STAG2 mutations regulate 3D genome organization, chromatin loops, and Polycomb signaling in glioblastoma multiforme.

Inactivating mutations of genes encoding the cohesin complex are common in a wide range of human cancers. STAG2 is the most commonly mutated subunit. Here we report the impact of stable correction of endogenous, naturally occurring STAG2 mutations on gene expression, 3D genome organization, chromatin loops, and Polycomb signaling in glioblastoma multiforme (GBM). In two GBM cell lines, correction of their STAG2 mutations significantly altered the expression of ∼10% of all expressed genes. Virtually all the most highly regulated genes were negatively regulated by STAG2 (i.e., expressed higher in STAG2-mutant cells), and one of them-HEPH-was regulated by STAG2 in uncultured GBM tumors as well. While STAG2 correction had little effect on large-scale features of 3D genome organization (A/B compartments, TADs), STAG2 correction did alter thousands of individual chromatin loops, some of which controlled the expression of adjacent genes. Loops specific to STAG2-mutant cells, which were regulated by STAG1-containing cohesin complexes, were very large, supporting prior findings that STAG1-containing cohesin complexes have greater loop extrusion processivity than STAG2-containing cohesin complexes and suggesting that long loops may be a general feature of STAG2-mutant cancers. Finally, STAG2 mutation activated Polycomb activity leading to increased H3K27me3 marks, identifying Polycomb signaling as a potential target for therapeutic intervention in STAG2-mutant GBM tumors. Together, these findings illuminate the landscape of STAG2-regulated genes, A/B compartments, chromatin loops, and pathways in GBM, providing important clues into the largely still unknown mechanism of STAG2 tumor suppression.

Long-term oncologic outcome of D3 lymph node dissection for clinical stage 2/3 right-sided colon cancer.

PURPOSE: To investigate oncologic outcomes including overall survival and disease-free survival depending on the extent of lymphadenectomy (D3 versus D2) by comparing D3 and D2 lymphadenectomy in patients with clinical stage 2/3 right colon cancer. METHODS: Consecutive series of patients who underwent radical resection for right colon cancer at our three hospitals between January 2015 and June 2018 were retrospectively analyzed. Study cohorts were divided into two groups: D3 group and D2 group. Oncologic, pathologic, and perioperative outcomes of the two groups were compared. RESULTS: A total of 295 patients (167 in the D2 group and 128 in the D3 group) were included in this study. Patients' characteristics showed no significant difference between the two groups. The median number of harvested lymph nodes was significantly higher in the D3 group than in the D2 group. The rate of complications was not significantly different between the two groups except for chyle leakage, which was more frequent in the D3 group. Five-year disease-free survival was 90.2% (95% CI: 84.8-95.9%) in the D3 group, which was significantly (p = 0.028) higher than that (80.5%, 95% CI: 74-87.5%) in the D2 group. There was no significant difference in overall survival between the two groups. CONCLUSION: Our results indicate that D3 lymphadenectomy is associated with more favorable 5-year disease-free survival than D2 lymphadenectomy for patients with stage 2/3 right-sided colon cancer. D3 lymphadenectomy might improve oncologic outcomes in consideration of the recurrence rate.

The effect of cognitive behavioral therapy on depression of people with epilepsy.

BACKGROUND: Depression, a common mental problem frequently detected in people with epilepsy (PWE), is a major factor that decreases the quality of life of PWE. The cognitive behavioral therapy (CBT) is the most commonly used non-pharmacological treatment for depressive disorders. The CBT for PWE with depression has not yet been studied in Korea. This study aimed to evaluate the effects of the CBT on depression in PWE in Korea. METHOD: This study included 16 PWE with depression who received CBT and 30 control PWE with depression who did not receive CBT. The mean number of CBT sessions per patient was 7.2 in the CBT group. The Beck Depression Inventory-II (BDI-II) and Patient Health Questionnaire-9 (PHQ-9) were administered before and after CBT sessions in the CBT group, whereas PHQ-9 was performed at baseline and follow-up in the control group. The difference in PHQ-9 and BDI-II scores were analyzed between the pre- and post-CBT periods in the CBT group. The difference between baseline and follow-up PHQ-9 scores was compared in the control group. RESULTS: There was no significant difference in baseline variables between the CBT and control groups. The PHQ-9 score significantly decreased after the CBT sessions in the CBT group (pre-CBT PHQ-9 = 13.56 vs. post-CBT PHQ-9 = 8.56) but it did not change in the control group (Baseline PHQ-9 = 13.83 vs. follow-up PHQ-9 = 14.67). Twelve PWE had undergone four or more CBT sessions with pre-CBT and post-CBT BDI-II. The BDI-II score significantly decreased after CBT sessions (pre-CBT BDI-II = 30.75 vs. post-CBT BDI-II = 21.5). The CBT decreased the sub-field scores of cognitive and physical-emotional factors as well as suicidal ideation, but the score of sleep problems did not significantly improve. The CBT did not significantly change the seizure frequency. CONCLUSION: The CBT significantly improved depression in Korean PWE. Therefore, it can be considered a treatment tool for depression in PWE. However, a study with more patients and a fixed number of CBT sessions is recommended to generalize this effect.

USP13 interacts with cohesin and regulates its ubiquitination in human cells.

Cohesin is a multiprotein ring complex that regulates 3D genome organization, sister chromatid cohesion, gene expression, and DNA repair. Cohesin is known to be ubiquitinated, although the mechanism, regulation, and effects of cohesin ubiquitination remain poorly defined. We previously used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in human HCT116 cells. Here we report that mass spectrometry analysis of dual-affinity purifications identified the USP13 deubiquitinase as a novel cohesin-interacting protein. Subsequent immunoprecipitation/Western blots confirmed the endogenous interaction in HCT116, 293T, HeLa, and RPE-hTERT cells; demonstrated that the interaction occurs specifically in the soluble nuclear fraction (not in the chromatin); requires the ubiquitin-binding domains (UBA1/2) of USP13; and occurs preferentially during DNA replication. Reciprocal dual-affinity purification of endogenous USP13 followed by mass spectrometry demonstrated that cohesin is its primary interactor in the nucleus. Ectopic expression and CRISPR knockout of USP13 showed that USP13 is paradoxically required for both deubiquitination and ubiquitination of cohesin subunits in human cells. USP13 was dispensable for sister chromatid cohesion in HCT116 and HeLa cells, whereas it was required for the dissociation of cohesin from chromatin as cells transit through mitosis. Together these results identify USP13 as a new cohesin-interacting protein that regulates the ubiquitination of cohesin and its cell cycle regulated interaction with chromatin.

Systematic proteomics of endogenous human cohesin reveals an interaction with diverse splicing factors and RNA-binding proteins required for mitotic progression.

The cohesin complex regulates sister chromatid cohesion, chromosome organization, gene expression, and DNA repair. Cohesin is a ring complex composed of four core subunits and seven regulatory subunits. In an effort to comprehensively identify additional cohesin-interacting proteins, we used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in cultured human cells, and we performed MS analyses on dual-affinity purifications. In addition to reciprocally identifying all known components of cohesin, we found that cohesin interacts with a panoply of splicing factors and RNA-binding proteins (RBPs). These included diverse components of the U4/U6.U5 tri-small nuclear ribonucleoprotein complex and several splicing factors that are commonly mutated in cancer. The interaction between cohesin and splicing factors/RBPs was RNA- and DNA-independent, occurred in chromatin, was enhanced during mitosis, and required RAD21. Furthermore, cohesin-interacting splicing factors and RBPs followed the cohesin cycle and prophase pathway of cell cycle-regulated interactions with chromatin. Depletion of cohesin-interacting splicing factors and RBPs resulted in aberrant mitotic progression. These results provide a comprehensive view of the endogenous human cohesin interactome and identify splicing factors and RBPs as functionally significant cohesin-interacting proteins.

CD4+ /CD8+ T-cell ratio correlates with the graft fate in pig-to-non-human primate islet xenotransplantation.

BACKGROUND: Xenogeneic islet transplantation using porcine pancreata has been a promising option for substituting human islet transplantation. Moreover, recent advances in pre-clinical results have put islet xenotransplantation closer to the possibility of clinical application. While preparing for the era of clinical xenotransplantation, developing non-invasive immune monitoring method which could predict the graft fate could benefit the patient. However, there are few reports showing predictive immune parameters associated with the fate of the graft in islet xenotransplantation. METHODS: The absolute number and ratio of T-cell subsets have been measured via flow cytometry from the peripheral blood of 16 rhesus monkeys before and after porcine islet xenotransplantation. The correlation between the graft survival and the absolute number or ratio of T cells was retrospectively analyzed. RESULTS: The ratio of CD4+ versus CD8+ T cells was significantly reduced due to CD8+ effector memory cells' increase. Correlation analyses revealed that CD4+ /CD8+ , CD4+ /CD8+ naïve, CD4+ naïve/CD8+ naïve, and CD4+ central memory/CD8+ naïve cell ratios negatively correlated with the duration of graft survival. Conversely, further analyses discovered strong, positive correlation of CD4+ /CD8+ cell ratios within the early graft-rejected monkeys (≤60 days). CONCLUSIONS: This retrospective study demonstrated that CD4+ /CD8+ ratios correlated with graft survival, especially in recipients which rejected the graft in early post-transplantation periods. CD4+ /CD8+ ratios could be used as a surrogate marker to predict the graft fate in pig-to-NHP islet xenotransplantation.

Development of a Novel Multi-Channel Thermocouple Array Sensor for In-Situ Monitoring of Ice Accretion.

A test was performed to determine the efficacy of a novel multi-channel thermocouple temperature sensor employing "N+1" array architecture for the in-situ detection of icing in cold climates. T-type thermoelements were used to fabricate a sensor with six independent temperature sensing points, capable of two-dimensional temperature mapping. The sensor was intended to detect the high latent heat of fusion of water (334 J/g) which is released to the environment during ice formation. The sensor was embedded on a plywood board and an aluminium plate, respectively by an epoxy resin. Three different ice accretion cases were considered. Ice accretion for all cases was achieved on the surface of the resin layer. In order to analyse the temperature variation for all three cases, the first 20 s response for each case was averaged between three cases. A temperature increase of (1.0 ± 0.1) °C and (0.9 ± 0.1) °C was detected by the sensors 20 s after the onset of icing, attributed to the latent heat of fusion of water. The results indicate that the sensor design is well-suited to cold temperature applications and that detection of the latent heat of fusion could provide a rapid and robust means of icing detection.

Vascularization of PLGA-based bio-artificial beds by hypoxia-preconditioned mesenchymal stem cells for subcutaneous xenogeneic islet transplantation.

BACKGROUND: Subcutaneous tissue is an attractive extra-hepatic heterotopic site for islet transplantation; however, poor oxygen tension and blood supply during early engraftment of implanted islets have limited the use of this site in clinical applications. METHODS: This study investigated the vascularization potential of hypoxia-preconditioned mesenchymal stem cells (3% O2 ; hypo-MSCs) in PLGA-based bio-artificial beds for subsequent subcutaneous islet transplantation. Sheet-typed polymeric PLGA scaffolds coated with hypo-MSCs or normo-MSCs (MSCs cultured under normoxia conditions, 21% O2 ) were implanted subcutaneously in mice. RESULTS: Compared to normo-MSCs, hypo-MSCs significantly enhanced vasculogenesis, both on the interior and exterior surfaces of the implanted PLGA devices, which peaked 4 weeks after implantation. Further, infusion of porcine islets inside the prevascularized PLGA bed restored normal glycemic control in 6 of 6 STZ-induced diabetic mice. The mass of the marginal islet was approximately 2000 IEQs, which is comparable to that required for the renal subcapsular space, a highly vascularized site. CONCLUSIONS: Therefore, PLGA-based bio-artificial devices prevascularized with hypo-MSCs could be a useful modality for successful subcutaneous islet transplantation, which is of high clinical relevance.

Degradation of dimethyl phthalate using a liquid phase plasma process with TiO2 photocatalysts.

The liquid phase plasma (LPP) method with a TiO2 photocatalyst and hydrogen peroxide was used to decompose dimethyl phthalate (DMP). As the applied voltage, pulse width, and frequency were increased, the rate of decomposition was increased and the decomposition rate was 63% for 180 min under plasma optimum conditions. The addition of TiO2 photocatalyst and hydrogen peroxide increased the DMP decomposition reaction rate, but an excess cause a decrease in decomposition rate due to a decrease in conductivity, blocking of ultraviolet light, and scavenger effect. When the TiO2 photocatalyst and hydrogen peroxide were used together, the decomposition reaction rate of DMP was greatly improved by using LPP single process alone. Also, when all the processes were used at the same time, the decomposition reaction rate was improved to about 2.8 times. DMP undergoes bond cleavage and ultimately decomposes into CO2 and H2O via dimethyl 4-hydroxyphthalate and methyl salicylates due to hydroxyl radicals and various active species generated by the LPP reaction.

Preparation and Characterization of Bimetallic Fe-Ni Oxide Nanoparticles Using Liquid Phase Plasma Process.

The Fe-Ni oxide bimetallic nanoparticles (FNOBNPs) were synthesized in the liquid phase plasma (LPP) method employed an iron chloride and nickel chloride as metal precursors. The sphericalshaped FNOBNPs were synthesized by the LPP process and, the size of particles was growing along with the progression of LPP reaction. The synthesized FNOBNPs were comprised of Fe3O4 and NiO. Iron had a higher reduction potential than nickel and resulted in higher iron composition in the synthesized FNOBNPs. The control of molar ratio of metal precursors in initial reactant solution was found that it could be employed as a means to control the composition of the elements in FNOBNP.

Intact Cohesion, Anaphase, and Chromosome Segregation in Human Cells Harboring Tumor-Derived Mutations in STAG2.

Somatic mutations of the cohesin complex subunit STAG2 are present in diverse tumor types. We and others have shown that STAG2 inactivation can lead to loss of sister chromatid cohesion and alterations in chromosome copy number in experimental systems. However, studies of naturally occurring human tumors have demonstrated little, if any, correlation between STAG2 mutational status and aneuploidy, and have further shown that STAG2-deficient tumors are often euploid. In an effort to provide insight into these discrepancies, here we analyze the effect of tumor-derived STAG2 mutations on the protein composition of cohesin and the expected mitotic phenotypes of STAG2 mutation. We find that many mutant STAG2 proteins retain their ability to interact with cohesin; however, the presence of mutant STAG2 resulted in a reduction in the ability of regulatory subunits WAPL, PDS5A, and PDS5B to interact with the core cohesin ring. Using AAV-mediated gene targeting, we then introduced nine tumor-derived mutations into the endogenous allele of STAG2 in cultured human cells. While all nonsense mutations led to defects in sister chromatid cohesion and a subset induced anaphase defects, missense mutations behaved like wild-type in these assays. Furthermore, only one of nine tumor-derived mutations tested induced overt alterations in chromosome counts. These data indicate that not all tumor-derived STAG2 mutations confer defects in cohesion, chromosome segregation, and ploidy, suggesting that there are likely to be other functional effects of STAG2 inactivation in human cancer cells that are relevant to cancer pathogenesis.

Construction of EMSC-islet co-localizing composites for xenogeneic porcine islet transplantation.

Pancreatic islet transplantation is an ultimate solution for treating patients with type 1 diabetes (T1D). The pig is an ideal donor of islets for replacing scarce human islets. Besides immunological hurdles, non-immunological hurdles including fragmentation and delayed engraftment of porcine islets need solutions to succeed in porcine islet xenotransplantation. In this study, we suggest a simple but effective modality, a cell/islet co-localizing composite, to overcome these challenges. Endothelial-like mesenchymal stem cells (EMSCs), differentiated from bone-marrow derived mouse mesenchymal stem cells (MSCs), and MSCs evenly coated the surface of porcine islets (>85%) through optimized culture conditions. Both MSCs and EMSCs significantly reduced the fragmentation of porcine islets and increased the islet masses, designated as islet equivalents (IEQs). In fibrin in vitro and in vivo angiogenesis analysis, constructed EMSC-islet composites showed higher angiogenic potentials than naked islets, MSC-islet composites, or human endothelial cell-islet composites. This novel delivery method of porcine islets may have beneficial effects on the engraftment of transplanted islets by prevention of fragmentation and enhancement of revascularization.

Pre-clinical results in pig-to-non-human primate islet xenotransplantation using anti-CD40 antibody (2C10R4)-based immunosuppression.

BACKGROUND: Islet transplantation is an effective therapy for selected patients with type 1 diabetes with labile glycemic control and hypoglycemic unawareness, but donor organs are limited. Islet xenotransplantation using porcine islets will potentially solve this problem. Although successful proof of concept studies using clinically inapplicable anti-CD154 monoclonal antibody (mAb) in pig-to-non-human primate (NHP) islet xenotransplantation has been demonstrated by several groups worldwide, potentially clinically applicable anti-CD40 (2C10R4) mAb-based studies have not been reported. METHODS: Nine streptozotocin (STZ)-induced diabetic rhesus monkeys were transplanted with adult porcine islets isolated from designated pathogen-free (DPF) miniature pigs. They were treated with anti-CD40 mAb-based immunosuppressive regimen and were divided into 3 groups: anti-CD40 only group (n = 2), belatacept group (anti-CD40 mAb+belatacept, n = 2), and tacrolimus group (anti-CD40 mAb+tacrolimus, n = 5). All monkeys received anti-thymocyte globulin (ATG), cobra venom factor (CVF), adalimumab, and sirolimus. Blood glucose levels (BGL) and serum porcine C-peptide concentrations were measured. Humoral and cellular immune responses were assessed by ELISA and ELISPOT, respectively. Liver biopsy and subsequent immunohistochemistry were conducted. RESULTS: All animals restored normoglycemia immediately after porcine islet transplantation and finished the follow-up without any severe adverse effects except for one animal (R092). Most animals maintained their body weight. Median survival, as defined by a serum porcine C-peptide concentration of >0.15 ng/mL, was 31, 27, and 60 days for anti-CD40 only, belatacept, and tacrolimus groups, respectively. Anti-αGal IgG levels in serum and the number of interferon-γ secreting T cells in peripheral blood mononuclear cells did not increase in most animals. CONCLUSION: These results showed that anti-CD40 mAb combined with tacrolimus was effective in prolonging porcine islet graft survival, but anti-CD40 mAb was not as effective as anti-CD154 mAb in terms of preventing early islet loss.

Effect of a Shortened Duration of FOLFOX Chemotherapy on the Survival Rate of Patients with Stage II and III Colon Cancer.

BACKGROUND: FOLFOX chemotherapy is widely used as an adjuvant treatment for advanced colon cancer. The duration of adjuvant chemotherapy is usually set to 6 months, which is based on a former study of 5-fluorouracil/leucovorin chemotherapy. However, the FOLFOX regimen is known to have complications, such as peripheral neuropathy. The aim of this study was to compare the survival rates and complications experienced by patients receiving either 4 or 6 months of FOLFOX chemotherapy. METHODS: Retrospective data analysis was performed for stage II and III patients who underwent radical resection of colon cancer. We compared the 5-year survival rates and the occurrence of complications in patients who completed only 8 cycles of FOLFOX chemotherapy with patients who completed 12 cycles of chemotherapy. RESULTS: Among 188 patients who underwent adjuvant FOLFOX chemotherapy for stage II or III colon cancer, 83 (44.1%) completed 6 months of FOLFOX chemotherapy and 64 (34.0%) patients discontinued after 4 months of chemotherapy. The 5-year overall survival and disease-free survival rates did not show a significant difference. Patients in the 6-month group had peripheral neuropathy more frequently (p = 0.028). CONCLUSIONS: Five-year overall and disease-free survival were not significantly different between the 2 groups. Large-scale prospective studies are necessary for the analysis of complications and survival rates.

Visible Light Photocatalytic Performance of In Situ Synthesized Graphite-SiO2-TiO2 Composite Towards Degradation of Benzene Gas.

Graphite-SiO2-TiO2 composites with optimum graphite and SiO2 loadings were prepared by a facile one-pot chemical route. The structural, morphological and physiochemical properties of the samples were investigated by analytical techniques. UV-Vis-DRS analysis confirmed light absorbance edge of composites was sharply red-shifted to the visible region with increasing graphite and SiO2 content. The prepared composites showed higher photocatalytic activity towards degradation of benzene gas under visible light. The contribution of graphite and SiO2 on the enchantment of visible light photocatalytic performance of the composites was discussed.

Precipitation of Nickel Oxide on TiO2 Photocatalysts for Enhanced Visible Degradation Activity.

The liquid phase plasma (LPP) synthetic process has been exploited to synthesize nickel oxide nanoparticles doped TiO2 photocatalyst (NOTP) that can respond to visible light. The physicochemical properties of NOTPs were studied by several analysis instruments. The nickel oxide nanoparticles precipitated uniformly on the TiO2 powder are mostly NiO. The band gap energy of the NOTP measured was 2.99 eV, which was smaller than that of bare TiO2, 3.12 eV. The NOTP synthesized in this work showed high photoactivity under visible blue light.

Estrogenicity of Octyl Glucoside Synthesized by Direct Glucosidation as Non-Endocrine Disruptive Surfactant.

The estrogenicity of octyl glucoside was studied with its preparation method using microporous zeolites. Its estrogenicity was estimated using E-assay method to confirm the possibility as non-endocrine disruptive surfactant. The octyl glucoside was synthesized from D-glucose with 1-octanol by direct glucosidation. The high conversion of D-glucose was obtained on H-FAU zeolite which has a mild acid strength. The conversion and yield were improved with increasing of acid site amount of the zeolite catalysts. The octyl glucopyranoside is more hydrophilic than nonylphenol and has a high wettability. The octyl glucosides represented extremely lower estrogenic cell proliferation compared with nonylphenol.

Facile Synthesis and Characterization of Zinc Oxide Nanoparticle on Activated Carbon Using Liquid Phase Plasma Method.

Zinc oxide/activated carbon nanocomposites were synthesized by impregnating zinc oxide nanoparticles onto activated carbon powder using liquid phase plasma (LPP) method. Zinc oxide nanoparticles on the surface of activated carbon were fabricated rapidly by the LPP method due to reducing the zinc ion in aqueous solution. The obtained zinc oxide/activated carbon nanocomposites were characterized by XPS, HRTEM, and EDS. The amount of zinc oxide nanoparticles impregnated increased with increasing initial precursor concentration. Approximately 150~300 nm sized spherical shaped nanoparticles were uniformly dispersed on the surface of activated carbon powder.

Cohesin mutations in human cancer.

Cohesin is a highly-conserved protein complex that plays important roles in sister chromatid cohesion, chromatin structure, gene expression, and DNA repair. In humans, cohesin is a ubiquitously expressed, multi-subunit protein complex composed of core subunits SMC1A, SMC3, RAD21, STAG1/2 and regulatory subunits WAPL, PDS5A/B, CDCA5, NIPBL, and MAU2. Recent studies have demonstrated that genes encoding cohesin subunits are somatically mutated in a wide range of human cancers. STAG2 is the most commonly mutated subunit, and in a recent analysis was identified as one of only 12 genes that are significantly mutated in four or more cancer types. In this review we summarize the findings reported to date and comment on potential functional implications of cohesin mutation in the pathogenesis of human cancer.

CD80CD86 deficiency disrupts regulatory CD4+FoxP3+T cell homoeostasis and induces autoimmune-like alopecia.

Alopecia areata (AA) is an autoimmune disease that results in spot baldness in humans. Adequate animal models for AA are currently lacking. The objective of this study was to elucidate the mechanism of autoimmune-like alopecia (ALA) in C57BL/6.CD80CD86-deficient (B6.CD80CD86-/- ) mice. Incidence and severity of alopecia were analysed in 58 B6.CD80CD86-/- mice using histological examination, flow cytometry, multiplex enzyme-linked immunosorbent assay, quantitative RT-PCR and CD25 inhibition test. Both male and female B6.CD80CD86-/- mice showed almost 100% incidence of hair loss at 40 weeks of age. Moreover, CD4+FoxP3+Treg (Treg) cell population in B6.CD80CD86-/- mice was significantly lower than in B6 mice, which presumably underlined autoimmune reaction. Histologically, B6.CD80CD86-/- mice showed CD4+ and CD8+ T-cell infiltration around terminal follicle region and exhibited hair follicle destruction in the anagen or catagen stage. Negative correlation between the number of CD4+FoxP3+ Tregs and ALA was confirmed by the CD25 depletion test in B6 mice, as follicle destruction was similar to that observed in B6.CD80CD86-/- animals. CD80CD86 deficiency disrupted CD4+FoxP3+ Treg homoeostasis and prompted the development of ALA. We demonstrated that B6.CD80CD86-/- mice might have several advantages as an ALA model, because they exhibited high incidence of disease phenotype and epipathogenesis similar to that observed in human AA.