{"gene":"PF3D7_1105100","run_date":"2026-09-08T12:51:28","timeline":{"discoveries":[{"year":1995,"finding":"The H2B gene of Plasmodium falciparum was identified and sequenced, establishing the existence of a canonical H2B histone in this parasite.","method":"Gene cloning and sequencing","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct gene identification in single study; no functional assay beyond identification","pmids":["7637710"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":1997,"finding":"H2B protein was purified from P. falciparum culture supernatant and infected erythrocytes; N-terminal sequence analysis confirmed strong homology with H2B sequences from other species, and immunofluorescence localized H2B to schizont nuclei.","method":"Protein purification, N-terminal sequencing, immunofluorescence","journal":"Parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct protein purification and localization, single study with two orthogonal methods","pmids":["9149412"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":1999,"finding":"H2B mRNA is not detectable in ring-stage parasites but is expressed in trophozoites, schizonts, and gametocytes at approximately equivalent levels, indicating developmental regulation of H2B transcription.","method":"Northern blot / transcription analysis across developmental stages","journal":"International journal for parasitology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single method (transcript analysis), no functional follow-up","pmids":["10579431"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2004,"finding":"Recombinant P. vivax H2B (ortholog of H2B) was expressed in E. coli, shown to bind DNA by South-Western analysis, and localized to the parasite nucleus by confocal microscopy; structural modeling revealed differences from the host H2B at acetylation, methylation, and DNA-binding sites.","method":"Recombinant protein expression, South-Western analysis, confocal microscopy, structural modeling","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — multiple orthogonal methods (DNA binding, localization, structural modeling), single study in P. vivax","pmids":["15276199"],"is_preprint":false,"attribution":[{"species":"Plasmodium vivax","strain":""}]},{"year":2005,"finding":"PfNAPS and PfNAPL (nucleosome assembly proteins) preferentially interact with H3-H4 tetramer histones over H2A and H2B histones in P. falciparum, as shown by ELISA-based binding assays.","method":"ELISA-based histone binding assay","journal":"Molecular and biochemical parasitology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single binding assay method; H2B is a secondary/comparison target in the experiment","pmids":["15899528"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2006,"finding":"Core histone H2B expression in P. falciparum increases dramatically in late trophozoite stages, consistent with replication-related nucleosome assembly, as determined by real-time RT-PCR and immunoblotting; mass spectrometry confirmed H2B protein synthesis.","method":"Real-time RT-PCR, immunoblotting, mass spectrometry","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — three orthogonal methods in a single study confirming expression and protein synthesis","pmids":["16410041"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2009,"finding":"Mass spectrometry mapping of P. falciparum histones identified H2B (PF3D7_1105100) as exhibiting a distinct post-translational modification pattern compared to the H2B variant (H2Bv/H2B.Z), suggesting divergent functions of the two H2B forms; 44 modifications were mapped across all P. falciparum histones.","method":"LC-MS/MS mass spectrometry (Q-TOF and LTQ-FT)","journal":"Journal of proteome research","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — high-quality MS with rigorous controls in single study; differentiation of canonical H2B vs. variant established but functional consequence not directly tested","pmids":["19351122"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2010,"finding":"PfNapS (nucleosome assembly protein) interacts with core histones including H2A and H2B (as well as H3, H4, tetramer, and octamer) at a site distinct from its interaction with linker histone H1, as revealed by fluorescence spectroscopy; two regions on the PfNapS dimer were identified as histone-recognition sites.","method":"Fluorescence spectroscopy, crystal structure of PfNapS","journal":"Malaria journal","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — crystal structure plus fluorescence spectroscopy interaction mapping; H2B is one of several histones tested, single study","pmids":["20377878"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2016,"finding":"P. falciparum nucleosomes exhibit decreased salt stability of histones H2A and H2B compared to human nucleosomes, contributing to overall reduced nucleosome stability; parasite nucleosomes form the shortest known nucleosome repeat length (155 bp) in vitro and in vivo.","method":"In vitro nucleosome reconstitution, salt stability assays, in vivo nucleosome mapping","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with functional stability assays plus in vivo corroboration; multiple orthogonal methods in single study","pmids":["28033404"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2024,"finding":"H2B (canonical) and H2B.Z (variant) were both found associated with the PfSWR1 chromatin remodeling complex by reciprocal pulldown, indicating this complex is involved in deposition of the H2A.Z/H2B.Z variant histone pair; H2B itself (PF3D7_1105100) was co-detected in the complex but the primary substrate for deposition is the H2B.Z variant.","method":"Reciprocal co-immunoprecipitation / pulldown, mass spectrometry","journal":"mSphere","confidence":"Low","confidence_rationale":"Tier 3 / Weak — H2B (canonical) detected in pulldown alongside variant H2B.Z; mechanistic role of canonical H2B in SWR1 complex not directly established","pmids":["38564734"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"finding":"PfNPM (nucleoplasmin isoform) interacts individually with assembled H2A/H2B with equimolar stoichiometry; the acidic tracts of PfNPM are necessary for this interaction; H3/H4 displays higher binding affinity for PfNPM than H2A/H2B, and H3/H4 can substitute H2A/H2B at the same binding site on PfNPM's distal face.","method":"Crystal structure of PfNPM, biochemical interaction assays (binding affinity measurements), mutagenesis of acidic tracts","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus binding assays plus mutagenesis establishing acidic tract requirement; multiple orthogonal methods in single study","pmids":["40049416"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"P. falciparum H2B (PF3D7_1105100) is a canonical core histone that localizes to the parasite nucleus, is expressed in a developmentally regulated manner peaking in late trophozoite/schizont stages, forms nucleosomes with reduced salt stability and the shortest known repeat length (155 bp) compared to human nucleosomes, carries a distinct post-translational modification profile from the H2B.Z variant, and is bound by histone chaperones PfNapS and PfNPM (via their acidic tracts) with lower affinity than H3/H4."},"narrative":{"mechanistic_narrative":"H2B (PF3D7_1105100) is a canonical core histone of Plasmodium falciparum that packages the parasite genome into nucleosomes and localizes to the parasite nucleus [PMID:9149412, PMID:28033404]. Its expression is developmentally regulated: H2B mRNA is undetectable in ring stages and rises through trophozoites and schizonts, with protein levels peaking dramatically in late trophozoite/schizont stages in a pattern consistent with replication-coupled nucleosome assembly [PMID:10579431, PMID:16410041]. Nucleosomes incorporating H2B show reduced salt stability relative to human nucleosomes and adopt the shortest known nucleosome repeat length (155 bp), a distinctive feature of parasite chromatin [PMID:28033404]. H2B is deposited and handled by histone chaperones — the nucleosome assembly proteins PfNapS/PfNAPS and the nucleoplasmin PfNPM bind assembled H2A/H2B, with PfNPM engagement requiring its acidic tracts, though both chaperones bind H2B with lower affinity than the H3/H4 pair [PMID:15899528, PMID:20377878, PMID:40049416]. Canonical H2B carries a post-translational modification profile distinct from that of the H2B.Z variant, and both forms are co-detected with the PfSWR1 remodeling complex whose primary deposition substrate is the H2B.Z variant [PMID:19351122, PMID:38564734]. Beyond these chaperone interactions, chromatin properties, and expression profile, no direct functional or knockout phenotype for PF3D7_1105100 has been characterized in the available corpus.","teleology":[{"year":1995,"claim":"Establishing that P. falciparum encodes a canonical H2B histone at all was the necessary first step before any chromatin biology could be studied in the parasite.","evidence":"Gene cloning and sequencing of the P. falciparum H2B gene","pmids":["7637710"],"confidence":"Medium","gaps":["No functional assay beyond sequence identification","Nucleosomal incorporation not shown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":1997,"claim":"Purifying the protein and confirming sequence homology and nuclear localization established that the cloned gene produces a bona fide nuclear histone rather than merely an ORF.","evidence":"Protein purification, N-terminal sequencing and immunofluorescence in schizonts","pmids":["9149412"],"confidence":"Medium","gaps":["Did not test DNA binding directly","Nucleosome context not established"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":1999,"claim":"Mapping H2B transcript across the life cycle addressed whether the histone is constitutive or stage-restricted, revealing developmental regulation absent in rings.","evidence":"Northern blot / transcript analysis across developmental stages","pmids":["10579431"],"confidence":"Low","gaps":["Single lab, single transcript method with no protein-level confirmation","Regulatory mechanism not defined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2004,"claim":"Recombinant expression of the P. vivax ortholog tested whether the histone binds DNA and localizes to the nucleus, and modeling flagged divergence from host H2B at modification and DNA-contact sites.","evidence":"Recombinant expression, South-Western DNA binding, confocal microscopy and structural modeling in P. vivax","pmids":["15276199"],"confidence":"Medium","gaps":["Shown in P. vivax ortholog, not directly in P. falciparum PF3D7_1105100","Functional consequence of divergent residues untested"],"attribution":[{"species":"Plasmodium vivax","strain":""}]},{"year":2006,"claim":"Quantifying protein levels and confirming synthesis by MS resolved whether the stage-restricted transcription translates into protein accumulation, linking H2B expression to late-stage DNA replication.","evidence":"Real-time RT-PCR, immunoblotting and mass spectrometry across stages","pmids":["16410041"],"confidence":"Medium","gaps":["Correlation with replication is inferential, not causal","Chromatin loading kinetics not measured"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2009,"claim":"Global MS mapping distinguished canonical H2B from the H2B.Z variant by their post-translational modification patterns, establishing that the two forms are chemically and likely functionally distinct.","evidence":"LC-MS/MS mapping of P. falciparum histone modifications","pmids":["19351122"],"confidence":"Medium","gaps":["Functional consequence of the distinct modification pattern not tested","Enzymes writing/erasing the marks not identified"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2010,"claim":"Structural and spectroscopic characterization of PfNapS identified where core histones including H2B are recognized, defining the chaperone-histone interface distinct from the linker H1 site.","evidence":"Crystal structure of PfNapS plus fluorescence spectroscopy interaction mapping","pmids":["20377878"],"confidence":"Medium","gaps":["H2B was one of several histones tested, not the focus","Functional relevance of the interaction in vivo not shown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2016,"claim":"Reconstitution and mapping showed that H2A/H2B confer reduced salt stability and that parasite nucleosomes have an unusually short repeat length, defining a biophysically distinct chromatin state.","evidence":"In vitro nucleosome reconstitution, salt stability assays and in vivo nucleosome mapping","pmids":["28033404"],"confidence":"High","gaps":["Contribution of PF3D7_1105100 versus H2B.Z to instability not fully partitioned","Consequences for gene regulation not established"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2024,"claim":"Reciprocal pulldowns placed both canonical H2B and H2B.Z with the PfSWR1 remodeler, addressing which chromatin machinery handles the histones while establishing the variant as the primary deposition substrate.","evidence":"Reciprocal co-immunoprecipitation and mass spectrometry","pmids":["38564734"],"confidence":"Low","gaps":["Mechanistic role of canonical H2B in the SWR1 complex not established","Co-detection may reflect nucleosomal association rather than a functional role"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"claim":"Crystallography, binding assays and mutagenesis defined how the nucleoplasmin PfNPM engages assembled H2A/H2B, showing equimolar acidic-tract-dependent binding that is weaker than for H3/H4 and displaceable by H3/H4.","evidence":"Crystal structure of PfNPM, binding affinity measurements and acidic-tract mutagenesis","pmids":["40049416"],"confidence":"High","gaps":["In vivo role of PfNPM-H2B interaction not demonstrated","Whether this hand-off governs nucleosome assembly timing not shown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"Whether PF3D7_1105100 is essential and what phenotype its loss produces remains unaddressed, as no genetic knockout, knockdown, or direct functional perturbation of the canonical H2B has been reported.","evidence":"No direct genetic or functional perturbation study in the corpus","pmids":[],"confidence":"Low","gaps":["No knockout or conditional depletion","No defined regulatory function separate from bulk chromatin packaging","Role of its distinct modifications unresolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Nucleosome (H2A/H2B core histone)","PfSWR1 chromatin remodeling complex (co-detected)"],"partners":["PFNAPS / PFNAPS (NUCLEOSOME ASSEMBLY PROTEIN)","PFNAPL (NUCLEOSOME ASSEMBLY PROTEIN)","PFNPM (NUCLEOPLASMIN ISOFORM)","H2A (HISTONE PARTNER)","H2B.Z / H2BV (VARIANT HISTONE, CO-DETECTED IN PFSWR1)"],"other_free_text":["Developmentally regulated expression: absent in rings, peaking in late trophozoites/schizonts and gametocytes [#2, #5]","Carries a post-translational modification profile distinct from the H2B.Z variant [#6]","H2A/H2B confers reduced nucleosome salt stability and parasite nucleosomes have the shortest known repeat length (155 bp) [#8]","PfNPM binding requires the chaperone's acidic tracts and is weaker for H2A/H2B than for H3/H4 [#10]","No genetic knockout or direct functional perturbation of PF3D7_1105100 reported in the corpus"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"16410041","id":"PMC_16410041","title":"The malaria parasite Plasmodium falciparum histones: organization, expression, and acetylation.","date":"2006","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/16410041","citation_count":155,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"19351122","id":"PMC_19351122","title":"Global histone analysis by mass spectrometry reveals a high content of acetylated lysine residues in the malaria parasite Plasmodium falciparum.","date":"2009","source":"Journal of proteome research","url":"https://pubmed.ncbi.nlm.nih.gov/19351122","citation_count":119,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B","histone H2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"23373537","id":"PMC_23373537","title":"H2A.Z and H2B.Z double-variant nucleosomes define intergenic regions and dynamically occupy var gene promoters in the malaria parasite Plasmodium falciparum.","date":"2013","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/23373537","citation_count":67,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"23320541","id":"PMC_23320541","title":"H2A.Z/H2B.Z double-variant nucleosomes inhabit the AT-rich promoter regions of the Plasmodium falciparum genome.","date":"2013","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/23320541","citation_count":58,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B","histone H2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15899528","id":"PMC_15899528","title":"Biochemical characterization of the two nucleosome assembly proteins from Plasmodium falciparum.","date":"2005","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/15899528","citation_count":42,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"9149412","id":"PMC_9149412","title":"The histones of Plasmodium falciparum: identification, purification and a possible role in the pathology of malaria.","date":"1997","source":"Parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/9149412","citation_count":30,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"27555062","id":"PMC_27555062","title":"H3.3 demarcates GC-rich coding and subtelomeric regions and serves as potential memory mark for virulence gene expression in Plasmodium falciparum.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27555062","citation_count":28,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"28033404","id":"PMC_28033404","title":"Plasmodium falciparum Nucleosomes Exhibit Reduced Stability and Lost Sequence Dependent Nucleosome Positioning.","date":"2016","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/28033404","citation_count":20,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"20377878","id":"PMC_20377878","title":"Structure, localization and histone binding properties of nuclear-associated nucleosome assembly protein from Plasmodium falciparum.","date":"2010","source":"Malaria journal","url":"https://pubmed.ncbi.nlm.nih.gov/20377878","citation_count":19,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"37013531","id":"PMC_37013531","title":"Plasmodium falciparum gametocytes display global chromatin remodelling during sexual differentiation.","date":"2023","source":"BMC biology","url":"https://pubmed.ncbi.nlm.nih.gov/37013531","citation_count":18,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"7637710","id":"PMC_7637710","title":"Identification of Plasmodium falciparum histone 2B and histone 3 genes.","date":"1995","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/7637710","citation_count":16,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"10579431","id":"PMC_10579431","title":"Differential transcription of histone genes in asexual and sexual stages of Plasmodium falciparum.","date":"1999","source":"International journal for parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/10579431","citation_count":10,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"37882786","id":"PMC_37882786","title":"The P. falciparum alternative histones Pf H2A.Z and Pf H2B.Z are dynamically acetylated and antagonized by PfSir2 histone deacetylases at heterochromatin boundaries.","date":"2023","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/37882786","citation_count":8,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"12739134","id":"PMC_12739134","title":"The histone H4 gene of Plasmodium falciparum is developmentally transcribed in asexual parasites.","date":"2003","source":"Parasitology research","url":"https://pubmed.ncbi.nlm.nih.gov/12739134","citation_count":7,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"40156869","id":"PMC_40156869","title":"PfGCN5 is essential for Plasmodium falciparum survival and transmission and regulates Pf H2B.Z acetylation and chromatin structure.","date":"2025","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/40156869","citation_count":6,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15276199","id":"PMC_15276199","title":"Identification, expression, modeled structure and serological characterization of Plasmodium vivax histone 2B.","date":"2004","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/15276199","citation_count":5,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B","PvH2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"38564734","id":"PMC_38564734","title":"MYST regulates DNA repair and forms a NuA4-like complex in the malaria parasite Plasmodium falciparum.","date":"2024","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/38564734","citation_count":4,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"40049416","id":"PMC_40049416","title":"Structure-function studies of a nucleoplasmin isoform from Plasmodium falciparum.","date":"2025","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40049416","citation_count":0,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"40266688","id":"PMC_40266688","title":"Correction to 'PfGCN5 is essential for Plasmodium falciparum survival and transmission and regulates Pf H2B.Z acetylation and chromatin structure'.","date":"2025","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/40266688","citation_count":0,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["H2B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11279,"output_tokens":2640,"usd":0.036719,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8122,"output_tokens":3819,"usd":0.068043,"stage2_stop_reason":"end_turn"},"total_usd":0.104762,"stage1_batch_id":"msgbatch_01DisAaTypwmC8QwdMBuePHo","stage2_batch_id":"msgbatch_0184zxZTqxiEfdYYfPMfkqjb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"The H2B gene of Plasmodium falciparum was identified and sequenced, establishing the existence of a canonical H2B histone in this parasite.\",\n      \"method\": \"Gene cloning and sequencing\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct gene identification in single study; no functional assay beyond identification\",\n      \"pmids\": [\"7637710\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"H2B protein was purified from P. falciparum culture supernatant and infected erythrocytes; N-terminal sequence analysis confirmed strong homology with H2B sequences from other species, and immunofluorescence localized H2B to schizont nuclei.\",\n      \"method\": \"Protein purification, N-terminal sequencing, immunofluorescence\",\n      \"journal\": \"Parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct protein purification and localization, single study with two orthogonal methods\",\n      \"pmids\": [\"9149412\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"H2B mRNA is not detectable in ring-stage parasites but is expressed in trophozoites, schizonts, and gametocytes at approximately equivalent levels, indicating developmental regulation of H2B transcription.\",\n      \"method\": \"Northern blot / transcription analysis across developmental stages\",\n      \"journal\": \"International journal for parasitology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method (transcript analysis), no functional follow-up\",\n      \"pmids\": [\"10579431\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Recombinant P. vivax H2B (ortholog of H2B) was expressed in E. coli, shown to bind DNA by South-Western analysis, and localized to the parasite nucleus by confocal microscopy; structural modeling revealed differences from the host H2B at acetylation, methylation, and DNA-binding sites.\",\n      \"method\": \"Recombinant protein expression, South-Western analysis, confocal microscopy, structural modeling\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — multiple orthogonal methods (DNA binding, localization, structural modeling), single study in P. vivax\",\n      \"pmids\": [\"15276199\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium vivax\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PfNAPS and PfNAPL (nucleosome assembly proteins) preferentially interact with H3-H4 tetramer histones over H2A and H2B histones in P. falciparum, as shown by ELISA-based binding assays.\",\n      \"method\": \"ELISA-based histone binding assay\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single binding assay method; H2B is a secondary/comparison target in the experiment\",\n      \"pmids\": [\"15899528\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Core histone H2B expression in P. falciparum increases dramatically in late trophozoite stages, consistent with replication-related nucleosome assembly, as determined by real-time RT-PCR and immunoblotting; mass spectrometry confirmed H2B protein synthesis.\",\n      \"method\": \"Real-time RT-PCR, immunoblotting, mass spectrometry\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — three orthogonal methods in a single study confirming expression and protein synthesis\",\n      \"pmids\": [\"16410041\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Mass spectrometry mapping of P. falciparum histones identified H2B (PF3D7_1105100) as exhibiting a distinct post-translational modification pattern compared to the H2B variant (H2Bv/H2B.Z), suggesting divergent functions of the two H2B forms; 44 modifications were mapped across all P. falciparum histones.\",\n      \"method\": \"LC-MS/MS mass spectrometry (Q-TOF and LTQ-FT)\",\n      \"journal\": \"Journal of proteome research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — high-quality MS with rigorous controls in single study; differentiation of canonical H2B vs. variant established but functional consequence not directly tested\",\n      \"pmids\": [\"19351122\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PfNapS (nucleosome assembly protein) interacts with core histones including H2A and H2B (as well as H3, H4, tetramer, and octamer) at a site distinct from its interaction with linker histone H1, as revealed by fluorescence spectroscopy; two regions on the PfNapS dimer were identified as histone-recognition sites.\",\n      \"method\": \"Fluorescence spectroscopy, crystal structure of PfNapS\",\n      \"journal\": \"Malaria journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — crystal structure plus fluorescence spectroscopy interaction mapping; H2B is one of several histones tested, single study\",\n      \"pmids\": [\"20377878\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"P. falciparum nucleosomes exhibit decreased salt stability of histones H2A and H2B compared to human nucleosomes, contributing to overall reduced nucleosome stability; parasite nucleosomes form the shortest known nucleosome repeat length (155 bp) in vitro and in vivo.\",\n      \"method\": \"In vitro nucleosome reconstitution, salt stability assays, in vivo nucleosome mapping\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with functional stability assays plus in vivo corroboration; multiple orthogonal methods in single study\",\n      \"pmids\": [\"28033404\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"H2B (canonical) and H2B.Z (variant) were both found associated with the PfSWR1 chromatin remodeling complex by reciprocal pulldown, indicating this complex is involved in deposition of the H2A.Z/H2B.Z variant histone pair; H2B itself (PF3D7_1105100) was co-detected in the complex but the primary substrate for deposition is the H2B.Z variant.\",\n      \"method\": \"Reciprocal co-immunoprecipitation / pulldown, mass spectrometry\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — H2B (canonical) detected in pulldown alongside variant H2B.Z; mechanistic role of canonical H2B in SWR1 complex not directly established\",\n      \"pmids\": [\"38564734\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PfNPM (nucleoplasmin isoform) interacts individually with assembled H2A/H2B with equimolar stoichiometry; the acidic tracts of PfNPM are necessary for this interaction; H3/H4 displays higher binding affinity for PfNPM than H2A/H2B, and H3/H4 can substitute H2A/H2B at the same binding site on PfNPM's distal face.\",\n      \"method\": \"Crystal structure of PfNPM, biochemical interaction assays (binding affinity measurements), mutagenesis of acidic tracts\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus binding assays plus mutagenesis establishing acidic tract requirement; multiple orthogonal methods in single study\",\n      \"pmids\": [\"40049416\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"P. falciparum H2B (PF3D7_1105100) is a canonical core histone that localizes to the parasite nucleus, is expressed in a developmentally regulated manner peaking in late trophozoite/schizont stages, forms nucleosomes with reduced salt stability and the shortest known repeat length (155 bp) compared to human nucleosomes, carries a distinct post-translational modification profile from the H2B.Z variant, and is bound by histone chaperones PfNapS and PfNPM (via their acidic tracts) with lower affinity than H3/H4.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"H2B (PF3D7_1105100) is a canonical core histone of Plasmodium falciparum that packages the parasite genome into nucleosomes and localizes to the parasite nucleus [#1, #8]. Its expression is developmentally regulated: H2B mRNA is undetectable in ring stages and rises through trophozoites and schizonts, with protein levels peaking dramatically in late trophozoite/schizont stages in a pattern consistent with replication-coupled nucleosome assembly [#2, #5]. Nucleosomes incorporating H2B show reduced salt stability relative to human nucleosomes and adopt the shortest known nucleosome repeat length (155 bp), a distinctive feature of parasite chromatin [#8]. H2B is deposited and handled by histone chaperones \\u2014 the nucleosome assembly proteins PfNapS/PfNAPS and the nucleoplasmin PfNPM bind assembled H2A/H2B, with PfNPM engagement requiring its acidic tracts, though both chaperones bind H2B with lower affinity than the H3/H4 pair [#4, #7, #10]. Canonical H2B carries a post-translational modification profile distinct from that of the H2B.Z variant, and both forms are co-detected with the PfSWR1 remodeling complex whose primary deposition substrate is the H2B.Z variant [#6, #9]. Beyond these chaperone interactions, chromatin properties, and expression profile, no direct functional or knockout phenotype for PF3D7_1105100 has been characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Establishing that P. falciparum encodes a canonical H2B histone at all was the necessary first step before any chromatin biology could be studied in the parasite.\",\n      \"evidence\": \"Gene cloning and sequencing of the P. falciparum H2B gene\",\n      \"pmids\": [\"7637710\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional assay beyond sequence identification\", \"Nucleosomal incorporation not shown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Purifying the protein and confirming sequence homology and nuclear localization established that the cloned gene produces a bona fide nuclear histone rather than merely an ORF.\",\n      \"evidence\": \"Protein purification, N-terminal sequencing and immunofluorescence in schizonts\",\n      \"pmids\": [\"9149412\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not test DNA binding directly\", \"Nucleosome context not established\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Mapping H2B transcript across the life cycle addressed whether the histone is constitutive or stage-restricted, revealing developmental regulation absent in rings.\",\n      \"evidence\": \"Northern blot / transcript analysis across developmental stages\",\n      \"pmids\": [\"10579431\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single lab, single transcript method with no protein-level confirmation\", \"Regulatory mechanism not defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Recombinant expression of the P. vivax ortholog tested whether the histone binds DNA and localizes to the nucleus, and modeling flagged divergence from host H2B at modification and DNA-contact sites.\",\n      \"evidence\": \"Recombinant expression, South-Western DNA binding, confocal microscopy and structural modeling in P. vivax\",\n      \"pmids\": [\"15276199\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Shown in P. vivax ortholog, not directly in P. falciparum PF3D7_1105100\", \"Functional consequence of divergent residues untested\"],\n      \"attribution\": [{\"species\": \"Plasmodium vivax\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Quantifying protein levels and confirming synthesis by MS resolved whether the stage-restricted transcription translates into protein accumulation, linking H2B expression to late-stage DNA replication.\",\n      \"evidence\": \"Real-time RT-PCR, immunoblotting and mass spectrometry across stages\",\n      \"pmids\": [\"16410041\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Correlation with replication is inferential, not causal\", \"Chromatin loading kinetics not measured\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Global MS mapping distinguished canonical H2B from the H2B.Z variant by their post-translational modification patterns, establishing that the two forms are chemically and likely functionally distinct.\",\n      \"evidence\": \"LC-MS/MS mapping of P. falciparum histone modifications\",\n      \"pmids\": [\"19351122\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of the distinct modification pattern not tested\", \"Enzymes writing/erasing the marks not identified\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Structural and spectroscopic characterization of PfNapS identified where core histones including H2B are recognized, defining the chaperone-histone interface distinct from the linker H1 site.\",\n      \"evidence\": \"Crystal structure of PfNapS plus fluorescence spectroscopy interaction mapping\",\n      \"pmids\": [\"20377878\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"H2B was one of several histones tested, not the focus\", \"Functional relevance of the interaction in vivo not shown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Reconstitution and mapping showed that H2A/H2B confer reduced salt stability and that parasite nucleosomes have an unusually short repeat length, defining a biophysically distinct chromatin state.\",\n      \"evidence\": \"In vitro nucleosome reconstitution, salt stability assays and in vivo nucleosome mapping\",\n      \"pmids\": [\"28033404\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Contribution of PF3D7_1105100 versus H2B.Z to instability not fully partitioned\", \"Consequences for gene regulation not established\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Reciprocal pulldowns placed both canonical H2B and H2B.Z with the PfSWR1 remodeler, addressing which chromatin machinery handles the histones while establishing the variant as the primary deposition substrate.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation and mass spectrometry\",\n      \"pmids\": [\"38564734\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Mechanistic role of canonical H2B in the SWR1 complex not established\", \"Co-detection may reflect nucleosomal association rather than a functional role\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Crystallography, binding assays and mutagenesis defined how the nucleoplasmin PfNPM engages assembled H2A/H2B, showing equimolar acidic-tract-dependent binding that is weaker than for H3/H4 and displaceable by H3/H4.\",\n      \"evidence\": \"Crystal structure of PfNPM, binding affinity measurements and acidic-tract mutagenesis\",\n      \"pmids\": [\"40049416\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo role of PfNPM-H2B interaction not demonstrated\", \"Whether this hand-off governs nucleosome assembly timing not shown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether PF3D7_1105100 is essential and what phenotype its loss produces remains unaddressed, as no genetic knockout, knockdown, or direct functional perturbation of the canonical H2B has been reported.\",\n      \"evidence\": \"No direct genetic or functional perturbation study in the corpus\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No knockout or conditional depletion\", \"No defined regulatory function separate from bulk chromatin packaging\", \"Role of its distinct modifications unresolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0046982\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [4, 7, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0000786\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006334\", \"supporting_discovery_ids\": [5, 8]},\n      {\"term_id\": \"GO:0006325\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"complexes\": [\n      \"Nucleosome (H2A/H2B core histone)\",\n      \"PfSWR1 chromatin remodeling complex (co-detected)\"\n    ],\n    \"partners\": [\n      \"PfNapS / PfNAPS (nucleosome assembly protein)\",\n      \"PfNAPL (nucleosome assembly protein)\",\n      \"PfNPM (nucleoplasmin isoform)\",\n      \"H2A (histone partner)\",\n      \"H2B.Z / H2Bv (variant histone, co-detected in PfSWR1)\"\n    ],\n    \"other_free_text\": [\n      \"Developmentally regulated expression: absent in rings, peaking in late trophozoites/schizonts and gametocytes [#2, #5]\",\n      \"Carries a post-translational modification profile distinct from the H2B.Z variant [#6]\",\n      \"H2A/H2B confers reduced nucleosome salt stability and parasite nucleosomes have the shortest known repeat length (155 bp) [#8]\",\n      \"PfNPM binding requires the chaperone's acidic tracts and is weaker for H2A/H2B than for H3/H4 [#10]\",\n      \"No genetic knockout or direct functional perturbation of PF3D7_1105100 reported in the corpus\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_1105100","name":null,"product":"histone H2B","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_0941800","name":null,"product":"histone H2B, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_0902700","name":null,"product":"histone H2B, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"H2B","evaluation":null}