Blocking junction repair may align defects across skin layers and increase penetration
Delayed blockade of transient receptor potential vanilloid 4 may align skin-layer defects despite matched itch relief. A barrier benefit confined to macrophage-containing models that rescues viable cells from engulfment would instead support immune-driven cell loss.
Stage of verification
- Hypothesis published2026-09-25
- Indirect evidenceAssessed at 5 of 10
- Direct testAwaited
Map of the hypothesis
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Lens
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Target map
Every target of every published hypothesis, each with the actions a hypothesis can propose on it. The targets and the actions of this hypothesis are drawn solid.

Signalling molecule
Interleukin-6
A secreted signalling molecule
Where this hypothesis actsSkin exposed to wet friction, with itch reduction matched between interventions
Hypotheses on this target 2
Lower level
Synthesis suppression1
Neutralisation1
Supplementation
Accelerated excretion

What is proposed
Neutralisation
Selectively suppress IL-31 while preserving junction repair
With whatNot stated in the record
HowIL-31 blockade that leaves TRPV4-dependent junction repair intact; the blocking agent is not stated
Possible result
Possible preservation of junction repair and better prevention of repeated skin damage
From the recordБлокада IL-31 сохраняет этот ремонтный процесс.

Receptor or channel
TRPV4
A protein involved in the formation and restoration of junctions between keratinocytes
Where this hypothesis actsKeratinocytes after wet friction, after the first protective response but before junction repair is complete
Hypotheses on this target 1
Lower level
Higher level
Blockade1
Agonism
Desensitisation
Function restoration
Function preservation

What is proposed
Blockade
Delay TRPV4 inhibition until after the first protective response
With whatControlled genetic model
HowDelayed TRPV4 blockade before junction repair is complete, with keratinocyte-specific TRPV4 inactivation as a comparison
Possible result
Expected increase in vertically aligned barrier defects and tracer penetration compared with IL-31 blockade
From the recordПредполагается, что отсроченная блокада TRPV4, начатая после первого защитного ответа, но до завершения восстановления соединений, увеличивает совпадение дефектов по глубине.
All targets of the lab
Every target read from the published hypotheses, each kind around its pictogram. A larger mark means more hypotheses act on that target. Point at a mark and the actions proposed on it branch out of it.
Solid and named: the targets of this hypothesis
Explore in depth
The logic
The train of thought that ends in this hypothesis. Each stage is the reason the next exists. The master question narrows to a goal, the goal to an unknown nobody has closed, the unknown to the hypothesis proposed here. Every step below says what it rests on and what carries it.
Skin protection may depend on where damage occurs across its layers, as well as how much damage there is. The unexpected move is to propose that two treatments providing equal itch relief could leave different routes open through the skin because one interrupts repair beneath existing surface gaps. This is a hypothesis generated by the pipeline, not a measured treatment comparison.
- Wet rubbing is proposed to create local gaps in the outer protective layer.
- TRPV4-dependent repair is proposed to restore connections between living cells beneath those gaps, keeping a surface opening from becoming a route through both layers.
- TRPV4 blockade after the first protective response but before repair finishes is proposed to change temporary gaps into gaps that remain open during the observation period.
- The resulting overlap between gaps in both layers is proposed to increase passage of an irritant.
- IL-31 blockade is proposed to preserve the deeper repair process, leaving fewer openings that coincide across layers.
- With itch relief and the early protective response held equal, fewer coinciding openings are predicted to reduce repeated injury.
Two overlapping sheets can each have holes yet still cover an opening if their holes sit in different places. Moving the holes into line creates a passage without increasing the number of holes.
Where the picture breaks: Skin repairs itself, and substances can spread sideways between layers. The proposal therefore has to establish whether aligned gaps are actually required for substantial passage of the chosen test substance during the chosen observation period.
- Master questionstep 01 of 04
A therapy would restore the functional condition of middle-aged people's skin toward that of young people.
Rests on: The stated goal is functional improvement toward a younger reference state.
Stated in the chain - Goal pillarstep 02 of 04
Skin would resist everyday stresses that amplify one another's damaging effects.
Rests on: Resistance to interacting everyday stresses is treated as one component of younger skin function.
AssumptionThe goal does not establish that this resistance differs between middle-aged and young skin, or define how restoring it would be measured.
- Gap questionstep 03 of 04
With equal itch relief, blocking interleukin-31 (IL-31), an immune signalling protein associated with itch, might preserve the protective response to wet rubbing and prevent repeated injury differently from delayed suppression of transient receptor potential (TRP) channels, a family of channels that carry charged particles across cell membranes.
Rests on: The previous stage identifies interacting everyday stresses as a target, but supplies no route from that target to this particular treatment comparison.
LeapThe supplied material does not establish why these interventions, their timing, or wet rubbing specifically resolve the preceding goal. Evidence about itch treatment alone does not supply the missing connection to protective responses and repeated injury.
- Hypothesisstep 04 of 04
Damage in the stratum corneum, the outer protective layer of dead skin cells, is proposed to become more permeable when it lines up with gaps between cells in the living epidermis, the living portion of the skin's outer covering. Delayed TRPV4 blockade is proposed to interrupt repair of those cell connections, whereas IL-31 blockade is proposed to preserve it, producing different levels of repeated injury despite equal itch relief and equal early protection.S1S4
Rests on: The preceding comparison supplies the need for a mechanism separating the treatments. S1, in Skin Pharmacology and Physiology in 2013, reports that TRPV4 activation strengthened the barrier associated with tight junctions, seals between neighbouring cells, in cultured human keratinocytes, the main cells of the epidermis; its screened abstract does not establish repair after wet rubbing or the effects of delayed blockade. S4, in Communicative & Integrative Biology in 2010, describes TRPV4 involvement in developing cell connections, but does not establish alignment of damage across layers or the comparison with IL-31 blockade.
Supported by literature
What is carried, and what is not. Screened sources speak to two of the six mechanism links at a broader level: mechanical stress affecting the skin barrier and TRPV4 involvement in cell connections; S6, in Acta Dermato-Venereologica in 2006, connects mechanical stress with water permeability in healthy volunteers but does not establish wet-rubbing damage or aligned gaps. None of the supplied sources establishes the sequence from delayed blockade through interrupted repair and aligned defects to greater penetration and repeated injury.S6
Where the reasoning is carried by something unstated · 2
- Goal pillar. The goal does not establish that this resistance differs between middle-aged and young skin, or define how restoring it would be measured.
- Gap question. The supplied material does not establish why these interventions, their timing, or wet rubbing specifically resolve the preceding goal. Evidence about itch treatment alone does not supply the missing connection to protective responses and repeated injury. Establish the missing link before relying on this step.
How a result here could mislead · 3
- Greater passage of a tracer, a detectable substance used to track penetration, could be attributed to alignment even if treatment simply creates more damage in one layer. Sideways tracer movement could also make entry appear to require vertically aligned gaps when it does not. What closes it: The proposed layer-by-layer maps must compare the damaged fraction of each layer alongside the frequency of overlapping defects and tracer passage. The region, observation period and rule for identifying overlap must be fixed in advance, and sideways spread must be measured separately.
- A treatment difference could be credited to repair timing when the treatments actually produce different itch relief or different early protective responses. Conversely, a negative result could reflect blockade beginning after repair was already complete. What closes it: Equal itch relief and equal early protection require verification wherever those responses are represented. The design must confirm that TRPV4 is inhibited during unfinished repair and separately assess blockade begun after confirmed repair; the supplied material gives no timing schedule or criteria for these conditions.
- A result in a model without macrophages could be read as excluding their contribution in intact skin, while a net benefit in their presence could conceal simultaneous harm to cell connections. The rival predicts that TRPV4 blockade prevents macrophages from engulfing still-recoverable skin cells. What closes it: Separating the routes requires comparisons with and without macrophages, the proposed TRPV4 shutdown restricted to keratinocytes, and measurements of both cell connections and engulfment of recoverable cells. The rival additionally requires confirmation that macrophages can physically reach the affected skin cells.
What would make this wrong. The central chain would be contradicted if, without macrophages and with confirmed TRPV4 inhibition during unfinished repair, delayed blockade did not impair cell connections, increase aligned gaps or increase tracer passage relative to IL-31 blockade. Its alignment-dependent explanation would also fail for the tested substance and observation period if penetration did not depend on overlap when the damaged fraction of each layer was comparable. Improvement confined to models containing macrophages, accompanied by prevention of engulfment of recoverable cells, would instead support the supplied rival.
What it would change. If the mechanism held, work toward restoring younger skin function would have to consider the spatial arrangement and repair timing of damage, alongside itch relief and the amount of damage. A treatment could reduce symptoms while leaving a more continuous route through the skin's protective layers. Even then, the supplied work would not establish an age-dependent difference, restoration to a young reference state, or lasting benefit in middle-aged people; its proposed stability outcome is not operationally defined.
Sources read · 10
Activation of TRPV4 strengthens the tight-junction barrier in human epidermal keratinocytes. · Skin pharmacology and physiology · 2013
“In conclusion, we demonstrated that the activation of TRPV4 strengthened the TJ-associated barrier of epidermal cells.”
Does not settle: Источник показывает усиление барьера плотных контактов при активации TRPV4 в культуре человеческих кератиноцитов. Он не устанавливает последствия блокады TRPV4 после влажного трения, пространственное совпадение дефектов между слоями кожи, проникновение раздражителя, роль IL-31, зуд или стабильность SPV_1.
TRPV4 Regulates Tight Junctions and Affects Differentiation in a Cell Culture Model of the Corneal Epithelium. · Journal of cellular physiology · 2017
“We showed that TRPV4 activity was necessary to establish the TJ.”
Does not settle: Источник описывает модель эпителия роговицы in vitro. Он не устанавливает последствия влажного трения в коже, пространственное совпадение дефектов по слоям эпидермиса, время блокады TRPV4, проникновение раздражителя, роль IL-31, зуд или SPV_1.
Bioactive Polysaccharides from Fermented Dendrobium officinale: Structural Insights and Their Role in Skin Barrier Repair. · Molecules (Basel, Switzerland) · 2025
“recovered levels of filaggrin (FLG), aquaporin 3 (AQP3), transient receptor potential vanilloid 4 (TRPV4), cathelicidin antimicrobial peptide (CAMP)/LL-37, and adiponectin (ADIPOQ);”
Does not settle: Источник не исследует влажное трение, межклеточные соединения, пространственное совпадение дефектов по глубине, проникновение раздражителя, блокаду TRPV4 или IL-31, зуд, повторное повреждение либо SPV_1.
The TRPV4 cation channel: A molecule linking skin temperature and barrier function. · Communicative & integrative biology · 2010
“TRPV4 associates with the E-cadherin complex via β-catenin, and thereby participates in the promotion of cell-cell junction development.”
Does not settle: Источник не исследует влажное трение, пространственное совпадение дефектов по глубине, проникновение раздражителя, отсроченную блокаду TRPV4, IL-31, зуд, повторное повреждение или SPV_1.
Altered stratum corneum barrier and enhanced percutaneous immune responses in filaggrin-null mice. · The Journal of allergy and clinical immunology · 2012
“increased desquamation under mechanical stress was demonstrated. Loss of keratin patterns, which are critical for corneocyte stabilization, is likely attributable to fragility in the Flg(-/-) SC. Antigens penetrated the Flg(-/-) SC more efficiently”
Does not settle: Источник описывает мышей с полным дефицитом филаггрина. Он не устанавливает эффект влажного трения, локальное совпадение дефектов по глубине, восстановление межклеточных соединений, роль TRPV4 или IL-31, время блокады, зуд, повторное повреждение и SPV_1.
Mechanical properties and barrier function of healthy human skin. · Acta dermato-venereologica · 2006
“Water permeability of the epidermis was significantly affected by the application of mechanical stress to the skin and vice versa, the mechanical properties of the skin were altered when compromising the barrier.”
Does not settle: Источник показывает связь механического воздействия с проницаемостью эпидермиса у здоровых добровольцев. В тексте остаются открытыми влажное трение, локальные дефекты рогового слоя, межклеточные соединения живого эпидермиса, TRPV4, IL-31, сроки вмешательства, проникновение раздражителя и SPV_1.
Photomechanical transcutaneous delivery of macromolecules. · The Journal of investigative dermatology · 1998
“A mechanical (stress) pulse generated by a single laser pulse was shown to transiently increase the permeability of the stratum corneum in vivo.”
Does not settle: This abstract does not establish effects of wet friction, TRPV4 or IL-31 blockade, junction repair in viable epidermis, spatial alignment of defects, itch, repeated damage, or SPV_1.
Assessment of non-invasive techniques and herbal-based products on dermatological physiology and intercellular lipid properties. · Heliyon · 2020
“Transepidermal water loss (TEWL) was measured regularly in order to provide further information on the epidermal permeability barrier—either normal, experimentally perturbed, or in diseased conditions [ ].”
Does not settle: Источник не исследует влажное трение, TRPV4, IL-31, межклеточные соединения живого эпидермиса, пространственное совпадение дефектов, проникновение раздражителя или SPV_1.
Interleukin-31: The "itchy" cytokine in inflammation and therapy. · Allergy · 2021
“In AD, IL-31 has been identified as one of the main "drivers" of its cardinal symptom, pruritus.”
Does not settle: Источник оставляет открытыми роль TRPV4, восстановление межклеточных соединений, пространственное совпадение дефектов по слоям кожи, проницаемость раздражителей, сроки блокады и сравнительную эффективность блокады IL-31.
IL-31/33 Axis in Atopic Dermatitis. · International journal of molecular sciences · 2025
“Anti-IL-31 receptor α (IL-31RA) agents (e.g., nemolizumab) demonstrate significant reductions in pruritus and disease severity across clinical trials.”
Does not settle: Источник не устанавливает влияние блокады IL-31 на восстановление межклеточных соединений, TRPV4-зависимый ремонт, пространственное совпадение дефектов, проникновение раздражителя, повторное повреждение или SPV_1.
The gap this hypothesis explains
Do targeted versus delayed blockers equally relieve itching yet differ in wet-rubbing protection and repeated skin damage?
Original wording · exactly as the pipeline generated it
При одинаковом уменьшении зуда позволяют ли избирательное подавление IL-31 и отсроченное подавление TRP-сигналов по-разному сохранить защитную реакцию на влажное трение и прекратить повторное повреждение кожи?
What this question is asking
The question concerns whether two ways of reducing itching also differ in preserving the skin’s response to potentially damaging rubbing when wet. It compares selectively suppressing interleukin-31 (IL-31), a chemical messenger involved in itching, with suppressing transient receptor potential (TRP) channel signals after a delay. With itch relief held equal, the outcomes are preservation of a protective response to wet rubbing and whether repeated skin damage stops. Equal itch relief is a condition of the comparison, not an established finding, and the question does not specify the channels, the delay, or what counts as a protective response. Its stated motivation concerns middle-aged skin, whereas the supplied evidence mainly concerns atopic dermatitis, an inflammatory skin condition.
- Interleukin-31 (IL-31)
- A chemical messenger involved in communication between cells and implicated in itching in the supplied sources. Suppressing it is the targeted intervention in the question [S2, S3].
- Selective suppression or targeted blocking
- Reducing the activity of a specified target, here interleukin-31. The wording identifies the intended target; it does not establish that all other skin or sensory functions remain unaffected.
- Transient receptor potential (TRP) channels
- A family of proteins that allow electrically charged particles to cross cell membranes and participate in cellular signals. This names a family rather than one interchangeable target; the question does not specify which members would be suppressed.
- Transient receptor potential vanilloid (TRPV) channels
- A subgroup of the transient receptor potential channel family. The supplied temperature-related source discusses possible effects of blocking this subgroup [S5].
- Transient receptor potential vanilloid 1 (TRPV1)
- A particular channel associated with transmission of both pain and itch signals. Its involvement in both sensations is relevant to whether itch relief could accompany changes in other sensory responses, but does not establish an effect on wet-rubbing protection [S7].
- Transient receptor potential vanilloid 3 (TRPV3)
- A particular channel whose activation in skin cells can lead to release of substances involved in itching and other skin functions. The supplied sources connect it both to inflammatory signaling and to functions including barrier formation and wound healing [S6, S8].
- Delayed suppression
- Starting to reduce a signal after some interval. The question provides neither the interval nor the event that starts it, so this does not identify a defined treatment schedule.
- Atopic dermatitis
- An inflammatory skin condition associated here with itching and impaired skin protection. It is the setting of most supplied findings, which do not establish equivalent effects in middle-aged skin generally.
- Sensory nerves and increased sensitivity
- Sensory nerves carry information about bodily stimuli. Increased sensitivity means stimuli can more readily provoke a response such as itching; the supplied sources associate this with scratching and worsening skin problems [S1, S4].
- Central nervous system
- The brain and spinal cord. The supplied source describes pain and itch signals reaching this system through sensory nerve cells [S7].
- Keratinocytes
- Cells of the skin’s outer layer. In the supplied sources, activation of one channel type in these cells causes release of substances that influence itching and other skin functions [S6, S8].
- Inflammation and inflammatory messengers
- Inflammation is a tissue response involving cellular activity and chemical signals; inflammatory messengers help initiate or regulate it. The supplied sources connect these processes to itching and worsening skin-barrier function [S4, S6].
- Skin barrier
- The protective function of the skin’s outer layers. Barrier impairment means this protection is weakened; the sources discuss it separately from the amount of itching [S4, S5].
- Wet rubbing and protective response
- Wet rubbing means friction involving wet skin or a wet contacting surface. A protective response would be a response that helps limit resulting harm, but the question does not specify what response would be measured.
- Repeated skin damage
- Skin injury that recurs rather than remaining a single event. The question asks whether this recurrence stops, which is a different outcome from reduced itching.
- Corns and calluses
- Localized areas of thickened skin discussed in the supplied source as consequences of pressure and friction. That finding does not address the proposed itch-treatment comparison [S9].
- Selective messenger suppression performs better At equal itch relief, selectively suppressing the messenger would preserve more of the response that limits harmful wet rubbing and reduce repeated damage more effectively. Under the question’s proposed mechanism, continued protection would distinguish this approach even when itching improves equally.
- Delayed channel suppression performs better At equal itch relief, delaying channel suppression would preserve more protection against wet rubbing and reduce repeated damage more effectively. Under the question’s proposed mechanism, the timing of suppression would matter for protection beyond its effect on itching.
- Neither approach performs better Equal itch relief would be accompanied by equivalent protection and repeated-damage outcomes. That could mean both preserve protection and stop injury, or that both leave injury unresolved; equivalence alone would not establish success.
- Protection and damage outcomes diverge One approach could preserve the measured protective response better without stopping repeated damage more effectively. That outcome would mean the protective-response measurement alone does not determine whether the skin continues to be injured.
The sources describe a sequence in which increased sensitivity to itch leads to scratching, which worsens inflammation and weakens the skin’s protective barrier [S4]. Reducing itch could interrupt that sequence, but itch relief alone does not establish that repeated injury has stopped. The channel family under discussion also includes members involved in pain signals and several skin functions, so the question concerns more than the amount of itching [S7, S8]. If protection against damaging rubbing differs between treatments, judging them solely by itch relief could miss that difference; the supplied sources do not establish whether it occurs.
The mechanism it proposes
The engine's own statement of the hypothesis, in full.
Различие вмешательств определяется пространственным совпадением повреждений рогового слоя и межклеточных соединений живого эпидермиса. После влажного трения верхний слой получает локальные дефекты. TRPV4-зависимое восстановление соединений под этими дефектами ограничивает образование сквозных путей проникновения раздражителя. Предполагается, что отсроченная блокада TRPV4, начатая после первого защитного ответа, но до завершения восстановления соединений, увеличивает совпадение дефектов по глубине. Блокада IL-31 сохраняет этот ремонтный процесс. Поэтому при одинаковом уменьшении зуда и одинаковой ранней защитной реакции подавление IL-31 лучше предотвращает повторное повреждение. Сохранение пространственной разобщённости дефектов стабилизирует SPV_1.
Where the idea comes from
The hypothesis borrows a result from another field. This is what it borrows, and from where.
Инженерия надёжности и проектирование резервирования: модель зависимых отказов двух защитных элементов и отказов по общей причине. Значение зависимости отказов рассмотрено в [материале NASA Common Cause Failures Dominate and Defeat Redundancy](https://ntrs.nasa.gov/citations/20240013667). Для данного исследования используется точное разложение совместной вероятности через корреляцию: P_AB = p_A × p_B + ρ × sqrt[p_A(1 − p_A)p_B(1 − p_B)]. A означает дефект рогового слоя в заданном участке и временном окне; B означает дефект соединений живого эпидермиса под тем же участком; p_A и p_B являются вероятностями этих событий; ρ является корреляцией их бинарных индикаторов; P_AB является вероятностью их совместного возникновения. Биологическое допущение модели: для выбранного трассера и короткого окна наблюдения выраженное проникновение требует совпадения обоих дефектов. Это допущение проверяют отдельно, включая боковое распространение трассера. Формула не является готовой моделью всей трансэпидермальной потери воды. Предлагаемая причинная связь состоит в том, что подавление восстановления соединений превращает общий эпизод трения в сопряжённый отказ двух слоёв.
Testing and possible results
The prediction that would tell it apart
A hypothesis that predicts what its rivals predict is not worth running an experiment over. This is the observation on which this one differs.
В модели без макрофагов отсроченная блокада TRPV4 увеличит частоту совпадающих по глубине дефектов и проникновение трассера по сравнению с блокадой IL-31. Эффект воспроизведёт выключение TRPV4 только в кератиноцитах. При сопоставимой доле дефектов каждого слоя различие проницаемости будет зависеть от их пространственной корреляции. Перенос начала блокады на момент после подтверждённого восстановления соединений уменьшит эффект. Если же блокада TRPV4 улучшит барьер исключительно при наличии макрофагов и спасёт поглощаемые жизнеспособные клетки, результат поддержит IH_Q_L3_M_G2_2_01.
Would tell it apart from at least one rival. The prediction specifies observable comparative changes in defects and tracer penetration, replication through cell-specific TRPV4 inactivation, and dependence on spatial correlation and blockade timing. No rival prediction was supplied. Only a bench experiment would settle it.
What testing it would take
The engine's own read on whether this is testable with methods that already exist.
Послойная микроскопия позволяет совместить карты дефектов рогового слоя, межклеточных соединений и проникновения трассера. Биологическое основание: активация TRPV4 поддерживала формирование межклеточных соединений человеческих кератиноцитов. [Kida et al., 2012](https://pubmed.ncbi.nlm.nih.gov/22374181/). Возрастную зависимость, пространственную корреляцию дефектов и результат отсроченной блокады необходимо установить экспериментально.
Other explanations
Every other hypothesis the engine wrote for the same gap, and the observation that would separate the two.
В модели без макрофагов отсроченная блокада TRPV4 увеличит частоту совпадающих по глубине дефектов и проникновение трассера по сравнению с блокадой IL-31. Эффект воспроизведёт выключение TRPV4 только в кератиноцитах. При сопоставимой доле дефектов каждого слоя различие проницаемости будет зависеть от их пространственной корреляции. Перенос начала блокады на момент после подтверждённого восстановления соединений уменьшит эффект. Если же блокада TRPV4 улучшит барьер исключительно при наличии макрофагов и спасёт поглощаемые жизнеспособные клетки, результат поддержит Macrophages may prolong skin damage by engulfing keratinocytes that could recover.
- Rival 01 of 01Macrophages may prolong skin damage by engulfing keratinocytes that could recover
Not yet published.
What would separate themMacrophages may prolong skin damage by engulfing keratinocytes that could recover predicts: В модели кожи с сенсорными нейронами и макрофагами отсроченное подавление TRPV4 уменьшит вторичную потерю кератиноцитов сильнее блокады IL-31 при сопоставимом подавлении ответа на зудовые стимулы и одинаковой механической нагрузке. Макрофагоспецифическое выключение TRPV4 воспроизведёт преимущество, а удаление макрофагов устранит его. Решающий результат: клетки, которые в контрольной группе поглощаются, после предотвращения поглощения сохраняют жизнеспособность, возвращают нормальную мембранную асимметрию и участвуют в восстановлении эпидермиса. Отсутствия активации каспаз перед поглощением для такого вывода недостаточно. Если преимущество сохраняется без макрофагов и определяется пространственным совпадением дефектов слоёв, результат поддержит this hypothesis.
What stands behind it
Which of the figures above have a study behind them, which are the engine's own, and what it would take to refute the hypothesis. This audit never judges the idea.
0 of 1 cited studies could be located, and 0 of 0 figures are not carried by one that resolved.
What it would take to refute it. Nothing already retrieved carries the prediction’s terms and it names no measurement this layer can route to a public dataset, so the bench is the residual — not a finding against it.
1 citation handle extracted; 3 Europe PMC searches run; 52 records examined; 0 sources stored for enrichment, 0 with full text. A citation that did not resolve is a bibliographic failure, not proof that no such paper exists, and no hypothesis is blocked by this audit.