Conserved and divergent gene regulatory programs of the mammalian neocortex.

Divergence of cis-regulatory elements drives species-specific traits1, but how this manifests in the evolution of the neocortex at the molecular and cellular level remains unclear. Here we investigated the gene regulatory programs in the primary motor cortex of human, macaque, marmoset and mouse using single-cell multiomics assays, generating gene expression, chromatin accessibility, DNA methylome and chromosomal conformation profiles from a total of over 200,000 cells. From these data, we show evidence that divergence of transcription factor expression corresponds to species-specific epigenome landscapes. We find that conserved and divergent gene regulatory features are reflected in the evolution of the three-dimensional genome. Transposable elements contribute to nearly 80% of the human-specific candidate cis-regulatory elements in cortical cells. Through machine learning, we develop sequence-based predictors of candidate cis-regulatory elements in different species and demonstrate that the genomic regulatory syntax is highly preserved from rodents to primates. Finally, we show that epigenetic conservation combined with sequence similarity helps to uncover functional cis-regulatory elements and enhances our ability to interpret genetic variants contributing to neurological disease and traits.

Single-cell analysis of chromatin accessibility in the adult mouse brain.

Recent advances in single-cell technologies have led to the discovery of thousands of brain cell types; however, our understanding of the gene regulatory programs in these cell types is far from complete1-4. Here we report a comprehensive atlas of candidate cis-regulatory DNA elements (cCREs) in the adult mouse brain, generated by analysing chromatin accessibility in 2.3 million individual brain cells from 117 anatomical dissections. The atlas includes approximately 1 million cCREs and their chromatin accessibility across 1,482 distinct brain cell populations, adding over 446,000 cCREs to the most recent such annotation in the mouse genome. The mouse brain cCREs are moderately conserved in the human brain. The mouse-specific cCREs-specifically, those identified from a subset of cortical excitatory neurons-are strongly enriched for transposable elements, suggesting a potential role for transposable elements in the emergence of new regulatory programs and neuronal diversity. Finally, we infer the gene regulatory networks in over 260 subclasses of mouse brain cells and develop deep-learning models to predict the activities of gene regulatory elements in different brain cell types from the DNA sequence alone. Our results provide a resource for the analysis of cell-type-specific gene regulation programs in both mouse and human brains.

De Novo Elastin Assembly Alleviates Development of Supravalvular Aortic Stenosis-Brief Report.

BACKGROUND: A series of incurable cardiovascular disorders arise due to improper formation of elastin during development. Supravalvular aortic stenosis (SVAS), resulting from a haploinsufficiency of ELN, is caused by improper stress sensing by medial vascular smooth muscle cells, leading to progressive luminal occlusion and heart failure. SVAS remains incurable, as current therapies do not address the root issue of defective elastin. METHODS: We use SVAS here as a model of vascular proliferative disease using both human induced pluripotent stem cell-derived vascular smooth muscle cells and developmental Eln+/- mouse models to establish de novo elastin assembly as a new therapeutic intervention. RESULTS: We demonstrate mitigation of vascular proliferative abnormalities following de novo extracellular elastin assembly through the addition of the polyphenol epigallocatechin gallate to SVAS human induced pluripotent stem cell-derived vascular smooth muscle cells and in utero to Eln+/- mice. CONCLUSIONS: We demonstrate de novo elastin deposition normalizes SVAS human induced pluripotent stem cell-derived vascular smooth muscle cell hyperproliferation and rescues hypertension and aortic mechanics in Eln+/- mice, providing critical preclinical findings for the future application of epigallocatechin gallate treatment in humans.

Short peripheral blood leukocyte telomere length in rheumatoid arthritis-interstitial lung disease.

Shortened telomere lengths (TLs) can be caused by single nucleotide polymorphisms and loss-of-function mutations in telomere-related genes (TRG), as well as ageing and lifestyle factors such as smoking. Our objective was to determine if shortened TL is associated with interstitial lung disease (ILD) in individuals with rheumatoid arthritis (RA). This is the largest study to demonstrate and replicate that shortened peripheral blood leukocytes-TL is associated with ILD in patients with RA compared with RA without ILD in a multinational cohort, and short PBL-TL was associated with baseline disease severity in RA-ILD as measured by forced vital capacity percent predicted.

Map-Based Cloning and Characterization of a Major QTL Gene, FfR1, Which Confers Resistance to Rice Bakanae Disease.

Bakanae disease (BD), caused by the fungal pathogen Fusarium fujikuroi, is a serious threat to rice production worldwide. Breeding elite rice varieties resistant to BD requires the identification of resistance genes. Previously, we discovered a resistant quantitative trait locus (QTL), qFfR1, in a Korean japonica rice variety, Nampyeong. In this study, we fine-mapped qFfR1 with a Junam*4/Nampyeong BC3F3 population and delimited its location to a 37.1 kb region on chromosome 1. Complementation experiments with seven candidate genes in this region revealed that OsI_02728 is the gene for qFfR1. This gene encodes a protein with a typical leucine-rich repeat (LRR) receptor-like protein structure. RNA-sequencing-based transcriptomic analysis revealed that FfR1 induces the transcription of defense genes, including lignin and terpenoid biosynthesis genes, pathogenesis-related genes, and thionin genes. These results may facilitate investigations into the molecular mechanisms underlying BD resistance, including molecular patterns of Fusarium fujikuroi interacting with FfR1 and players working in signal transduction pathways downstream of FfR1, and the breeding of new BD-resistant varieties by providing a BD resistance gene with its precise selection marker. This will contribute to efficient control of BD, which is becoming more prevalent according to temperature rises due to climate change.

Gendered Trends in Formal and Informal Care Utilization Among Older Adults in South Korea.

Drawing on data from two waves of the Korean Longitudinal Study of Ageing (2010 and 2018), this study examined how community-dwelling older men and women (65+) with functional limitations utilized formal and informal sources of care and how their patterns of care utilization changed over time. The usage patterns of formal and informal caregiving services were categorized into three groups: (a) informal help only, (b) formal-informal mix, and (c) no help from either. More men and women used both formal and informal help for their care needs in 2018 than in 2010 (15% compared to 7%). The proportion of older men who relied on informal help only remained similar across survey years, whereas a smaller proportion of older women relied on informal help only in 2018. Although formal care use has been expanded in South Korea, older men continue to utilize help from their families. However, for older women, the proportion who did not receive any help increased - despite an increase in formal care utilization. These findings highlight the importance of considering gendered resources in caregiving in Korea.

The relationship between elastin cross linking and alveolar wall rupture in human pulmonary emphysema.

To better define the role of mechanical forces in pulmonary emphysema, we employed methods recently developed in our laboratory to identify microscopic level relationships between airspace size and elastin-specific desmosine and isodesmosine (DID) cross links in normal and emphysematous human lungs. Free DID in wet tissue (a biomarker for elastin degradation) and total DID in formalin-fixed, paraffin-embedded (FFPE) tissue sections were measured using liquid chromatography-tandem mass spectrometry and correlated with alveolar diameter, as determined by the mean linear intercept (MLI) method. There was a positive correlation between free lung DID and MLI (P < 0.0001) in formalin-fixed lungs, and elastin breakdown was greatly accelerated when airspace diameter exceeded 400 µm. In FFPE tissue, DID density was markedly increased beyond 300 µm (P < 0.0001) and leveled off around 400 µm. Elastic fiber surface area similarly peaked at around 400 µm, but to a much lesser extent than DID density, indicating that elastin cross linking is markedly increased in response to early changes in airspace size. These findings support the hypothesis that airspace enlargement is an emergent phenomenon in which initial proliferation of DID cross links to counteract alveolar wall distention is followed by a phase transition involving rapid acceleration of elastin breakdown, alveolar wall rupture, and progression to an active disease state that is less amenable to therapeutic intervention.NEW & NOTEWORTHY The current findings support the hypothesis that airspace enlargement is an emergent phenomenon in which initial proliferation of DID cross links to counteract alveolar wall distention is followed by a phase transition involving rapid acceleration of elastin breakdown, alveolar wall rupture, and progression to an active disease state that is less amenable to therapeutic intervention.

Molecular Mechanisms of Neuroprotection by Ketone Bodies and Ketogenic Diet in Cerebral Ischemia and Neurodegenerative Diseases.

Ketone bodies (KBs), such as acetoacetate and ß-hydroxybutyrate, serve as crucial alternative energy sources during glucose deficiency. KBs, generated through ketogenesis in the liver, are metabolized into acetyl-CoA in extrahepatic tissues, entering the tricarboxylic acid cycle and electron transport chain for ATP production. Reduced glucose metabolism and mitochondrial dysfunction correlate with increased neuronal death and brain damage during cerebral ischemia and neurodegeneration. Both KBs and the ketogenic diet (KD) demonstrate neuroprotective effects by orchestrating various cellular processes through metabolic and signaling functions. They enhance mitochondrial function, mitigate oxidative stress and apoptosis, and regulate epigenetic and post-translational modifications of histones and non-histone proteins. Additionally, KBs and KD contribute to reducing neuroinflammation and modulating autophagy, neurotransmission systems, and gut microbiome. This review aims to explore the current understanding of the molecular mechanisms underpinning the neuroprotective effects of KBs and KD against brain damage in cerebral ischemia and neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease.

Safety evaluation of oral calcitonin-gene-related peptide receptor antagonists in patients with acute migraine: a systematic review and meta-analysis.

OBJECTIVE: Calcitonin gene-related peptide (CGRP) receptor antagonists have been suggested as novel treatments for acute migraine. This study aimed to use meta-analysis to compare the safety and tolerability of five existing oral CGRP receptor antagonists (BI44370TA, MK-3207, rimegepant, telcagepant, and ubrogepant) with that of a placebo or triptans against acute migraine. METHODS: Five prominent databases were searched to identify randomized controlled trials on this topic. The primary safety outcomes of interest were any adverse events (AEs) and treatment-related adverse events (TRAEs), and secondary outcomes were individual events, namely diarrhea, dizziness, dry mouth, fatigue, nausea, paresthesia, somnolence, upper abdominal pain, and vomiting. RESULTS: Fifteen studies met the eligibility criteria and were examined in detail. Although, compared to placebo, oral CGRP receptor antagonists significantly increased the incidence of any AEs (risk ratio [RR] = 1.15; 95% confidence interval [CI] = 1.07-1.23), there was no difference in the incidence of TRAEs (RR = 1.18; 95% CI = 1.00-1.38). Moreover, CGRP receptor antagonists were safer than triptans with respect to primary safety outcomes, such as any AEs (RR = 0.78; 95% CI = 0.63-0.98) and TRAEs (RR = 0.68; 95% CI = 0.58-0.79). CONCLUSION: Despite oral CGRP receptor antagonists posing a significantly higher risk of AEs when compared to placebo, CGRP receptor antagonists have a favorable safety profile compared to triptans. Our findings inform strategies to enhance safety and tolerability in the treatment of acute migraine.

Effect of Human Peripheral Blood Mononuclear Cells on Mouse Endometrial Cell Proliferation: A Potential Therapeutics for Endometrial Regeneration.

OBJECTIVES: The persistently thin endometrium is a major cause of repeated implantation failure; however, there is no definite treatment for it yet. This study aimed to confirm the potential of human peripheral blood mononuclear cells (hPBMCs) as a therapeutic agent for endometrial regeneration. DESIGN: An experimental study was carried out. PARTICIPANTS/MATERIALS, SETTING, METHODS: To assess the in vitro effect of hPBMC, the human primary endometrial epithelial cell lines SNU-685 and SNU-1077 were co-cultured with or without 1 × 105 hPBMCs for 24 h. To evaluate the in vivo effect, either 1 × 105 hPBMCs in PBS or PBS alone were injected into the left uterine horn of nonobese diabetic-severe combined immune-deficient mice, and the right untreated uterine horn was used as control. RESULTS: Co-culture with hPBMCs stimulated significant proliferation in both SNU-685 and SNU-1077 cell lines (p = 0.002 and 0.044, respectively). Moreover, treatment with hPBMCs significantly increased the thickness in all parts of the endometrium compared with that in the untreated control uterine horn (proximal: 1.69 ± 0.19 vs. 1.00 ± 0.10, p = 0.009; middle: 1.51 ± 0.14 vs. 1.00 ± 0.12, p = 0.010; distal: 1.72 ± 0.22 vs. 1.00 ± 0.12, p = 0.003, respectively). Compared with the PBS injection group, the hPBMC injection group had significantly thickened endometrium in the middle (p = 0.036) and distal segments (p = 0.002) of the uterine horn. Immunohistochemical analysis revealed the presence of exogenously injected hPBMCs in the uterus of recipient mice. hPBMC-recipient mice had cyclic uterus with normal histology in the endometrium. LIMITATIONS: hPBMCs were not applied directly to a mouse model with thin endometrium, so further study is needed. CONCLUSION: The beneficial effect of hPBMCs on endometrium may suggest their clinical feasibility for the safe treatment of infertile patients with persistently thin endometrium.

The MicroRNA-92a/Sp1/MyoD Axis Regulates Hypoxic Stimulation of Myogenic Lineage Differentiation in Mouse Embryonic Stem Cells.

Hypoxic microenvironments exist in developing embryonic tissues and determine stem cell fate. We previously demonstrated that hypoxic priming plays roles in lineage commitment of embryonic stem cells. In the present study, we found that hypoxia-primed embryoid bodies (Hyp-EBs) efficiently differentiate into the myogenic lineage, resulting in the induction of the myogenic marker MyoD, which was not mediated by hypoxia-inducible factor 1α (HIF1α) or HIF2α, but rather by Sp1 induction and binding to the MyoD promoter. Knockdown of Sp1 in Hyp-EBs abrogated hypoxia-induced MyoD expression and myogenic differentiation. Importantly, in the cardiotoxin-muscle injury mice model, Hyp-EB transplantation facilitated muscle regeneration in vivo, whereas transplantation of Sp1-knockdown Hyp-EBs failed to do. Moreover, we compared microRNA (miRNA) expression profiles between EBs under normoxia versus hypoxia and found that hypoxia-mediated Sp1 induction was mediated by the suppression of miRNA-92a, which directly targeted the 3' untranslated region (3' UTR) of Sp1. Further, the inhibitory effect of miRNA-92a on Sp1 in luciferase assay was abolished by a point mutation in specific sequence in the Sp1 3' UTR that is required for the binding of miRNA-92a. Collectively, these results suggest that hypoxic priming enhances EB commitment to the myogenic lineage through miR-92a/Sp1/MyoD regulatory axis, suggesting a new pathway that promotes myogenic-lineage differentiation.

Synergistic Antitumor Activity of SH003 and Docetaxel via EGFR Signaling Inhibition in Non-Small Cell Lung Cancer.

Epidermal growth factor receptor (EGFR) is overexpressed in lung cancer patients. Despite treatment with various EGFR tyrosine kinase inhibitors, recurrence and metastasis of lung cancer are inevitable. Docetaxel (DTX) is an effective conventional drug that is used to treat various cancers. Several researchers have studied the use of traditional herbal medicine in combination with docetaxel, to improve lung cancer treatment. SH003, a novel herbal mixture, exerts anticancer effects in different cancer cell types. Here, we aimed to investigate the apoptotic and anticancer effects of SH003 in combination with DTX, in human non-small-cell lung cancer (NSCLC). SH003, with DTX, induced apoptotic cell death, with increased expression of cleaved caspases and cleaved poly (ADP-ribose) polymerase in NSCLC cells. Moreover, SH003 and DTX induced the apoptosis of H460 cells via the suppression of the EGFR and signal transducer and activator of transcription 3 (STAT3) signaling pathways. In H460 tumor xenograft models, the administration of SH003 or docetaxel alone diminished tumor growth, and their combination effectively killed cancer cells, with increased expression of apoptotic markers and decreased expression of p-EGFR and p-STAT3. Collectively, the combination of SH003 and DTX may be a novel anticancer strategy to overcome the challenges that are associated with conventional lung cancer therapy.

Spatial distribution of salinity and heavy metals in surface soils on the Mugan Plain, the Republic of Azerbaijan.

The Republic of Azerbaijan suffers from low agricultural productivity caused by soil salinization and erosion, and limited and insufficient soil data are available for economic and political reasons. In this study, soil salinity and heavy metal levels were assessed. Environmental risk assessment was conducted to evaluate the potential risk posed by soils to human health. Soil guideline values were proposed to monitor soil pollution in the Republic of Azerbaijan. Water extraction and spatial variability analysis were conducted to understand soil salinization and heavy metal pollution. Among the 20 studied elements, the elements Ca, Cl, and S and the heavy metals Cr, Ni, and Pb were classified as problematic on the basis of the geoaccumulation index, and As was also identified as posing a possible risk on the basis of the potential ecological risk index. Based on the developed soil guideline values for agricultural soil, the As, Cr, and Ni in the soil samples exceeded their respective guidelines by 31.3, 41.8, and 61.6%, respectively. Water extraction results confirmed that 99% of the leached ions were cationic salts, and the most problematic ion was Na, followed by Ca, Cl, and S. The extractability values of Cr and Ni were significantly lower than those of other heavy metals, which implies that their actual leaching potential may be overestimated. The linear regression and spatial variability analysis confirmed that leachable salts have accumulated in lowland areas due to the capillary rise of water and evaporation, but the distribution of heavy metals confirmed that As, Cr, and Ni were abundant in agricultural soils. Our results clearly showed that heavy metal soil contamination and high salinity levels are major problems that should be considered when assessing food safety and health hazards in the Mugan Plain of Azerbaijan. Therefore, future studies should be performed for additional environmental risk assessment, detailed hazard identification, and health risk assessment.

AIM2 inflammasome contributes to brain injury and chronic post-stroke cognitive impairment in mice.

Although over one-third of stroke patients may develop post-stroke cognitive impairment (PSCI), the mechanisms underlying PSCI remain unclear. We explored here, the involvement of post-stroke inflammasomes in long-term PSCI development, using a 45 min-middle cerebral artery occlusion (MCAO)/reperfusion-induced PSCI model. Immunohistological assessment on day 1, 3, and 7 was followed by cognitive function test 28 days post-stroke. Evaluation of inflammasome sensor gene expression in aged mouse brains showed dominant expression of absent in melanoma 2 (Aim2) in 6-, 12-, and 18-month-old mouse brains. AIM2 mRNA and protein increased until 7 days post-stroke. PSCI decreased anxiety in elevated plus maze test and impaired spatial learning and memory functions in Morris water maze test 28 days post-stroke. AIM2 and other inflammasome subunit immunoreactivities, including those for caspase-1, interleukin (IL)-1ß, and IL-18, were higher in the hippocampus and cortex of the PSCI than in those of the sham group 7 days post-stroke. AIM2 immunoreactivity of the PSCI group was primarily co-localized with Iba-1 (microglial marker) and CD31 (endothelial cell marker) immunoreactivities but not NeuN (neuronal marker) and GFAP (astrocyte marker) immunoreactivities, suggesting that microglia or endothelial cell-induced AIM2 production mediated PSCI pathogenesis. Additionally, inflammasome-induced pyroptosis might contribute to acute and chronic neuronal death after stroke. AIM2 knockout (KO) and Ac-YVAD-CMK-induced caspase-1 inhibition in mice significantly improved cognitive function and reversed brain volume in the hippocampus relative to those in stroke mice. Conclusively, AIM2 inflammasome-mediated inflammation and pyroptosis likely aggravated PSCI; therefore, targeting and controlling AIM2 inflammasome could potentially treat PSCI.

3D Bioprinted Vascularized Tumour for Drug Testing.

An in vitro screening system for anti-cancer drugs cannot exactly reflect the efficacy of drugs in vivo, without mimicking the tumour microenvironment (TME), which comprises cancer cells interacting with blood vessels and fibroblasts. Additionally, the tumour size should be controlled to obtain reliable and quantitative drug responses. Herein, we report a bioprinting method for recapitulating the TME with a controllable spheroid size. The TME was constructed by printing a blood vessel layer consisting of fibroblasts and endothelial cells in gelatine, alginate, and fibrinogen, followed by seeding multicellular tumour spheroids (MCTSs) of glioblastoma cells (U87 MG) onto the blood vessel layer. Under MCTSs, sprouts of blood vessels were generated and surrounding MCTSs thereby increasing the spheroid size. The combined treatment involving the anti-cancer drug temozolomide (TMZ) and the angiogenic inhibitor sunitinib was more effective than TMZ alone for MCTSs surrounded by blood vessels, which indicates the feasibility of the TME for in vitro testing of drug efficacy. These results suggest that the bioprinted vascularized tumour is highly useful for understanding tumour biology, as well as for in vitro drug testing.

Isolinderalactone Induces Cell Death via Mitochondrial Superoxide- and STAT3-Mediated Pathways in Human Ovarian Cancer Cells.

The mortality rate of ovarian cancer (OC) worldwide increases with age. OC is an often fatal cancer with a curative rate of only 20-30%, as symptoms often appear after disease progression. Studies have reported that isolinderalactone (ILL), a furanosesquiterpene derivative extracted from the dried root of Lindera aggregata, can inhibit several cancer cell lines' growth. However, the molecular mechanisms underlying ILL activities in human OC cells remain unexplored. This study investigated the antitumor activities of ILL in human OC cells by inducing mitochondrial superoxide (mtSO) and JAK-signal transducer and activator of transcription 3 (STAT3)-dependent cell death. ILL caused cell death in SKOV-3 and OVCAR-3 cells and increased the cell proportion in the subG1 phase. Additionally, ILL significantly induced mtSO production and reduced ROS production. Moreover, ILL downregulated mitochondrial membrane potential and the expression levels of anti-apoptotic Bcl-2 family proteins and superoxide dismutase (SOD)2. Results showed that ILL decreased phosphorylation of serine 727 and tyrosine 705 of STAT3 and expression of survivin, a STAT3-regulated gene. Furthermore, ILL-induced cell death was reversed by pretreatment of Mito-TEMPO, a mitochondria-specific antioxidant. These results suggest that ILL induces cell death by upregulation of mtSO, downregulation of mitochondrial SOD2, and inactivation of the STAT3-mediated pathway.

Hepatocyte Growth Factor Improves the Therapeutic Efficacy of Human Bone Marrow Mesenchymal Stem Cells via RAD51.

Human embryonic stem cell-derived mesenchymal stem cells (hE-MSCs) have greater proliferative capacity than other human mesenchymal stem cells (hMSCs), suggesting that they may have wider applications in regenerative cellular therapy. In this study, to uncover the anti-senescence mechanism in hE-MSCs, we compared hE-MSCs with adult bone marrow (hBM-MSCs) and found that hepatocyte growth factor (HGF) was more abundantly expressed in hE-MSCs than in hBM-MSCs and that it induced the transcription of RAD51 and facilitated its SUMOylation at K70. RAD51 induction/modification by HGF not only increased telomere length but also increased mtDNA replication, leading to increased ATP generation. Moreover, HGF-treated hBM-MSCs showed significantly better therapeutic efficacy than naive hBM-MSCs. Together, the data suggest that the RAD51-mediated effects of HGF prevent hMSC senescence by promoting telomere lengthening and inducing mtDNA replication and function, which opens the prospect of developing novel therapies for liver disease.

Differential ubiquitin binding by the acidic loops of Ube2g1 and Ube2r1 enzymes distinguishes their Lys-48-ubiquitylation activities.

The ubiquitin E2 enzymes, Ube2g1 and Ube2r1, are able to synthesize Lys-48-linked polyubiquitins without an E3 ligase but how that is accomplished has been unclear. Although both E2s contain essential acidic loops, only Ube2r1 requires an additional C-terminal extension (184-196) for efficient Lys-48-ubiquitylation activity. The presence of Tyr-102 and Tyr-104 in the Ube2g1 acidic loop enhanced both ubiquitin binding and Lys-48-ubiquitylation and distinguished Ube2g1 from the otherwise similar truncated Ube2r1(1-183) (Ube2r1C). Replacement of Gln-105-Ser-106-Gly-107 in the acidic loop of Ube2r1C (Ube2r1C(YGY)) by the corresponding residues from Ube2g1 (Tyr-102-Gly-103-Tyr-104) increased Lys-48-ubiquitylation activity and ubiquitin binding. Two E2∼UB thioester mimics (oxyester and disulfide) were prepared to characterize the ubiquitin binding activity of the acidic loop. The oxyester but not the disulfide derivative was found to be a functional equivalent of the E2∼UB thioester. The ubiquitin moiety of the Ube2r1C(C93S)-[(15)N]UB(K48R) oxyester displayed two-state conformational exchange, whereas the Ube2r1C(C93S/YGY)-[(15)N]UB(K48R) oxyester showed predominantly one state. Together with NMR studies that compared UB(K48R) oxyesters of the wild-type and the acidic loop mutant (Y102G/Y104G) forms of Ube2g1, in vitro ubiquitylation assays with various mutation forms of the E2s revealed how the intramolecular interaction between the acidic loop and the attached donor ubiquitin regulates Lys-48-ubiquitylation activity.