bluuebunny/crossref_metadata_2025 huggingface dataset:
Supplementary Data from Characterization of KRAS Mutation Subtypes in Non–small Cell Lung Cancer
Authors: Judd Julia , Abdel Karim Nagla , Khan Hina , Naqash Abdul Rafeh , Baca Yasmine , Xiu Joanne , VanderWalde Ari M. , Mamdani Hirva , Raez Luis E. , Nagasaka Misako , Pai Sachin Gopalkrishna , Socinski Mark A. , Nieva Jorge J. , Kim Chul , Wozniak Antoinette J. , Ikpeazu Chukwuemeka , de Lima Lopes Gilberto , Spira Alexander I. , Korn W. Michael , Kim Edward S. , Liu Stephen V. , Borghaei Hossein
Published: June 2024

Data set used for analysis.
Figure S1 from Characterization of KRAS Mutation Subtypes in Non–small Cell Lung Cancer
Authors: Judd Julia , Abdel Karim Nagla , Khan Hina , Naqash Abdul Rafeh , Baca Yasmine , Xiu Joanne , VanderWalde Ari M. , Mamdani Hirva , Raez Luis E. , Nagasaka Misako , Pai Sachin Gopalkrishna , Socinski Mark A. , Nieva Jorge J. , Kim Chul , Wozniak Antoinette J. , Ikpeazu Chukwuemeka , de Lima Lopes Gilberto , Spira Alexander I. , Korn W. Michael , Kim Edward S. , Liu Stephen V. , Borghaei Hossein
Published: June 2024

Supplemental Figure 1. Key Biomarkers for KRAS mutation subtypes in adenocarcinoma and squamous cell NSCLC histologies. Frequency of the 10 most common co-occurring mutations including TP53, STK11, ATM, KEAP1, CDKN2A, U2AF1, BRAF, NFE3L2, RAF1 and EGFR are displayed for each KRAS mutation subtype in adenocarcinoma and squamous cell NSCLC cohorts.
Data from Characterization of KRAS Mutation Subtypes in Non–small Cell Lung Cancer
Authors: Judd Julia , Abdel Karim Nagla , Khan Hina , Naqash Abdul Rafeh , Baca Yasmine , Xiu Joanne , VanderWalde Ari M. , Mamdani Hirva , Raez Luis E. , Nagasaka Misako , Pai Sachin Gopalkrishna , Socinski Mark A. , Nieva Jorge J. , Kim Chul , Wozniak Antoinette J. , Ikpeazu Chukwuemeka , de Lima Lopes Gilberto , Spira Alexander I. , Korn W. Michael , Kim Edward S. , Liu Stephen V. , Borghaei Hossein
Published: June 2024

Abstract
KRAS is the most commonly mutated oncogene in NSCLC and development of direct KRAS inhibitors has renewed interest in this molecular variant. Different KRAS mutations may represent a unique biologic context with different prognostic and therapeutic impact. We sought to characterize genomic landscapes of advanced, KRAS-mutated non–small cell lung cancer (NSCLC) in a large national cohort to help guide future therapeutic development.

Molecular profiles of 17,095 NSCLC specimens were obtained using DNA next-generation sequencing of 592 genes (Caris Life Sciences) and classified on the basis of presence and subtype of KRAS mutations. Co-occurring genomic alterations, tumor mutational burden (TMB), and PD-L1 expression [22C3, tumor proportion score (TPS) score] were analyzed by KRAS mutation type.

Across the cohort, 4,706 (27.5%) samples harbored a KRAS mutation. The most common subtype was G12C (40%), followed by G12V (19%) and G12D (15%). The prevalence of KRAS mutations was 37.2% among adenocarcinomas and 4.4% in squamous cell carcinomas. Rates of high TMB (≥10 mutations/Mb) and PD-L1 expression varied across KRAS mutation subtypes. KRAS G12C was the most likely to be PD-L1 positive (65.5% TPS ≥ 1%) and PD-L1 high (41.3% TPS ≥ 50%). STK11 was mutated in 8.6% of KRAS wild-type NSCLC but more frequent in KRAS-mutant NSCLC, with the highest rate in G13 (36.2%). TP53 mutations were more frequent in KRAS wild-type NSCLC (73.6%).

KRAS mutation subtypes have different co-occurring mutations and a distinct genomic landscape. The clinical relevance of these differences in the context of specific therapeutic interventions warrants investigation.
Table S6 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Table S6. TAAgs regulated by proteasome activation or inhibition. Shown is the peptide sequence, gene accession number, mutation or PTM fold change in that peptide antigen following treatment with either tubastatin-A, ACY-738, or bortezomib. Green indicates upregulation and red indicates downregulation. Numbers indicate the fold-change relative to untreated RPMI-8226 cells. Arrows indicate direction of change for peptide sequences that were not detected in untreated cells. ND (blue) indicates that the peptide was not detected under the indicated condition. Blue asterisks indicate that the antigen was not detected in untreated cells and unmasked after treatment with the HDAC6 inhibitor.
Table S5 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Table S5. NeoAgs regulated by proteasome activation or inhibition. Shown is the peptide sequence, gene accession number, mutation or PTM fold change in that peptide antigen following treatment with either tubastatin-A, ACY-738, or bortezomib. Green indicates upregulation and red indicates downregulation. Numbers indicate the fold-change relative to untreated RPMI-8226 cells. Arrows indicate the direction of change for peptide sequences that were not detected in untreated cells. ND (blue) indicates that the peptide was not detected under the indicated condition. Blue asterisks indicate that the antigen was not detected in untreated cells and unmasked after treatment with the HDAC6 inhibitor.
Table S4 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Table S4. Proteasomal Fluorogenic Peptide Substrates. Shown are the fluorogenic substrates, catalytic site preferences and excitation and emission values used for detection of substrate hydrolysis.
Table S2 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Table S2. Top inhibitors of proteasomal ChT-like activity. Shown are the top pharmacologics that decreased proteasome ChT-like in the HTS.
Table S1 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Table S1. Top activators of proteasomal ChT-Like activity. Shown are the top pharmacologics that increased proteasome ChT-like in the HTS.
Figure S7 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Fig. S7. E.G7-Ova system to quantitate the SIINFEKL-MHC class I complex.
Figure S6 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Fig. S6. Dose-dependent effect of ACY-1215 and tubastatin-A on the biochemical activity of the HDACs and SIRTs.
Figure S5 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Fig. S5. Effect of top pharmacologics on a. inhibition of proteasome ChT-like activity in MM cells and b. MM cell viability.
Figure S4 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Fig. S4. a. Effect of hits from the HTS on proteasome ChT-like activity on ARH-77 and U266 MMCLs. Proteasome ChT-like activity was determined with 50,000 ARHG-77 or U266 cells/well after 24 h of incubation with 50 uM LLVY-R110. b. Effect of proteasome inhibitors on proteasome ChT-like activity in three different MMCLs. Bortezomib, carfilzomib and ixazomib were added to MM cells at indicated concentrations. Proteasome ChT-like activity was then determined after 24 h incubation.
Figure S20 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Fig. S20. SDS-PAGE of HDAC6 KO cells. MM cells were either untreated or treated with sgRNA scrambled control or sgRNA specific to HDAC6. Following selection, cell lysates were prepared and electrophoresed on SDS gels, transferred to PVDF. Membranes were probed for HDAC6 using a knockout verified antibody (catalog number ab133493, Abcam, Waltham, MA) a dilution of 1:10,000.
Figure S2 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Fig. S2. a. Correlation of proteasome ChT-like activity with cell number. Proteasome ChT-like activity was determined with indicated number of RPMI-8226 cells at 24 hrs. b. Correlation of proteasome ChT-like activity with incubation time. Proteasome ChT-like activity was determined with 50,000 RPMI-8226 cells/well at indicated time points.
c. Correlation of proteasome ChT-like activity with LLVY-R110 concentration. Proteasome ChT-like activity was determined with 50,000 RPMI-8226 cells/well after 24 h of incubation. d. Effect of DMSO (%) on proteasome activity in three MMCLs.
Figure S19 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Fig. S19. Graphical representation to depict the effect of HDAC6 inhibitors on proteasome activity. HDAC6 inhibitors release HR23B which is bound to the HDAC6 BUZ domain. Free HR23B binds and shuttles ubiquitinated cargo proteins to the proteasome. Rad23 binds the proteasome through a UbL (ubiquitin-like) domain and contains UBA (ubiquitin-associated) motifs that bind multi-ubiquitin chains. These domains allow Rad23 to function as a substrate shuttle-factor. Shown is the association of HR23B with the 26S proteasome through interaction with the non-ATPase regulatory subunit 14, also known as Rpn11. The ability of HDAC6 to downregulate HR23B occurs independently of its deacetylase activity.
Figure S18 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Fig. S18. Correlation of the effect of HDAC6 inhibitors on proteasome activity with the effect on MHC-I antigen presentation. a.) Effect of tubastatin-A on proteasome activity correlated with the effect on pan HLA-ABC antigen presentation. b.) Effect of ACY-738 on proteasome activity correlation with the effect on pan HLA-ABC antigen presentation c.) Effect of ACY-1215 on proteasome activity correlated with the effect on pan HLA-ABC antigen presentation.
Figure S17 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Fig. S17. Effect of HDAC6 inhibitors on the expansion of MM spheroids cultured alone or with T-cells. MM patient CD138+ cells (50,000/ sample) were cultured in 50 μL of Matrigel (Corning) in 96-well plates according to the manufacturer’s protocol. Spheroids were allowed to form and maintained at 37 °C. Cells were then treated with HDAC6 inhibitors (1 uM) for 24 h followed by co-culture with T-cells (E:T 2;1) for another 24 h. Values represent the average of triplicate measurements.
Figure S16 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Fig. S16. Effect of anti-HLA antibody on the generation of apoptotic MM cells after treatment with HDCA6 inhibitors and co-culture with autologous T-cells. MM patient CD138+ cells (20,000/ sample) were treated with each HDAC6 inhibitor (1 uM) for 24 h followed by incubated with 0.5 ug/mL mouse anti-HLA ABC antibody W6/32 (catalog number 14-9983-82, Fisher Scientific, Pittsburgh, PA) followed by co-culture with T-cells (E:T 2:1). The percent of apoptotic cells was quantitated by flow cytometry. Values represent the average of triplicate measurements. Error bars represent the SD of the mean.
Figure S15 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Fig. S15. Effect of HDAC6 inhibitors on a. aggresome formation and b. autophagosome formation. RPMI8226 cells were treated with bortezomib and HDAC6 at the indicated concentrations for 16 h. Aggresomes were quantitated by flow cytometry using the cell-based Proteostat® aggresome detection kit (Enzo Life Sciences, Farmingdale, NY). The kit utilizes a molecular rotor dye which while in solution is prevented from fluorescing by free intramolecular rotation along a single central bond. Specifically intercalation of the dye into the cross-β spine of quaternary protein structures typically found in misfolded and aggregated proteins, inhibits the dye’s rotation and leads to a strong fluorescence. b. Autophagosome formation. RPMI8226 cells were treated with bortezomib and HDAC6 at the indicated concentrations for 16 h. Autophagosomes were quantitated by flow cytometry using the Cyto-ID autophagosome detection kit which measures autophagic vacuoles and monitors autophagic flux in lysosomally inhibited live cells using a novel dye that selectively labels accumulated autophagic vacuoles (Enzo Life Sciences). The 488nm-excitable green dye allows for minimal staining of lysosomes while exhibiting bright fluorescence upon incorporation into pre-autophagosomes, autophagosomes, and autolysosomes (autophagolysosomes). All assays were performed in triplicate. Error bars represent the SD of the mean.
Figure S12 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Fig. S12. a. Effect of the top pharmacologics alone on E.G7-Ova cell viability as measured by LDH release into the culture medium (green). Also shown is the effect of pre-treating the B3Z cells with the pharmacologics then co-culturing with E.G7-Ova cells (red). b. Effect of pre-
treatment of B3Z cells with pharmacologics followed by co-culture with E.G7-Ova cells. Cell viability was measured by annexin-V+ staining as above. c. Effect of HDAC6 inhibitors on E.G7-Ova viability. d. Effect of HDAC6 inhibitors on B3Z viability.
e. Effect of HDAC6 inhibitors on E.G7-Ova viability after co-culture with B3Z cells at E:T 2:1.
Figure S11 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Fig. S11. Effect of the SGC-UBD253N (catalog number SML-3542, Sigma-Aldrich/Millipore-Sigma, Burlington, MA) is a negative control) probe on SIINFEKL-H2Kb presentation on E.G7-Ova cells (a) and effect of SGC-UBD253N on pan HLA-ABC presentation on three different MM cells (b). SGC-UBD253N is a closely related negative control for SGC-UBD253, a chemical probe for the HDAC6 UBD. E.G7-Ova (a) and MM cells (b) were treated with SGC-UBD253N at indicated concentration for 72h and the relative cell surface of the SIINFEKL-H2Kb complex detected by flow cytometry. Cells were then stained with a monoclonal antibody to SIINFEKL-H2Kb and quantitated using a BD-LSRII sorter interfaced with FlowJo software as above. MMCLs were treated with SGC-UBD253N at the indicated concentrations for 72 h. Cells were then stained with an anti- HLA-ABC (W6/32) antibody and quantitated using a BD-LSRII sorter interfaced with FlowJo software as above. Values represent the average of triplicate measurements. Error bars represent the SD.
Figure S10 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Fig. S10. Effect of proteasome inhibitors on presentation of the SIINFEKL-MHC class I molecule complex. E.G7-Ova cells were treated with pharmacologics at the indicated concentration for 16 h. Cells were then stained with a monoclonal antibody to Ova 257-264 (SIINFEKL) peptide bound to H2Kb and quantitated using a BD-LSRII sorter interfaced with FlowJo software as above.
Figure S1 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Fig. S1. Conversion of the constitutive proteasome to immunoproteasome by IFN-γ-induced subunit substitution.
Figure 13 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Fig. S13. MS workflow used to identify MHC class I peptides increased by treatment with HDAC6 inhibitors or bortezomib.
FIGURE 7 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Pan-cancer cell effect of HDAC6 inhibitors on proteasomes and MHC class I antigen presentation. A, Effect of HDAC6 inhibitors on proteasome activity in cancer cell lines. Cells were treated with tubastatin-A, ACY-738 and ACY-1215 at 1 µmol/L concentration for 72 hours. Posttreatment cells were incubated for 21 hours with proteasome substrate LLVY-R110 and substrate cleavage was monitored as above. Cell lines were ranked on the basis of their effect on proteasome activity. B, Effect of HDAC6 inhibitors on pan MHC-I (HLA-ABC) in a panel of cancer cell lines. Cells were treated with tubastatin-A, ACY-738, and ACY-1215 for 72 hours at 1 µmol/L final concentration. Posttreatment the cells were washed with PBS and stained with mouse anti-human HLA-ABC (BD Pharmingen, 55555) as per manufacturer's guidelines. Cells that stained positively with an anti-human HLA-ABC were analyzed on Attune Nxt and quantified using Flow Jo version 10.9.0. The cells are rank ordered according to their MHC-I presentation. Error bars represent MADP of the average. C, Effect of HDAC6 inhibitors on healthy human T cells. CD8+ T cells were cultured for 18 hours with HDAC6 inhibitors, PBS washed, stained, and analyzed by flow cytometry to quantitate annexin-V+/propidium iodide+ cells. Shown is the average of triplicate measurements. D, Effect of HDAC6 inhibitors on CD8+ T-cell cytotoxicity of tumor cells. OCI-AML3 (leukemia), OV81 (ovarian), BT549 (breast), DU145 (prostate), MiaPaCa-2 (pancreatic), and PC3 (prostate) cells (1 × 106 cells/treatment) were treated with tubastatin-A, ACY-738, or ACY-1215 for 48 hours (1 µmol/L). Cells were washed and cultured alone or with healthy human T cells (E:T 2.5:1) for 24 hours. Cells were PBS washed, stained, and analyzed by flow cytometry to quantitate CD8neg/annexin-V+/propidium iodide+ cells. Shown is the average of triplicate measurements. A P-value ≤ 0.05 is flagged with one star (*) and ns indicates a difference that was not significant.
FIGURE 4 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Effect of HDAC6 inhibitors on the myeloma immunopeptidome. A, Number of peptides eluted from human MHC-I molecules at each indicated peptide lengths (range = 6 to 15 amino acids). RPMI-8226 cells were treated with tubastatin-A, ACY-738 or bortezomib. B, Number of peptides and their fold change after treatment of RPMI-8226 cells with tubastatin-A, ACY-738, or bortezomib. C, The terminal amino acids of the top 50 peptides upregulated by tubastatin-A or ACY-738 treatment were analyzed. Shown is the relative percent of each terminal amino acid for the top 50 peptides upregulated by HDAC6 inhibitor treatment. D, Shown is the number of peptides reduced by treatment of E.G7-Ova cells with bortezomib and the fold decrease relative to untreated cells. E, Shown is the peptide sequence, fold decrease, and gene accession for the 20 peptides most downregulated by bortezomib treatment. Red asterisks indicate peptides upregulated by treatment with both HDAC6 inhibitors. F, The terminal amino acids of the top 50 peptides upregulated by tubastatin-A or ACY-738 treatment were analyzed. Shown is the relative percent of each terminal amino acid for the top 50 peptides upregulated following treatment with each HDAC6 inhibitor. G, Shown are gene accession numbers for the top 20 peptides most downregulated by bortezomib treatment. H, Shown are gene accession numbers for the top 20 peptides most upregulated by either tubastatin-A or ACY-738. Red asterisks indicate genes and peptides that were upregulated following treatment with both of the HDAC6 inhibitors.
FIGURE 2 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Effect of HDAC6 inhibitors on SIINFEKL presentation. A, Effect of HDAC6 inhibitors on SIINFEKL presentation. EL.4 lymphoma cells were transfected with the plasmid pAc-neo-Ova to generate the cell line E.G7-Ova. pAc-neo-Ova carries a complete copy of chicken OVA mRNA and the neomycin (G418) resistance gene. Proteasome degradation of Ova generates the peptide SIINFEKL that is then presented in a complex with the MHC-I molecule on E.G7-Ova cells. Shown is the relative effect of each HDAC6 inhibitor on the amount of SIINFEKL-H-2Kb molecule presented on the surface of treated cells normalized to untreated E.G7-Ova cells. Cells were also treated with bortezomib which reduced SIINFEKL-H-2Kb presentation and demonstrated that SIINFEKL presentation was proteasome dependent. B, Effect of HDAC6 inhibitors on LDH release. E.G7-Ova cells were treated with each agent at 3 µmol/L for 72 hours. Vehicle indicates E.G7-Ova cells treated with media alone. Positive control indicates E.G7-Ova cells treated with bortezomib at 10 nmol/L for 16 hours. After drug treatment, E.G7-Ova cells were incubated with B3Z cells at an E:T ratio of 2:1. Shown is the relative percent of LDH detected in culture media after 24 hours. C, Effect of HDAC6 inhibitors on CTL-mediated tumor lysis. E.G7-Ova cells were treated with each HDAC6 inhibitor at 1 µmol/L for 72 hours. Cells were also treated with bortezomib (10 nmol/L) for 16 hours. Untreated indicates E.G7-Ova cells treated with media alone. Cells were then washed, fresh media added and cells then cocultured with B3Z cells (E:T 2:1). CD8neg/annexin-V+/propidium iodide+ cells were then quantitated by flow cytometry. D, Effect of the HDAC6-BUZ domain specific inhibitor SGC-UBD253 on SIINFEKL-H-2Kb presentation on E.G7-Ova cells. Cells were treated with SGC-UBD253 at indicated concentrations and the level of SIINFEKL-H-2Kb quantitated by flow cytometry. All bioassays were performed in triplicate. Shown are averages of multiple experiments. Error bars represent the relative SD of the mean. E, Model of the HDAC6 domain structure and function and inhibitor binding sites. A P-value ≤ 0.05 is flagged with one star (*) and P-value ≤ 0.01 is flagged with two stars (**).
FIGURE 1 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity
Authors: Rana Priyanka S. , Ignatz-Hoover James J. , Kim Byung-Gyu , Malek Ehsan , Federov Yuriy , Adams Drew , Chan Timothy , Driscoll James J.
Published: June 2024

Cell-based HTS to identify pharmacologics that increased proteasome ChT-like activity. A, HTS scheme using the cell-permeable substrate LLVY-R110 to measure proteasome activity. LLVY-R110 is specifically cleaved by the proteasome to release the fluorogenic substrate R110 which fluoresces at an excitation of 490 nmol/L and emission 525 nmol/L. RPMI-8226 cells were incubated under standard culture conditions in 96-well plates in complete RPMI media that lacked phenol red. Pharmacologics (3,400) were then individually injected into each well at a final concentration of 3 µmol/L. Cells were incubated with pharmacologics for 21 hours. LLVY-R110 at a final concentration of 100 µmol/L was then added and incubated with cells for 18 hours under the same conditions. B, Rank-order effect of the top pharmacologics detected in the HTS that increased proteasome ChT-like activity. RPMI-8226 cells were incubated in 96-well plates with each pharmacologic at 1 µmol/L for 72 hours. Bioassays were performed in triplicate. Shown is the arithmetic average of triplicate measurements. Error bars indicate the SD of the mean. C, Effect of the top pharmacologics identified in the HTS on cell viability. Pharmacologics were added at 1 µmol/L for 16 hours and cell viability determined using the XTT assay (53). Shown is the arithmetic average of proteasome hydrolysis of LLVY-R110. D, Plot of the effect of pharmacologics on proteasome activity versus the effect on cell viability. Pharmacologics were prioritized based upon arbitrary cutoffs of >30% increase in proteasome activity and 75% cell viability. E, Chemical structure of clinically relevant HDAC6 inhibitors. F, Effect of HDAC6 inhibitors on proteasome ChT-like activity in MMCLs. Cells were incubated in 96-well plates with pharmacologics for 72 hours. Lysates were prepared in CelLyticM (Sigma) containing 20 mmol/L Tris-HCl (pH 7.6), 20 mmol/L NaCl, 10% glycerol, 3 mmol/L ATP, 5 mmol/L MgCl2, and 1 mmol/L DTT. Shown is the arithmetic average of triplicate measurements. Error bars indicate the SD of the mean. G, Effect of HDAC6 inhibitors on individual proteasome peptide-hydrolyzing activities. RPMI-8226 cells were incubated with HDAC6 inhibitors at 1 µmol/L for 72 hours, pelleted, washed thrice in cold PBS and lysed in CelLyticM containing 20 mmol/L Tris-HCl (pH 7.6), 20 mmol/L NaCl, 10% glycerol, 3 mmol/L ATP, 5 mmol/L MgCl2, and 1 mmol/L DTT. Fluorogenic peptide substrates were incubated with 5 µg of lysate for 1 hour at 37°C. All assays were performed in triplicate. Error bars indicate the relative SD.
Epidemiology and Outcomes of Neonatal Sepsis: Experience from a Tertiary Australian NICU
Authors: Mackay Cheryl Anne , Nathan Elizabeth A. , Porter Michelle Claire , Shrestha Damber , Kohan Rolland , Strunk Tobias
Published: June 2024

Introduction: Neonatal sepsis is associated with significant mortality and morbidity. Low-middle-income countries are disproportionately affected, but late-onset sepsis (LOS) still occurs in up to 20% of infants <28 weeks in high-income countries. Understanding site-specific data is vital to guide management. Methods: A retrospective cohort study was conducted at King Edward Memorial Hospital (KEMH), Perth. Infants admitted between January 2012 and June 2022 were included. Data were extracted from routine electronic databases. Incidence and aetiology of sepsis were determined and the association of sepsis with neonatal outcomes analysed. Results: During the study period, 23,395 newborns were admitted with a median gestation of 37 weeks and birth weight of 2,800 g. There were 370 sepsis episodes in 350 infants; 102 were early-onset sepsis (EOS) (1.6 per 1,000 live births), predominantly Streptococcus agalactiae (35, 34.3%) and Escherichia coli (27, 26.5%); 268 were LOS (0.9 per 1,000 inpatient days), predominantly coagulase-negative staphylococci (CONS) (156, 57.6%) and E. coli (30, 11.1%). The incidence of LOS declined from 2012 to 2022 (p = 0.002). Infants with EOS had increased brain injury (25.7% vs. 4.1%; p = 0.002) and mortality (18.8% vs. 1.6%; p < 0.001). Those with LOS had increased hospital stay (median 95 vs. 15 days; p < 0.001), mortality (15.3% vs. 1.6%; p = 0.018), necrotising enterocolitis (NEC) (7.4% vs. 0.5%; p < 0.001), and chronic lung disease (CLD) (58.1% vs. 5.9%; p = 0.005). Infants <28 weeks with sepsis were at increased risk of neurodevelopmental impairment compared to those without infection (43.2% vs. 30.9%, p = 0.027). Conclusions: While we observed a reduction in LOS incidence, sepsis remains associated with higher mortality, and in survivors with longer hospital stay and increased risk of brain injury, NEC, CLD, and neurodevelopmental impairment.
Air or Oxygen for Infant Resuscitation: A Prospective Cohort Study of Moderate-Late Preterm Infants Requiring Delivery Room Resuscitation
Authors: Sotiropoulos James X. , Binoy Sheeba , Pham Thy A.N. , Yates Kylie , Allgood Catherine L. , Kunjunju Ansar , Tracy Mark , Smyth John , Oei Ju Lee
Published: June 2024

Introduction: Due to concerns of oxidative stress and injury, most clinicians currently use lower levels of fractional inspired oxygen (FiO2, 0.21–0.3) to initiate respiratory support for moderate to late preterm (MLPT, 32–36 weeks gestation) infants at birth. Whether this practice achieves recommended oxygen saturation (SpO2) targets is unknown. Methods: We aimed to determine SpO2 trajectories of MLPT infants requiring respiratory support at birth. We conducted a prospective, opportunistic, observational study with consent waiver. Preductal SpO2 readings were obtained during the first 10 min of life from infants between 32 and 36 weeks gestation requiring respiratory support in the delivery room. Primary outcome was reaching a minimum SpO2 80% at 5 min of life. The study was prospectively registered (ACTRN12620001252909). Results: A total of 76 eligible infants were recruited between February 2021 and March 2022 from 5 hospitals in Australia. Most (n = 58, 76%) had respiratory support initiated with FiO2 0.21 (range 0.21–1.0) using CPAP (92%). Median SpO2 at 5 min was 81% (interquartile range [IQR] 67–90) and 93% (IQR 86–96) at 10 min. At 5 min, 18/43 (42%) infants had SpO2 below 80% and only 8/43 (19%) reached SpO2 80–85%. Conclusions: Many MLPT infants requiring respiratory support do not achieve recommended SpO2 targets. In very preterm infants, SpO2 <80% at 5 min of life increases risk of death, intraventricular haemorrhage, and neurodevelopmental impairment. The implications on this practice on the health outcomes of MLPT infants are unclear and require further research.
Late-Onset Sepsis among Extremely Preterm Infants of 24–28 Weeks Gestation: An International Comparison in 10 High-Income Countries
Authors: Klinger Gil , Reichman Brian , Norman Mikael , Kusuda Satoshi , Battin Malcolm , Helenius Kjell , Isayama Tetsuya , Lui Kei , Adams Mark , Vento Maximo , Hakansson Stellan , Beltempo Marc , Poggi Chiara , San Feliciano Laura , Lehtonen Liisa , Bassler Dirk , Yang Junmin , Shah Prakesh S. ,   on behalf of the International Network for Evaluating Outcomes of Neonates (iNeo)
Published: June 2024

Introduction: Despite advances in neonatal care, late-onset sepsis remains an important cause of preventable morbidity and mortality. Neonatal late-onset sepsis rates have decreased in some countries, while in others they have not. Our objective was to compare trends in late-onset sepsis rates in 9 population-based networks from 10 countries and to assess the associated mortality within 7 days of late-onset sepsis. Methods: We performed a retrospective population-based cohort study. Infants born at 24–28 weeks’ gestation between 2007 and 2019 were eligible for inclusion. Late-onset sepsis was defined as a positive blood or cerebrospinal fluid culture. Late-onset sepsis rates were calculated for 3 epochs (2007–11, 2012–15, and 2016–19). Adjusted risk ratios (aRRs) for late-onset sepsis were calculated for each network. Results: Of a total of 82,850 infants, 16,914 (20.4%) had late-onset sepsis, with Japan having the lowest rate (7.1%) and Spain the highest (44.6%). Late-onset sepsis rates decreased in most networks and remained unchanged in a few. Israel, Sweden, and Finland showed the largest decrease in late-onset sepsis rates. The aRRs for late-onset sepsis showed wide variations between networks. The rate of mortality temporally related to late-onset sepsis was 10.9%. The adjusted mean length of stay for infants with late-onset sepsis was increased by 5–18 days compared to infants with no late-onset sepsis. Conclusions: One in 5 neonates of 24–28 weeks’ gestation develops late-onset sepsis. Wide variability in late-onset sepsis rates exists between networks with most networks exhibiting improvement. Late-onset sepsis was associated with increased mortality and length of stay.
Kratom (Mitragyna speciosa) Use and Mental Health: A Systematic Review and Multilevel Meta-Analysis
Authors: Yang Yuting , Müller Christian P. , Singh Darshan
Published: June 2024

Introduction: Kratom (Mitragyna speciosa) is a medicinal tree native to Southeast Asia. The present multilevel meta-analysis describes the association between kratom use and the positive and negative indicators of mental health. Methods: A total of thirty-six articles were included in the meta-analysis to examine the associations, using a random-effects model. Results: The pooled effect size showed a very small positive association between kratom use and negative indicators of mental health {r = 0.092, 95% confidence interval (CI) = [0.020, 0.164], p < 0.05}, while no significant association was found with positive indicators of mental health (r = −0.031, 95% CI = [−0.149, 0.087], p > 0.05). Pooled effect sizes of specific mental health outcomes indicated that kratom use showed only a small positive correlation with externalizing disorders (r = 0.201, 95% CI = [0.107, 0.300], p < 0.001). No significant association was found between kratom use and quality of life (r = 0.069, 95% CI = [−0.104, 0.242], p > 0.05) and internalizing disorders (r = −0.001, 95% CI = [−0.115, 0.095], p > 0.05). Multilevel moderator analysis showed that the pooled effect size of the association between kratom use and substance use disorder was stronger in Malaysia (r = 0.347, 95% CI = [0.209, 0.516], p < 0.001), and with the mean age (β1 = −0.035, 95% CI = [−0.055, −0.014], p = 0.003), and the drug profile of those who were not co-using other drugs (r = 0.347, 95% CI = [0.209, 0.516], p < 0.001). Conclusion: The meta-analysis supports the kratom instrumentalization concept, in that a positive gain from kratom consumption can be achieved without any significant adverse associations with mental health.
Trichoscopy of Frontal Fibrosing Alopecia in Caucasians: A Review
Authors: Quadrelli Federico , Cedirian Stephano , Piraccini Bianca Maria
Published: June 2024

Background: Frontal fibrosing alopecia (FFA) is a scarring hair loss condition primarily affecting the frontotemporal hairline and often leading to partial or complete loss of eyebrows in a significant number of cases. It is characterized by slow progression and typically shows poor response to available treatments. Diagnosis relies on clinical, trichoscopic, and histopathological assessments, with specific criteria proposed by Vañó‐Galván et al. in 2018 [J Am Acad Dermatol. 2018;78(1):e21‐2]. Summary: This literature review examines trichoscopic observations of FFA in Caucasians, focusing on both scalp and eyebrow manifestations. A comprehensive search of the PubMed database from 2010 to 2023 yielded 6 relevant articles. Trichoscopic findings varied across studies but highlighted common features such as empty follicles, perifollicular erythema, follicular hyperkeratosis, and the presence of “lonely hair” on the scalp. Additional research is warranted to thoroughly characterize demographic traits and FFA peculiarities within the Caucasian population, given the limited existing data in the literature. Key Messages: Distinct trichoscopic features: trichoscopic examination of FFA among Caucasians reveals specific markers such as empty follicles, perifollicular erythema, and follicular hyperkeratosis, aiding in diagnosis and monitoring. Limited literature data: more investigation is needed to fully understand the demographic characteristics and FFA peculiarities among Caucasians, considering the scarcity of available data in existing literature.
Case report - Rabies in a dog, São Paulo city, 2023
Authors: Brandão Paulo Eduardo , Agostinho Washington Carlos , Silva Sheila Oliveira de Souza , Miyagi Sueli Akemi Taniwaki , Mori Enio , Oliveira Rafael de Novaes , Barone Gisely Toledo , Conselheiro Juliana Amorim
Published: June 2024

This publication reports on a rabid dog in São Paulo city, Brazil, with the AgV3 variant of rabies virus related to vampire and fruit bats, which is of significant importance to the surveillance of rabies in a region where the typical canine AgV2 rabies virus has been eradicated.
Procedure Volume and Outcomes With WATCHMAN Left Atrial Appendage Occlusion
Authors: Friedman Daniel J. , Du Chengan , Zimmerman Sarah , Tan Zhen , Lin Zhenqiu , Vemulapalli Sreekanth , Kosinski Andrzej S. , Piccini Jonathan P. , Pereira Lucy , Minges Karl E. , Faridi Kamil F. , Masoudi Frederick A. , Curtis Jeptha P. , Freeman James V.
Published: June 2024

BACKGROUND:

Procedure volumes are associated with outcomes for many cardiovascular procedures, leading to guidelines on minimum volume thresholds for certain procedures; however, the volume-outcome relationship with left atrial appendage occlusion is poorly understood. As such, we sought to determine the relationship between hospital and physician volume and WATCHMAN left atrial appendage occlusion procedural success overall and with the new generation WATCHMAN FLX device.

METHODS:

We performed an analysis of WATCHMAN procedures (January 2019 to October 2021) from the National Cardiovascular Data Registry LAAO Registry. Three-level hierarchical generalized linear models were used to assess the adjusted relationship between procedure volume and procedural success (device released with peridevice leak <5 mm, no in-hospital major adverse events).

RESULTS:

Among 87 480 patients (76.2±8.0 years; 58.8% men; mean CHA
2
DS
2
-VASc score, 4.8±1.5) from 693 hospitals, the procedural success rate was 94.2%. With hospital volume Q4 (greatest volume) as the reference, the likelihood of procedural success was significantly less among Q1 (odds ratio [OR], 0.66 [CI, 0.57–0.77]) and Q2 (OR, 0.78 [CI, 0.69–0.90]) but not Q3 (OR, 0.95 [CI, 0.84–1.07]). With physician volume Q4 (greatest volume) as the reference, the likelihood of procedural success was significantly less among Q1 (OR, 0.72 [CI, 0.63–0.82]), Q2 (OR, 0.79 [CI, 0.71–0.89]), and Q3 (OR, 0.88 [CI, 0.79–0.97]). Among WATCHMAN FLX procedures, there was attenuation of the volume-outcome relationships, with statistically significant but modest absolute differences of only ≈1% across volume quartiles.

CONCLUSIONS:

In this contemporary national analysis, greater hospital and physician WATCHMAN volumes were associated with increased procedure success. The WATCHMAN FLX transition was associated with increased procedural success and less heterogeneity in outcomes across volume quartiles. These findings indicate the importance of understanding the volume-outcome relationship for individual left atrial appendage occlusion devices.
Natural History of Coronary Atherosclerosis in Patients With Aortic Stenosis Undergoing Transcatheter Aortic Valve Replacement: The Role of Quantitative Flow Ratio
Authors: Colaiori Iginio , Paolucci Luca , Mangiacapra Fabio , Barbato Emanuele , Ussia Gian Paolo , Grigioni Francesco , Demola Pierluigi , Vitolo Marco , Benatti Giorgio , Vignali Luigi , Gabbieri Davide , Magnavacchi Paolo , Sgura Fabio Alfredo , Boriani Giuseppe , Guiducci Vincenzo
Published: June 2024

BACKGROUND:

The prognostic impact of functionally significant coronary artery disease, as assessed with quantitative flow ratio (QFR), in patients with severe aortic stenosis treated with transcatheter aortic valve replacement is unknown.

METHODS:

This is a retrospective study with blind analysis of angiographic data, enrolling consecutive patients with severe aortic stenosis treated with transcatheter aortic valve replacement at 4 Italian centers. None of the patients enrolled received pre-transcatheter aortic valve replacement or concomitant coronary revascularization, either for the absence of significant coronary stenoses or by clinical decision. Visual estimation of diameter stenosis and QFR analysis were performed in all coronary arteries. The end point was all-cause mortality at a 3-year follow-up.

RESULTS:

A total of 318 patients were enrolled. At visual estimation, 140 patients (44%) presented a diameter stenosis ≥50% in at least 1 coronary artery, whereas 78 patients (24.5%) had at least 1 vessel with QFR <0.80 and, therefore, included in the positive QFR group. Overall, 69 (21.7%) patients died during the follow-up. In the Kaplan-Meier analysis, patients with positive QFR experienced significantly higher rates of death during follow-up compared with those without (51.1% versus 12.1%;
P
<0.001), whereas no significant difference was evident in terms of death between patients with or without significant coronary artery disease according to angiographic evaluation (24.3% versus 19.7%;
P
=0.244). In a multivariate regression model, positive QFR was an independent predictor of all-cause death during follow-up (hazard ratio, 5.31 [95% CI, 3.21–8.76]).

CONCLUSIONS:

Coronary QFR can predict mortality in patients with severe aortic stenosis treated with transcatheter aortic valve replacement without revascularization.
Creating diverse and inclusive scientific practices for research datasets and dissemination
Authors: Kam Julia W.Y. , Badhwar AmanPreet , Borghesani Valentina , Lee Kangjoo , Noble Stephanie , Raamana Pradeep Reddy , Ratnanather J. Tilak , Tan Davynn G.H. , Oestreich Lena K.L. , Lee Hyang Woon , Marzetti Laura , Nakua Hajer , Rippon Gina , Olsen Rosanna , Pozzobon Alyssa , Uddin Lucina Q. , Yanes Julio Alejandro , Tzovara Athina
Published: June 2024

Diversity, equity, and inclusivity (DEI) are important for scientific innovation and progress. This widespread recognition has resulted in numerous initiatives for enhancing DEI in recent years. Although progress has been made to address gender and racial disparities, there remain biases that limit the opportunities for historically under-represented researchers to succeed in academia. As members of the Organization for Human Brain Mapping (OHBM) Diversity and Inclusivity Committee (DIC), we identified the most challenging and imminent obstacles toward improving DEI practices in the broader neuroimaging field. These obstacles include the lack of diversity in and accessibility to publicly available datasets, barriers in research dissemination, and/or barriers related to equitable career advancements. In order to increase diversity and promote equity and inclusivity in our scientific endeavors, we suggest potential solutions that are practical and actionable to overcome these barriers. We emphasize the importance of the enduring and unwavering commitment required to advance DEI initiatives consistently. By doing so, the OHBM and perhaps other neuroscience communities will strive toward a future that is not only marked by scientific excellence but also characterized by diverse, inclusive, and equitable opportunities for all, including historically under-represented individuals around the world.
Misperceived Social Norms and Willingness to Act Against Climate Change
Authors: Andre Peter , Boneva Teodora , Chopra Felix , Falk Armin
Published: June 2024

We document the individual willingness to act against climate change and study the role of social norms in a large sample of US adults. Individual beliefs about social norms positively predict pro-climate donations, comparable in strength to universal moral values and economic preferences. However, we document systematic misperceptions of social norms. Respondents vastly underestimate the prevalence of climate-friendly behaviors and norms. Correcting these misperceptions in an experiment causally raises individual willingness to act against climate change and individual support for climate policies. The effects are strongest for individuals who are skeptical about the existence and threat of global warming.