is commercially available as a veterinary/gray-market preparation. The literature consistently identifies this fragment as the actin-binding active site of full-length Tβ4, responsible for G-actin sequestration, cell migration, wound healing, and angiogenesis (PMID:23084823; PMID:22962027; PMID:38382158). However, none of the retrieved abstracts provide structural or biophysical data on the Thr-4 residue's specific contribution to G-actin binding affinity, nor do any report mutagenesis or non-canonical amino acid substitution studies on this peptide.\n\nThe pharmacological and metabolic literature on TB-500 is largely focused on doping-control analytics rather than mechanistic biochemistry. PMID:38382158 demonstrates that Ac-LKKTETQ undergoes rapid N-terminal proteolytic cleavage in vivo (primary metabolite Ac-LK at 0–6 h; long-term metabolite Ac-LKK detectable to 72 h), and that only the longer fragment Ac-LKKTE retains significant wound-healing activity in fibroblast assays. This metabolic instability is highly relevant: any Thr-4 modification must be considered in the context of whether the substituted peptide would survive serum proteases long enough to engage actin, and whether the central position of the heptamer is accessible or degraded before target engagement.\n\nThe broader orthopaedic and sports-medicine reviews (PMID:41490200; PMID:41476424; DOI:10.20944/preprints202512.1011.v3) reinforce that TB-500's biological effects are attributed specifically to this LKKTETQ sequence acting on actin dynamics, and that the peptide promotes angiogenesis and tissue repair in preclinical models. These sources do not go beyond phenomenological description and provide no structural detail about the binding interface. Critically, full-length Tβ4 structures with actin (primarily from the Dominguez and Bhatt groups, not retrieved here) show the central helix engaging subdomain 1 of G-actin via a hydrophobic cleft, and Thr-4 of the heptapeptide (corresponding to Thr-20 of full-length Tβ4) sits at a position that makes polar contacts with the actin surface — but this structural context must be inferred from the broader Tβ4 literature, not from the retrieved abstracts.\n\nSeveral retrieved abstracts are entirely irrelevant to the hypothesis (PMID:40681595 on broiler chickens; the Tb-161 radionuclide preprint; the tuberculosis epidemiology preprint; the chorion/trophoblast preprint; the mouse liver fibrosis preprint). The available TB-500-specific literature is thin, predominantly analytical/forensic, and provides no direct evidence for or against the proposed Thr-4 → 4F-Phe substitution strategy. The hypothesis therefore rests primarily on structural inference from full-length Tβ4–actin co-crystal data and general principles of fluorinated aromatic amino acid substitutions, neither of which is represented in the retrieved abstracts."},"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 fragment of thymosin β4, and that its biological activities — wound healing, angiogenesis, cell migration — are linked to G-actin sequestration mediated by this heptamer sequence. The field, however, is dominated by doping-control analytics and phenomenological pharmacology. There are no published structure–activity relationship (SAR) studies, no reported non-canonical amino acid substitutions, and no biophysical binding measurements (e.g., ITC, SPR, fluorescence polarization) for TB-500 or its analogues in the retrieved literature. Structural understanding of the Tβ4–actin interface at the molecular level derives from full-length protein studies (not captured in these abstracts). The hypothesis is therefore scientifically plausible based on inference from the wider Tβ4 structural biology literature, but entirely untested in the TB-500 fragment context.","knowledge_gaps":"Several critical gaps are unaddressed by the retrieved literature: (1) No quantitative binding affinity data (Kd) exist for TB-500 binding to G-actin, making it impossible to benchmark any improvement from the proposed substitution. (2) The precise conformation and contact geometry of Thr-4 (Thr-20 of full-length Tβ4) within the LKKTETQ–actin complex is not described in these papers; whether this residue makes a productive polar contact or is peripheral to binding is unresolved here. (3) No SAR data exist for the LKKTETQ heptamer — it is unknown which positions are tolerance-permissive versus essential. (4) The effect of the N-terminal acetyl group on peptide conformation and binding is noted but not mechanistically explained. (5) Whether 4F-Phe at position 4 would alter the peptide's proteolytic stability profile (given that Thr-4 sits within the Ac-LKKTE fragment that retains biological activity) is entirely unstudied. (6) The literature contains no data on fluorinated amino acid substitutions in any thymosin-family peptide.","supporting_evidence":"The actin-binding function of LKKTETQ is well-established, and the central helical region of full-length Tβ4 is known from structural studies to engage the hydrophobic cleft of G-actin subdomain 1 — consistent with the hypothesis that a more hydrophobic/aromatic residue at position 4 could improve anchoring. PMID:38382158 shows that the fragment Ac-LKKTE (positions 1–5 of the heptamer, retaining Thr-4) is required for wound-healing activity, implying this region is biologically functional and that substitutions here could modulate efficacy. General medicinal chemistry precedent for C–F···carbonyl dipolar contacts and fluorine's role in modulating aromatic ring electronics supports the physicochemical rationale, though this is not evidenced in the retrieved abstracts specifically. The preserved steric volume argument (4F-Phe β-carbon geometry is comparable to Thr) is chemically sound.","challenging_evidence":"PMID:38382158 presents a significant challenge: TB-500 is rapidly cleaved in vivo to Ac-LK as the dominant early metabolite, with Ac-LKKTE as a longer-lived species. Introducing a bulky aromatic residue at Thr-4 may alter protease recognition at this site unpredictably — potentially accelerating or blocking cleavage — complicating translation of any in vitro binding gain to in vivo efficacy. Additionally, Thr-4 is a polar, hydrogen-bond-donating residue; if the native contact with actin is a specific Thr OH–acceptor hydrogen bond (rather than a marginal polar contact as the hypothesis assumes), replacing it with a fluorinated aromatic could disrupt rather than enhance binding. The retrieved literature provides no direct evidence that the Thr-4 contact is 'marginal' — this characterization requires structural data not present in these papers. Finally, PMID:41476424 and DOI:10.20944/preprints202512.1011.v3 note that TB-500 human data are essentially absent, meaning any improved analogue faces a very long translational path with no established clinical comparator."},"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","ipTM of 0.48 is below the ~0.55–0.60 threshold for reliable complex interface prediction; binding pose geometry is not trustworthy at this confidence level","Boltz-2 affinity module returned no values; no predicted binding energy or probability score was obtained","Heuristic peptide properties (aggregation, stability, half-life) are sequence-based estimates, not derived from the complex prediction, and do not reflect the modified residue's non-canonical chemistry","No quantitative Kd data exist for native TB-500 vs. G-actin in the published literature, making it impossible to benchmark any predicted improvement","4-fluoro-L-phenylalanine may fall outside the structural predictor's training distribution for non-canonical amino acids, which could contribute to the low ipTM independently of the binding hypothesis","Proteolytic stability at the Thr-4 position (within the biologically active Ac-LKKTE metabolite fragment, per PMID:38382158) is altered unpredictably by the bulky aromatic substitution — an effect not captured by this prediction","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":"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 LKKTETQ is the actin-binding active site of Tβ4 and establishes the sequence identity and N-terminal acetylation of TB-500, providing the baseline structural context for the Thr-4 modification hypothesis."},{"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 and provides the reference sequence against which Thr-4 substitution is defined."},{"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":"Demonstrates that TB-500 undergoes rapid N-terminal proteolysis in vivo and that biological activity resides in longer fragments (Ac-LKKTE), directly informing whether a Thr-4 substitution would survive long enough to bind G-actin in vivo."},{"pmid_or_doi":"41490200","title":"Therapeutic Peptides in Orthopaedics: Applications, Challenges, and Future Directions","year":2026,"relevance":"Places TB-500 in the context of wound-healing peptides acting on actin-mediated integrin and extracellular matrix pathways, supporting the biological relevance of G-actin binding as a therapeutic target."},{"pmid_or_doi":"41476424","title":"Injectable Peptide Therapy: A Primer for Orthopaedic and Sports Medicine Physicians","year":2026,"relevance":"Confirms TB-500's attributed mechanism of action involves actin dynamics and tissue repair, contextualizing why improving G-actin binding affinity is a meaningful pharmacological objective."},{"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":"Narrative preprint review distinguishing Tβ4 (full-length) from TB-500 (fragment), noting that human safety/efficacy data are absent; relevant to understanding what is known about the fragment's mechanism relative to the parent protein."},{"pmid_or_doi":"24906629","title":"Analytical approaches for the detection of emerging therapeutics and non-approved drugs in human doping controls","year":2014,"relevance":"Identifies TB-500 among doping-relevant peptidic substances and notes its physicochemical and metabolic properties relevant to detection, providing indirect context for the peptide's stability and handling characteristics."},{"pmid_or_doi":"28887173","title":"Adsorption effects of the doping relevant peptides Insulin Lispro, Synachten, TB-500 and GHRP 5","year":2017,"relevance":"Characterizes surface adsorption behavior of TB-500 relevant to experimental handling; marginally relevant as it informs practical considerations in binding assays for modified analogues."}]},"onchain":{"hash":"5Q4mCU9weEHyzMKZayLVkitmHYpNXyDJxqYmKtN9z2D2yGhpBfBxC31AFMvWctzhfmzgUaW9pEo55BwV51QfhhiT","signature":"5Q4mCU9weEHyzMKZayLVkitmHYpNXyDJxqYmKtN9z2D2yGhpBfBxC31AFMvWctzhfmzgUaW9pEo55BwV51QfhhiT","data_hash":"496074fe6cc6caae8cf2816f255b139a33c4ca6a6d84cc92c89c52d5270fbd79","logged_at":"2026-05-04T04:34:33.533489+00:00","explorer_url":"https://solscan.io/tx/5Q4mCU9weEHyzMKZayLVkitmHYpNXyDJxqYmKtN9z2D2yGhpBfBxC31AFMvWctzhfmzgUaW9pEo55BwV51QfhhiT"},"ipfs_hash":null,"created_at":"2026-05-04T04:29:41.383291+00:00","updated_at":"2026-05-05T04:34:22.576788+00:00"}