Epigenetic small-molecule screen for inhibition and reversal of acinar ductal metaplasia in mouse pancreatic organoids.

Background: Acinar ductal metaplasia (ADM) is among the earliest initiating events in pancreatic ductal adenocarcinoma (PDAC) development. Methods: We developed a novel morphology-based screen using organoids from wildtype and p48Cre/+ (Cre) mice to discover epigenetic modulators that inhibit or reverse pancreatic ADM more effectively than the broad-spectrum HDAC inhibitor trichostatin A (TSA). Results: Of the 144 compounds screened, nine hits and two additional natural product HDAC inhibitors were validated by dose-response analysis. The class I HDAC inhibitors apicidin and FK228, and the histone methyltransferase inhibitor chaetocin demonstrated pronounced ADM inhibition and reversal without inducing significant cytotoxicity at 1 µM. Thioester prodrug class I HDAC inhibitor largazole attenuated ADM while its disulfide homodimer was effective in both ADM inhibition and reversal. Prioritized compounds were validated for ADM reversal in p48Cre/+; LSL-KrasG12D/+ (KC) mouse organoids using both morphological and molecular endpoints. Molecular index analysis of ADM reversal in KC mouse organoids demonstrated improved activity compared to TSA. Improved prodrug stability translated into a stronger phenotypic and molecular response. RNA-sequencing indicated that angiotensinogen was the top inhibited pathway during ADM reversal. Conclusion: Our findings demonstrate a unique epigenetic mechanism and suggest that the phenotypic screen developed here may be applied to discover potential treatments for PDAC.

Peptide-based capture-and-release purification of extracellular vesicles and statistical algorithm enabled quality assessment.

Circulating extracellular vesicles (EVs) have gained significant attention for discovering tumor biomarkers. However, isolating EVs with well-defined homogeneous populations from complex biological samples is challenging. Different isolation methods have been found to derive different EV populations carrying different molecular contents, which confounds current investigations and hinders subsequent clinical translation. Therefore, standardizing and building a rigorous assessment of isolated EV quality associated with downstream molecular analysis is essential. To address this need, we introduce a statistical algorithm (ExoQuality Index, EQI) by integrating multiple EV characterizations (size, particle concentration, zeta potential, total protein, and RNA), enabling direct EV quality assessment and comparisons between different isolation methods. We also introduced a novel capture-release isolation approach using a pH-responsive peptide conjugated with NanoPom magnetic beads (ExCy) for simple, fast, and homogeneous EV isolation from various biological fluids. Bioinformatic analysis of next-generation sequencing (NGS) data of EV total RNAs from pancreatic cancer patient plasma samples using our novel EV isolation approach and quality index strategy illuminates how this approach improves the identification of tumor associated molecular markers. Results showed higher human mRNA coverage compared to existing isolation approaches in terms of both pancreatic cancer pathways and EV cellular component pathways using gProfiler pathway analysis. This study provides a valuable resource for researchers, establishing a workflow to prepare and analyze EV samples carefully and contributing to the advancement of reliable and rigorous EV quality assessment and clinical translation.

Epigenetic Small-Molecule Screen for Inhibition and Reversal of Acinar Ductal Metaplasia in Mouse Pancreatic Organoids.

Background: Acinar ductal metaplasia (ADM) is among the earliest initiating events in pancreatic ductal adenocarcinoma (PDAC) development. Methods: We developed a novel morphology-based screen using organoids from wildtype and p48 Cre/+ (Cre) mice to discover epigenetic modulators that inhibit or reverse pancreatic ADM more effectively than the broad-spectrum HDAC inhibitor trichostatin A (TSA). Results: Of the 144 compounds screened, nine hits and two additional natural product HDAC inhibitors were validated by dose-response analysis. The class I HDAC inhibitors apicidin and FK228, and the histone methyltransferase inhibitor chaetocin demonstrated pronounced ADM inhibition and reversal without inducing significant cytotoxicity at 1 µM. Thioester prodrug class I HDAC inhibitor largazole attenuated ADM while its disulfide homodimer was effective in both ADM inhibition and reversal. Prioritized compounds were validated for ADM reversal in p48 Cre/+ ;LSL-Kras G12D/+ (KC) mouse organoids using both morphological and molecular endpoints. Molecular index analysis of ADM reversal in KC mouse organoids demonstrated improved activity compared to TSA. Improved prodrug stability translated into a stronger phenotypic and molecular response. RNA-sequencing indicated that angiotensinogen was the top inhibited pathway during ADM reversal. Conclusion: Our findings demonstrate a unique epigenetic mechanism and suggest that the phenotypic screen developed here may be applied to discover potential treatments for PDAC.

Florida-California Cancer Research, Education and Engagement (CaRE2) Health Equity Center: Structure, Innovations, and Initial Outcomes.

INTRODUCTION: The Florida-California Cancer Research, Education, and Engagement (CaRE2) Health Equity Center is a triad partnership committed to increasing institutional capacity for cancer disparity research, the diversity of the cancer workforce, and community empowerment. This article provides an overview of the structure, process innovations, and initial outcomes from the first 4 years of the CaRE2 triad partnership. METHODS: CaRE2 serves diverse populations in Florida and California using a "molecule to the community and back" model. We prioritize research on the complex intersection of biological, environmental, and social determinants health, working together with scientific and health disparities communities, sharing expertise across institutions, bidirectional training, and community outreach. Partnership progress and outcomes were assessed using mixed methods and four Program Steering Committee meetings. RESULTS: Research capacity was increased through development of a Living Repository of 81 cancer model systems from minority patients for novel cancer drug development. CaRE2 funded 15 scientific projects resulting in 38 publications. Workforce diversity entailed supporting 94 cancer trainees (92 URM) and 34 ESIs (32 URM) who coauthored 313 CaRE2-related publications and received 48 grants. Community empowerment was promoted via outreaching to more than 3000 individuals, training 145 community cancer advocates (including 28 Community Scientist Advocates), and publishing 10 community reports. CaRE2 members and trainees together have published 639 articles, received 61 grants, and 57 awards. CONCLUSION: The CaRE2 partnership has achieved its initial aims. Infrastructure for translational cancer research was expanded at one partner institution, and cancer disparities research was expanded at the two cancer centers.

Transcriptional Profile of Human Pancreatic Acinar Ductal Metaplasia.

BACKGROUND AND AIMS: Aberrant acinar to ductal metaplasia (ADM), one of the earliest events involved in exocrine pancreatic cancer development, is typically studied using pancreata from genetically engineered mouse models. METHODS: We used primary, human pancreatic acinar cells from organ donors to evaluate the transcriptional and pathway profiles during the course of ADM. RESULTS: Following 6 days of three-dimensional culture on Matrigel, acinar cells underwent morphological and molecular changes indicative of ADM. mRNA from 14 donors' paired cells (day 0, acinar phenotype and day 6, ductal phenotype) was subjected to whole transcriptome sequencing. Acinar cell specific genes were significantly downregulated in the samples from the day 6 cultures while ductal cell-specific genes were upregulated. Several regulons of ADM were identified including transcription factors with reduced activity (PTF1A, RBPJL, and BHLHA15) and those ductal and progenitor transcription factors with increased activity (HNF1B, SOX11, and SOX4). Cells with the ductal phenotype contained higher expression of genes increased in pancreatic cancer while cells with an acinar phenotype had lower expression of cancer-associated genes. CONCLUSION: Our findings support the relevancy of human in vitro models to study pancreas cancer pathogenesis and exocrine cell plasticity.

Disclosing quantitative RT-PCR raw data during manuscript submission: a call for action.

Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT-qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT-qPCR raw data. The Real-time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose.

Pharmacological inhibition and reversal of pancreatic acinar ductal metaplasia.

Pancreatic acinar cells display a remarkable degree of plasticity and can dedifferentiate into ductal-like progenitor cells by a process known as acinar ductal metaplasia (ADM). ADM is believed to be one of the earliest precursor lesions toward the development of pancreatic ductal adenocarcinoma and maintaining the pancreatic acinar cell phenotype suppresses tumor formation. The effects of a novel pStat3 inhibitor (LLL12B) and the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) were investigated using 3-D cultures from p48Cre/+ and p48Cre/+LSL-KrasG12D/+ (KC) mice. LLL12B and TSA inhibited ADM in both KC and p48Cre/+ mouse pancreatic organoids. Furthermore, treatment with LLL12B or TSA on dedifferentiated acini from p48Cre/+ and KC mice that had undergone ADM produced morphologic and gene expression changes that suggest a reversal of ADM. Validation experiments using qRT-PCR (p48Cre/+ and KC) and RNA sequencing (KC) of the LLL12B and TSA treated cultures showed that the ADM reversal was more robust for the TSA treatments. Pathway analysis showed that TSA inhibited Spink1 and PI3K/AKT signaling during ADM reversal. The ability of TSA to reverse ADM was also observed in primary human acinar cultures. We report that pStat3 and HDAC inhibition can attenuate ADM in vitro and reverse ADM in the context of wild-type Kras. Our findings suggest that pharmacological inhibition or reversal of pancreatic ADM represents a potential therapeutic strategy for blocking aberrant ductal reprogramming of acinar cells.

Method for Isolating Extracellular Vesicles from Human Neural Stem Cells Expanded Under Neurosphere Culture.

Neural stem cells (NSCs) transplantation enhances plasticity and restores functions in neurological diseases. Therapeutic benefits of NSCs are due to their ability to replace the lost neurons and glial cells and also secreting a wide array of free and membrane-bound bioactive molecules that can reduce the hostility of diseased microenvironment, resolve inflammation, and rescue damaged neural cells. Membrane-encircled spherical nanostructures that are collectively known as extracellular vesicles (EVs) contain mRNA, miRNA, lipids, and specific proteins that affect different biological processes in cells located nearby or at far distances. Using EVs as an alternative non-cell-based therapy has gained huge attention, and developing methods for large-scale production of EVs is of great clinical importance. Here, we describe an efficient method to yield significant quantity of EVs from human NSCs that are expanded under free floating neurosphere assay culture system. Using the neurosphere assay in bioreactors under GMP-compliant conditions can result in scalable NSC-EVs required for human trials.

Role of non-coding RNAs in tumor progression and metastasis in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer with an overall 5-year survival rate of less than 10%. The 1-year survival rate of patients with locally advanced or metastatic disease is abysmal. The aggressive nature of cancer cells, hypovascularization, extensive desmoplastic stroma, and immunosuppressive tumor microenvironment (TME) endows PDAC tumors with multiple mechanisms of drug resistance. With no obvious genetic mutation(s) driving tumor progression or metastatic transition, the challenges for understanding the biological mechanism(s) of these processes are paramount. A better understanding of the molecular and cellular mechanisms of these processes could lead to new diagnostic tools for patient management and new targets for therapeutic intervention. microRNAs (miRNAs) are an evolutionarily conserved gene class of short non-coding regulatory RNAs. miRNAs are an extensive regulatory layer that controls gene expression at the posttranscriptional level. This review focuses on preclinical models that functionally dissect miRNA activity in tumor progression or metastatic processes in PDAC. Collectively, these studies suggest an influence of miRNAs and RNA-RNA networks in the processes of epithelial to mesenchymal cell transition and cancer cell stemness. At a cell-type level, some miRNAs mainly influence cancer cell-intrinsic processes and pathways, whereas other miRNAs predominantly act in distinct cellular compartments of the TME to regulate fibroblast and immune cell functions and/or influence other cell types' function via cell-to-cell communications by transfer of extracellular vesicles. At a molecular level, the influence of miRNA-mediated regulation often converges in core signaling pathways, including TGF-ß, JAK/STAT, PI3K/AKT, and NF-κB.

Enrichment of the erythrocyte miR-451a in brain extracellular vesicles following impairment of the blood-brain barrier.

Extracellular RNAs (exRNAs) are present in all biofluids and incorporate many types of RNAs including miRNA. To enhance their stability outside of the cell, exRNAs are bound within ribonucleoprotein complexes or packaged into extracellular vesicles (EVs). The blood-brain barrier (BBB) is a dynamic interface between the systemic circulation and the CNS and is responsible for maintaining a stable extracellular environment for CNS cells. The intent of this study was to determine if EVs and their contents are transferred from the peripheral circulation to the CNS under conditions of an impaired BBB. The BBB of mice was disrupted by unilateral intracarotid artery infusion with hyperosmolar mannitol solution. To validate barrier opening, the uptake clearance of [13C12]-sucrose in the left forebrain (i.e. the ipsilateral, mannitol injected hemisphere) was quantified and revealed a 14-fold increase in the mannitol perfused hemisphere compared to sham treated mice. EVs were isolated from the extracellular spaces of the left forebrain following gentle tissue lysis and differential ultracentrifugation. EVs were confirmed using nanotracking analysis, electron microscopy and western blotting. qRT-PCR showed that the erythrocyte-enriched miR-451a in brain tissue EVs increased with mannitol treatment by 24-fold. Small RNA sequencing performed on the EVs isolated from the sham and mannitol treated mice showed that miR-9-5p was the most abundant miRNA contained within the brain EVs. qRT-PCR analysis of plasma EVs did not produce a statistically significant difference in the expression of the CNS-enriched miR-9-5p or miR-9-3p, suggesting that transfer of CNS EVs to the peripheral circulation did not occur under the conditions of our experiment. We demonstrate that EVs containing miR-451a, a highly abundant miRNA present within erythrocytes and erythrocyte EVs, are enhanced in the CNS upon BBB disruption.

Method for improved integrity of RNA isolated from Matrigel cultures.

Matrigel is a commercially available substrate that is derived from the extracellular matrix. Matrigel is widely used in cell culture experiments such as the transdifferentiation of primary pancreatic acini to ductal epithelial-like cells. Difficulty arises during gene expression analysis for cells cultured on Matrigel because residual RNA in the Matrigel will not only contribute to the poor integrity of RNA isolated from Matrigel cultures, but also will impact the gene expression data. We report here a simple method of removing Matrigel from primary cultures of human or mouse pancreatic acini. Following the experiment, the cultures are placed on wet ice to liquefy the Matrigel. The cell and Matrigel mixture is then centrifuged at low speed to separate the pancreatic cells from the Matrigel solution that resides in the supernatant. RNA isolated from the pelleted cells has high integrity and may be readily used for gene expression analysis such as quantitative reverse transcription PCR.

Alterations in mouse spinal cord and sciatic nerve microRNAs after the chronic constriction injury (CCI) model of neuropathic pain.

Pain is one of the most common reasons to seek medical attention and chronic pain is a worldwide epidemic. There are currently no relevant biomarkers for the diagnosis of chronic pain, and new therapeutic strategies for chronic pain treatment are desperately needed. The chronic constriction injury (CCI) of the sciatic nerve is a widely used preclinical model of pathological neuropathic pain. Over the past decade, investigators have come to appreciate the many contributions of noncoding RNA including microRNA (miRNA), and other long and short noncoding (nc) RNAs. The development and/or maintenance of chronic pain could be controlled epigenetically through ncRNAs. Here we seek to characterize CNS tissues in a mouse model of neuropathic pain as this may serve to elucidate potential biomarkers relevant to pathological pain in humans. Male C57BL6/J mice (6 CCI and 6 sham procedure) underwent surgery for sciatic nerve ligation with chromic gut sutures. Following 7 days, mechanical allodynia was quantified using the von Frey assay. Mice were then euthanized for collection of spinal cord and sciatic nerve. cDNA was synthesized to 627 unique mature miRNAs from the total RNA. In the CCI mice that displayed mechanical allodynia, 11 and 125 miRNAs were differentially expressed (i.e., greater than 1.5-fold increase or decrease; P < 0.05) in the spinal cord and sciatic nerve, respectively, as compared to sham controls. Among those differentially expressed miRNAs in the sciatic nerve of CCI mice, the following passed the more stringent Bonfferoni correction: miR-138-3p, miR-138-5p and miR-676-3p, reduced and miR-142-5p, increased. Our data support miRNAs as promising therapeutic targets for the treatment of pathological pain.

Loss of RE-1 silencing transcription factor accelerates exocrine damage from pancreatic injury.

Regulation of pancreas plasticity is critical for preventing injury and promoting regeneration upon tissue damage. The intricate process of pancreatic differentiation is governed by an orchestrated network of positive and negative transcription factors for appropriate gene expression. While the transcriptional repressor REST is well characterized as a silencer of neuronal genes in non-neuronal cells, the role of REST in regulating exocrine pancreas cell identity remains largely unexplored. Rest expression is increased upon injury in the mouse pancreas, such as induced acute and chronic pancreatitis and ductal adenocarcinoma. At the cellular level, Rest expression is lower in mature acinar cells compared with pancreas progenitor and ductal cells. To investigate the role of REST activity in pancreatic transdifferentiation and homeostasis, we developed a novel mouse model (Cre/RESTfl/fl) with conditional knockout (KO) of Rest expression within pancreas cells. The high Cre-mediated excision efficiency of Rest exon two KO caused decreased Rest expression and activity within the pancreas. Short-term organoid cultures of pancreatic acini to undergo acinar-to-ductal metaplasia (ADM) showed that loss of REST impedes induced ADM, while overexpression of REST increases ADM. Interestingly, REST ablation accelerated acute pancreatitis in mice treated with the cholecystokinin analog caerulein, as indicated by cellular morphology, elevated serum amylase levels and pancreatic edema. Furthermore, Cre/RESTfl/fl mice were more sensitive to acute pancreatitis injury and displayed augmented tissue damage and cellular lesions. These results suggest REST has a novel protective role against pancreatic tissue damage by acting as a regulator of exocrine cell identity.

Human Colon Mucosal Biofilms and Murine Host Communicate via Altered mRNA and microRNA Expression during Cancer.

Disrupted interactions between host and intestinal bacteria are implicated in colorectal cancer (CRC) development. However, activities derived from these bacteria and their interplay with the host are unclear. Here, we examine this interplay by performing mouse and microbiota RNA sequencing on colon tissues and 16S and small RNA sequencing on stools from germfree (GF) and gnotobiotic ApcMin Δ 850/+ ;Il10-/- mice associated with microbes from biofilm-positive human CRC tumor (BF+T) and biofilm-negative healthy (BF-bx) tissues. The bacteria in BF+T mice differentially expressed (DE) >2,900 genes, including genes related to bacterial secretion, virulence, and biofilms but affected only 62 host genes. Small RNA sequencing of stools from these cohorts revealed eight significant DE host microRNAs (miRNAs) based on biofilm status and several miRNAs that correlated with bacterial taxon abundances. Additionally, computational predictions suggest that some miRNAs preferentially target bacterial genes while others primarily target mouse genes. 16S rRNA sequencing of mice that were reassociated with mucosa-associated communities from the initial association revealed a set of 13 bacterial genera associated with cancer that were maintained regardless of whether the reassociation inoculums were initially obtained from murine proximal or distal colon tissues. Our findings suggest that complex interactions within bacterial communities affect host-derived miRNA, bacterial composition, and CRC development.IMPORTANCE Bacteria and bacterial biofilms have been implicated in colorectal cancer (CRC), but it is still unclear what genes these microbial communities express and how they influence the host. MicroRNAs regulate host gene expression and have been explored as potential biomarkers for CRC. An emerging area of research is the ability of microRNAs to impact growth and gene expression of members of the intestinal microbiota. This study examined the bacteria and bacterial transcriptome associated with microbes derived from biofilm-positive human cancers that promoted tumorigenesis in a murine model of CRC. The murine response to different microbial communities (derived from CRC patients or healthy people) was evaluated through RNA and microRNA sequencing. We identified a complex interplay between biofilm-associated bacteria and the host during CRC in mice. These findings may lead to the development of new biomarkers and therapeutics for identifying and treating biofilm-associated CRCs.

Enhancing African American Participation in Biospecimens: A Case in Point for Pancreatic Cancer.

Diseases of the pancreas (i.e. chronic pancreatitis, diabetes, and pancreatic cancer) disproportionally affect the African American community. Challenges associated with engaging the African American community in biospecimen research are longstanding. We surveyed a number of pancreas-related biobanks, and data repositories for African American representation. While some of the biobanks and databases surveyed contain biospecimens and data from African American donors at levels that reflect minority representation among the general population, others do not. A number of factors have historically contributed to reduced participation of the African Americans community in biospecimen donation including medical mistrust, lack of transparency, fear, and a poor knowledge and understanding about the use of biospecimens for research. Suggestions for increasing African American participation in organ and biospecimen donation include educational interventions, particularly in community groups, and providing printed and online recruitment materials to patients, patient advocates, and care partners. Increasing awareness of the many benefits of biospecimen donation among African Americans will positively affect health disparities research into pancreatic cancer and other diseases.

Knockout of Acinar Enriched microRNAs in Mice Promote Duct Formation But Not Pancreatic Cancer.

The pancreatic acinar-enriched miR-216a, miR-216b and miR-217 are encoded within the miR217HG. These miRNAs have been purported to play a tumor suppressive role as their expression is reduced in both human and mouse pancreatic ductal adenocarcinoma (PDAC). To examine this possibility, we generated individual, germline knockout (KO) mice of miR-216a, miR-216b or miR-217. Unlike our previous study showing germline deletion of the miR217HG was embryonic lethal, CRISPR-Cas9 deleted portions of the 5' seed region of the miRNAs produced live births. To investigate possible phenotypes during pancreatic acinar ductal metaplasia (ADM), pancreatic acini from wild type and KO mice were plated on collagen and allowed to transdifferentiate over 4 days. Acini from each of the three miRNA KO mice produced greater numbers of ducts compared to controls. Evaluation of the gene expression during in vitro ADM demonstrated an increase in Krt19 and a reduction in acinar genes (Carboxypeptidase A1, Amylase2a) on day 4 of the transdifferentiation. Recovery was delayed for the miR-216a and miR-216b KOs following caerulein-induced acute pancreatitis. Also predominate in the caerulein treated miR-216a and miR-216b KO mice was the presence of pancreatic duct glands (PDGs). To further establish a phenotype, miRNA KO mice were crossed with EL-KRASG12D (EK) mice and followed up to 13 months of age. While all mice developed severe dysplasia and cystic papillary neoplasms, there existed no apparent phenotypic difference in the miRNA KO/EK mice compared to EK mice. Our data does not support a tumor suppressor role for miR-216a, miR-216b or miR-217 in PDAC and emphasizes the need for phenotypic evaluation of miRNAs in complex in vivo models beyond that performed using cell culture.

Exosomal miRNA Cargo as Mediator of Immune Escape Mechanisms in Neuroblastoma.

Both natural killer (NK) cells and exosomes released from these cells induce tumor cell cytotoxicity by way of the cell killing proteins perforin and granzyme. TGFß1 protein in the tumor microenvironment generates an immune escape mechanism rendering NK cells inactive. The tumor-suppressive miR-186 that is downregulated in neuroblastoma and in TGFß-treated NK cells represses oncogenic proteins in neuroblastoma (MYCN and AURKA) and components of the TGFß pathway. Restoration of miR-186 levels in neuroblastoma through NK cell-derived exosomes or by nanoparticle delivery reduces tumor burden, promotes survival, and restores the cell-killing abilities of NK cells, demonstrating the therapeutic potential of tumor-suppressive miRNAs in neuroblastoma.See related article by Neviani and colleagues; Cancer Res 79(6):1151-64.

Pancreatic Cancer Related Health Disparities: A Commentary.

We summarize the risk factors that may significantly contribute to racial disparities in pancreatic cancer, which is now the third leading cause of cancer deaths and projected to be second around 2030 in 12 years. For decades, the incidence rate of pancreatic cancer among Blacks has been 30% to 70% higher than other racial groups in the United States and the 5-year survival rate is approximately 5%. Diabetes and obesity have been identified as potentially predisposing factors to pancreatic cancer and both are more common among Blacks. Smoking continues to be one of the most important risk factors for pancreatic cancer and smoking rates are higher among Blacks compared to other racial groups. The overall risk of pancreatic cancer due to changes in DNA is thought to be the same for most racial groups; however, DNA methylation levels have been observed to be significantly different between Blacks and Whites. This finding may underlie the racial disparities in pancreatic cancer. Identification and prevention of these factors may be effective strategies to reduce the high incidence and mortality rates for pancreatic cancer among Blacks.