Stellate cells are in utero markers of pancreatic disease in cystic fibrosis.

BACKGROUND: Pancreatic fibrosis is an early diagnostic feature of the common inherited disorder cystic fibrosis (CF). Many people with CF (pwCF) are pancreatic insufficient from birth and the replacement of acinar tissue with cystic lesions and fibrosis is a progressive phenotype that may later lead to diabetes. Little is known about the initiating events in the fibrotic process though it may be a sequela of inflammation in the pancreatic ducts resulting from loss of CFTR impairing normal fluid secretion. Here we use a sheep model of CF (CFTR-/-) to examine the evolution of pancreatic disease through gestation. METHODS: Fetal pancreas was collected at six time points from 50-days of gestation through to term, which is equivalent to ~ 13 weeks to term in human. RNA was extracted from tissue for bulk RNA-seq and single cells were prepared from 80-day, 120-day and term samples for scRNA-seq. Data were validated by immunochemistry. RESULTS: Transcriptomic evidence from bulk RNA-seq showed alterations in the CFTR-/- pancreas by 65-days of gestation, which are accompanied by marked pathological changes by 80-days of gestation. These include a fibrotic response, confirmed by immunostaining for COL1A1, αSMA and SPARC, together with acinar loss. Moreover, using scRNA-seq we identify a unique cell population that is significantly overrepresented in the CFTR-/- animals at 80- and 120-days gestation, as are stellate cells at term. CONCLUSION: The transcriptomic changes and cellular imbalance that we observe likely have pivotal roles in the evolution of CF pancreatic disease and may provide therapeutic opportunities to delay or prevent pancreatic destruction in CF.

A systematic review on the role of mitochondrial dysfunction/disorders in neurodevelopmental disorders and psychiatric/behavioral disorders.

Introduction: Mitochondrial diseases are known inborn errors affecting energy metabolism and are as common as chronic diseases such as diabetes, affecting approximately 1 in 5,000 people. The role of mitochondrial diseases/dysfunction has been highlighted in neurodevelopmental disorders like ASD, ADHD, intellectual disability, and speech delay, as well as various psychiatric conditions. Neurodevelopmental disorders are increasingly recognized as having behavioral and psychiatric symptoms. Our study aimed to investigate reports of mitochondrial disorders, noting neurodevelopmental disorders and psychiatric/behavioral conditions. Methods: This was done through a systematic review of literature from PubMed/MEDLINE, Scopus, and Cochrane Library up to November 2022. Results: We found 277 publications, of which 139 met the inclusion criteria. We mostly found review articles with mention of mitochondrial dysfunction/disorder in relation to ASD with brief mentions of psychiatric/behavioral comorbidities. Discussion: This suggests a need for broader research efforts beyond ASD to understand the relationship between mitochondrial disorder or dysfunction and various neurodevelopmental and psychiatric/behavioral comorbidities.

A promoter-dependent upstream activator augments CFTR expression in diverse epithelial cell types.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an anion-selective channel found in epithelial cell membranes. Mutations in CFTR cause cystic fibrosis (CF), an inherited disorder that impairs epithelial function in multiple organs. Most men with CF are infertile due to loss of intact genital ducts. Here we investigated a novel epididymis-selective cis-regulatory element (CRE), located within a peak of open chromatin at -9.5 kb 5' to the CFTR gene promoter. Activation of the -9.5 kb CRE alone by CRISPRa had no impact on CFTR gene expression. However, CRISPRa co-activation of the -9.5 kb CRE and the CFTR gene promoter in epididymis cells significantly augmented CFTR mRNA and protein expression when compared to promoter activation alone. This increase was accompanied by enhanced chromatin accessibility at both sites. Furthermore, the combined CRISPRa strategy activated CFTR expression in other epithelial cells that lack open chromatin at the -9.5 kb site and in which the locus is normally inactive. However, the -9.5 kb CRE does not function as a classical enhancer of the CFTR promoter in transient reporter gene assays. These data provide a novel mechanism for activating/augmenting CFTR expression, which may have therapeutic utility for mutations that perturb CFTR transcription.

Investigating adverse genomic and regulatory changes caused by replacement of the full-length CFTR cDNA using Cas9 and AAV.

A "universal strategy" replacing the full-length CFTR cDNA may treat >99% of people with cystic fibrosis (pwCF), regardless of their specific mutations. Cas9-based gene editing was used to insert the CFTR cDNA and a truncated CD19 (tCD19) enrichment tag at the CFTR locus in airway basal stem cells. This strategy restores CFTR function to non-CF levels. Here, we investigate the safety of this approach by assessing genomic and regulatory changes after CFTR cDNA insertion. Safety was first assessed by quantifying genetic rearrangements using CAST-seq. After validating restored CFTR function in edited and enriched airway cells, the CFTR locus open chromatin profile was characterized using ATAC-seq. The regenerative potential and differential gene expression in edited cells was assessed using scRNA-seq. CAST-seq revealed a translocation in ∼0.01% of alleles primarily occurring at a nononcogenic off-target site and large indels in 1% of alleles. The open chromatin profile of differentiated airway epithelial cells showed no appreciable changes, except in the region corresponding to the CFTR cDNA and tCD19 cassette, indicating no detectable changes in gene regulation. Edited stem cells produced the same types of airway cells as controls with minimal alternations in gene expression. Overall, the universal strategy showed minor undesirable genomic changes.

The impact of genomic distance on enhancer-promoter interactions at the CFTR locus.

We identified and characterized multiple cell-type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the -35 kb airway enhancer and the CFTR promoter in the 16HBE14o- airway cell line, or between the intron 1 (185 + 10 kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line. The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and -35 kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o- cells.

Functional genomics of the human epididymis: Further characterization of efferent ducts and model systems by single-cell RNA sequencing analysis.

BACKGROUND: The human epididymis is poorly studied due to the lack of availability of tissue samples. Our understanding of its structure and function depends upon anatomical and histological observations of archived material. OBJECTIVES: Here we used single-cell RNA sequencing (scRNA-seq) technologies to elucidate the identity of cells within the human efferent ducts (EDs) and compared them to caput epididymis cells. We also compared the cellularity of primary tissues with those of 2D and 3D (organoid) culture models used for functional studies. MATERIALS AND METHODS: Human epididymis tissue was dissected to separate different anatomical regions and digested to release single cells for processing on the 10X Genomics Chromium platform. Primary human epididymis epithelial (HEE) cells and HEE organoids were grown as described previously and subjected to scRNA-seq. scRNA-seq data were processed by standard bioinformatics pipelines and used for comparative analysis. RESULTS: We define the cell types in the EDs which include specialized epithelial cells, connective tissue stromal cells, vascular endothelial cells, smooth muscle cells, and immune cells, but lack basal cells that are seen in the caput epididymis. Furthermore, we identify a sub-population of epithelial cells which have marker genes found in the bladder and urothelium. Comparative genomics analysis of the 2D and 3D culture models shows cellular identities adapted to the culture environment while still maintaining similarity to the primary tissue. DISCUSSION: Our data suggest that EDs are lined with a transitional epithelium, which like the urothelium is able to stretch and contract depending on luminal volume. This is consistent with its primary role in seminal fluid resorption and sperm concentration. Moreover, we describe the cellularity of models to study the human epididymis epithelium in vitro. CONCLUSION: Single-cell RNA-seq data from the human epididymis make a valuable contribution to our understanding of this highly specialized organ.

Cellular heterogeneity in the 16HBE14o- airway epithelial line impacts biological readouts.

The airway epithelial cell line, 16HBE14o- , is an important cell model for studying airway disease. 16HBE14o- cells were originally generated from primary human bronchial epithelial cells by SV40-mediated immortalization, a process that is associated with genomic instability through long-term culture. Here, we explore the heterogeneity of these cells, with respect to expression of the cystic fibrosis transmembrane conductance regulator (CFTR) transcript and protein. We isolate clones of 16HBE14o- with stably higher and lower levels of CFTR in comparison to bulk 16HBE14o- , designated CFTRhigh and CFTRlow . Detailed characterization of the CFTR locus in these clones by ATAC-seq and 4C-seq showed open chromatin profiles and higher order chromatin structure that correlate with CFTR expression levels. Transcriptomic profiling of CFTRhigh and CFTRlow cells showed that the CFTRhigh cells had an elevated inflammatory/innate immune response phenotype. These results encourage caution in interpreting functional data from clonal lines of 16HBE14o- cells, generated after genomic or other manipulations.

Transcriptomic analysis of lung development in wildtype and CFTR-/- sheep suggests an early inflammatory signature in the CF distal lung.

The precise molecular events initiating human lung disease are often poorly characterized. Investigating prenatal events that may underlie lung disease in later life is challenging in man, but insights from the well-characterized sheep model of lung development are valuable. Here, we determine the transcriptomic signature of lung development in wild-type sheep (WT) and use a sheep model of cystic fibrosis (CF) to characterize disease associated changes in gene expression through the pseudoglandular, canalicular, saccular, and alveolar stages of lung growth and differentiation. Using gene ontology process enrichment analysis of differentially expressed genes at each developmental time point, we define changes in biological processes (BP) in proximal and distal lung from WT or CF animals. We also compare divergent BP in WT and CF animals at each time point. Next, we establish the developmental profile of key genes encoding components of ion transport and innate immunity that are pivotal in CF lung disease and validate transcriptomic data by RT-qPCR. Consistent with the known pro-inflammatory phenotype of the CF lung after birth, we observe upregulation of inflammatory response processes in the CF sheep distal lung during the saccular stage of prenatal development. These data suggest early commencement of therapeutic regimens may be beneficial.

Early developmental phenotypes in the cystic fibrosis sheep model.

Highly effective modulator therapies for cystic fibrosis (CF) make it a treatable condition for many people. However, although CF respiratory illness occurs after birth, other organ systems particularly in the digestive tract are damaged before birth. We use an ovine model of CF to investigate the in utero origins of CF disease since the sheep closely mirrors critical aspects of human development. Wildtype (WT) and CFTR -/- sheep tissues were collected at 50, 65, 80, 100, and 120 days of gestation and term (147 days) and used for histological, electrophysiological, and molecular analysis. Histological abnormalities are evident in CFTR-/- -/-  animals by 80 days of gestation, equivalent to 21 weeks in humans. Acinar and ductal dilation, mucus obstruction, and fibrosis are observed in the pancreas; biliary fibrosis, cholestasis, and gallbladder hypoplasia in the liver; and intestinal meconium obstruction, as seen at birth in all large animal models of CF. Concurrently, cystic fibrosis transmembrane conductance regulator (CFTR)-dependent short circuit current is present in WT tracheal epithelium by 80 days gestation and is absent from CFTR -/- tissues. Transcriptomic profiles of tracheal tissues confirm the early expression of CFTR and suggest that its loss does not globally impair tracheal differentiation.

An ectopic enhancer restores CFTR expression through de novo chromatin looping.

Transcription of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is regulated by both ubiquitous and cell-type selective cis-regulatory elements (CREs). These CREs include extragenic and intronic enhancers that bind lineage-specific transcription factors, and architectural protein-marked structural elements. Deletion of the airway-selective -35 kb enhancer in 16HBE14o- lung epithelial cells was shown earlier to disrupt normal enhancer-promoter looping at the CFTR locus and nearly abolish its expression. Using a 16HBE14o- clone that lacks the endogenous -35 kb CRE, we explore the impact of relocating the functional core of this element to an ectopic site in intron 1. The -35 kb sequence establishes a de novo enhancer signature in chromatin at the insertion site, and augments CFTR expression, albeit not fully restoring WT levels. The relocated -35 kb enhancer also initiates de novo chromatin contacts with the CFTR promoter and other known CFTR CREs. These results are broadly relevant to therapeutic gene editing.

Dual SMAD inhibition enhances the longevity of human epididymis epithelial cells.

Primary human epididymis epithelial (HEE) cells are valuable reagents for functional studies on the human epididymis. We used them previously to determine the transcriptional networks that establish cell identity along the length of the epididymis from caput, corpus, and cauda. These studies on HEE cells and organoids derived from them revealed important cellular properties. However, similar to other primary cells, HEE cells undergo replicative senescence and de-differentiation in culture. A cocktail of small molecules was shown elsewhere to extend longevity of epithelial basal cells. The components included transforming growth factor ß (TGF-ß)/bone morphogenetic protein (BMP) antagonists, WNT agonist, and Rho-associated and coiled-coil containing protein kinase (ROCK) inhibitor (ROCKi), which together prevented the senescence-related upregulation of TGF-ß signaling pathway members. Here, we treat HEE cells with the same cocktail and observed enhanced replicative potential and prolonged expression of markers of HEE differentiation. This treatment expands the differentiated HEE cell population available from individual epididymis tissue samples that can be used for molecular, cellular, and functional studies.

A Distracted Scientist: The Life and Contributions of John Senders.

OBJECTIVE: To provide an evaluative and personal overview of the life and contributions of Professor John Senders and to introduce this Special Issue dedicated to his memory. BACKGROUND: John Senders made many profound contributions to HF/E. These various topics are exemplified by the range of papers which compose the Special Issue. Collectively, these works document and demonstrate the impact of his many valuable research works. METHOD: The Special Issue serves to summarize Senders' collective body of work as can be extracted from archival sources. This introductory paper recounts a series of remembrances derived from personal relationships, as well as the products of cooperative investigative research. RESULTS: This collective evaluative process documents Senders' evident and deserved status in the highest pantheon of HF/E pioneers. It records his extraordinary life, replete with accounts of his insights and joie de vivre in exploring and explaining the world which surrounded him. APPLICATIONS: Senders' record of critical contributions provides the example, par excellence, of the successful and fulfilling life in science. It encourages all, both researchers and practitioners alike, in their own individual search for excellence.

Enhancement of airway epithelial cell differentiation by pulmonary endothelial cell co-culture.

Cross-talk between lung epithelial cells and their microenvironment has an important physiological role in development. Using an in vitro model of differentiation of human induced pluripotent stem cells (iPSCs) to air-liquid interface (ALI)-cultured lung epithelial cells, we investigated the contribution of the microenvironment to maintenance of the lung progenitor cell state. Our protocol modeled in vivo cell-to-matrix and cell-to-cell interactions. These included growth of iPSCs on inserts coated with different basement membrane proteins (collagen I, IV, fibronectin, heparan sulfate or Matrigel plus collagen IV) and co-culture with human pulmonary microvascular endothelial cells (HPMECs). Marker gene expression was measured by RT-qPCR and protein expression and localization was confirmed by immunocytochemistry. The results showed that iPSCs grown on collagen IV had the highest success rate in terms of differentiation to robust ALI-cultured lung epithelial cells, followed by fibronectin, collagen I and heparan sulfate. Coating with Matrigel mixed with collagen IV further increased the success rate to > 97 %. Co-culture of iPSCs with HPMECs enhanced the expression of key airway lineage markers (NKX2.1, KRT5, TP63, MUC5AC, MUC16, FOXJ1, CFTR and SCGB1A1) during ALI culture. Cross-talk between iPSCs and their microenvironment during cell differentiation had a significant effect on lung epithelial cell differentiation in these 3D in vitro models. Both matrix proteins and endothelial cells play critical roles in the differentiation of lung progenitor cells.

Cell function and identity revealed by comparative scRNA-seq analysis in human nasal, bronchial and epididymis epithelia.

The evolutionary relationship of cells within tissues having a similar function but located in different anatomical sites is of considerable biological interest. The development of single-cell RNA sequencing (scRNA-seq) protocols has greatly enhanced opportunities to address this topic. Here we focus on cells in the epithelium which lines two regions of the human respiratory tract and the male genital ducts to delineate the shared, differentiated functions of the different cell populations. Transcriptomic data were used to assess the gene expression profiles of human bronchial, nasal, and epididymal epithelium (HBE, HNE, and HEE). Bulk RNA-seq showed many shared genes expressed in cells from the nasal and bronchial epithelium and highlighted their divergence from the epididymal epithelium. ScRNA-seq in HBE and HNE cells demonstrated overlapping gene expression patterns within basal and secretory cell populations. Moreover, the distribution of cell types was altered in HNE cells derived from donors with cystic fibrosis (CF) when compared to cells from healthy donors. Next, the HBE and HNE datasets were merged and confirmed intersection of cell type gene expression profiles from the two sites. However, secretory and ciliated cells were the most abundant types in the HBE samples, while more basal cells were seen in the HNE populations. We then merged single-cell data from the epididymis to determine if overlapping functions of these cells corresponded to those in the airway. Of note, only the pulmonary ionocytes/epididymis clear cells showed a strongly conserved identity, which was confirmed by imputation in bulk RNA-seq datasets from the same cells.

Sexual Well-Being After Nipple-Sparing Mastectomy: Does Preservation of the Nipple Matter?

INTRODUCTION: The primary aim of this study was to evaluate patient-reported outcome measures in patients undergoing mastectomy with and without breast reconstruction (immediate or delayed) with and without nipple preservation. METHODS: All female patients undergoing mastectomy between 2011 and 2015 at Mayo Clinic Rochester were identified and were mailed the BREAST-Q survey. Breast satisfaction, psychosocial well-being, and sexual well-being were evaluated and compared by surgery type using Wilcoxon rank-sum tests for univariate analysis and linear regression for multivariable analysis adjusting for potential confounders. RESULTS: Of 1547 patients, 771 completed the BREAST-Q survey (response rate 50%). Of these 771 respondents, 237 (31%) did not have reconstruction, 198 (26%) had nipple-sparing mastectomy with reconstruction (NSM), and 336 (44%) had skin-sparing mastectomy with reconstruction (SSM) ± nipple-areolar complex (NAC) reconstruction (via surgery ± tattoo). Patients with breast reconstruction had consistently higher BREAST-Q scores versus those without. Comparing NSM with all SSMs, there was no difference in satisfaction with breasts (mean 71.8 vs. 70.2, p = 0.21) or psychosocial well-being (mean 81.9 vs. 81.3, p = 0.47); however, sexual well-being was significantly higher in the NSM group on univariate (mean 64.5 vs. 58.0, p = 0.002) and multivariable (ß = -4.69, p = 0.03) analysis. Sexual well-being scores were similar for NSM and the SSM subgroups with any type of NAC reconstruction. CONCLUSIONS: This study demonstrates that NSM positively impacts patient sexual well-being after breast reconstruction compared with SSM, particularly SSM without nipple reconstruction or tattoo. SSM with any type of NAC reconstruction achieved similar satisfaction and sexual well-being to those undergoing NSM.

Krüppel-Like Factor 5 Regulates CFTR Expression Through Repression by Maintaining Chromatin Architecture Coupled with Direct Enhancer Activation.

Single cell RNA-sequencing has accurately identified cell types within the human airway that express the Cystic Fibrosis Transmembrane Conductance regulator (CFTR) gene. Low abundance CFTR transcripts are seen in many secretory cells, while high levels are restricted to rare pulmonary ionocytes. Here we focus on the mechanisms coordinating basal CFTR expression in the secretory compartment. Cell-selective regulation of CFTR is achieved within its invariant topologically associating domain by the recruitment of cis-regulatory elements (CREs). CRE activity is coordinated by cell-type-selective transcription factors. One such factor, Krüppel-Like Factor 5 (KLF5), profoundly represses CFTR transcript and protein in primary human airway epithelial cells and airway cell lines. Here we reveal the mechanism of action of KLF5 upon the CFTR gene. We find that depletion or ablation of KLF5 from airway epithelial cells changes higher order chromatin structure at the CFTR locus. Critical looping interactions that are required for normal gene expression are altered, the H3K27ac active chromatin mark is redistributed, and CTCF occupancy is modified. However, mutation of a single KLF5 binding site within a pivotal airway cell CRE abolishes CFTR expression. Hence, KLF5 has both direct activating and indirect repressive effects, which together coordinate CFTR expression in the airway.

Cross-talk between enhancers, structural elements and activating transcription factors maintains the 3D architecture and expression of the CFTR gene.

Robust protocols to examine 3D chromatin structure have greatly advanced knowledge of gene regulatory mechanisms. Here we focus on the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which provides a paradigm for validating models of gene regulation built upon genome-wide analysis. We examine the mechanisms by which multiple cis-regulatory elements (CREs) at the CFTR gene coordinate its expression in intestinal epithelial cells. Using CRISPR/Cas9 to remove CREs, individually and in tandem, followed by assays of gene expression and higher-order chromatin structure (4C-seq), we reveal the cross-talk and dependency of two cell-specific intronic enhancers. The results suggest a mechanism whereby the locus responds when CREs are lost, which may involve activating transcription factors such as FOXA2. Also, by removing the 5' topologically-associating domain (TAD) boundary, we illustrate its impact on CFTR gene expression and architecture. These data suggest a multi-layered regulatory hierarchy that is highly sensitive to perturbations.

Sheep models of F508del and G542X cystic fibrosis mutations show cellular responses to human therapeutics.

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The F508del and G542X are the most common mutations found in US patients, accounting for 86.4% and 4.6% of all mutations, respectively. The F508del causes deletion of the phenylalanine residue at position 508 and is associated with impaired CFTR protein folding. The G542X is a nonsense mutation that introduces a stop codon into the mRNA, thus preventing normal CFTR protein synthesis. Here, we describe the generation of CFTRF508del / F508del and CFTRG542X / G542X lambs using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT). First, we introduced either F508del or G542X mutations into sheep fetal fibroblasts that were subsequently used as nuclear donors for SCNT. The newborn CF lambs develop pathology similar to CFTR -/- sheep and CF patients. Moreover, tracheal epithelial cells from the CFTRF508del / F508del lambs responded to a human CFTR (hCFTR) potentiator and correctors, and those from CFTRG542X / G542X lambs showed modest restoration of CFTR function following inhibition of nonsense-mediated decay (NMD) and aminoglycoside antibiotic treatments. Thus, the phenotype and electrophysiology of these novel models represent an important advance for testing new CF therapeutics and gene therapy to improve the health of patients with this life-limiting disorder.

BACH1, the master regulator of oxidative stress, has a dual effect on CFTR expression.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene lies within a topologically associated domain (TAD) in which multiple cis-regulatory elements (CREs) and transcription factors (TFs) regulate its cell-specific expression. The CREs are recruited to the gene promoter by a looping mechanism that depends upon both architectural proteins and specific TFs. An siRNA screen to identify TFs coordinating CFTR expression in airway epithelial cells suggested an activating role for BTB domain and CNC homolog 1 (BACH1). BACH1 is a ubiquitous master regulator of the cellular response to oxidative stress. Here, we show that BACH1 may have a dual effect on CFTR expression by direct occupancy of CREs at physiological oxygen (∼8%), while indirectly modulating expression under conditions of oxidative stress. Hence BACH1, can activate or repress the same gene, to fine tune expression in response to environmental cues such as cell stress. Furthermore, our 4C-seq data suggest that BACH1 can also directly regulate CFTR gene expression by modulating locus architecture through occupancy at known enhancers and structural elements, and depletion of BACH1 alters the higher order chromatin structure.

Human Molecular Genetics and the long road to treating cystic fibrosis.

The causative gene in cystic fibrosis (CF) was identified in 1989, 3 years before the publication of the first issue of Human Molecular Genetics. The cystic fibrosis transmembrane conductance regulator (CFTR) gene was among the first underlying a common inherited disorder to be cloned, and hence, its subsequent utilization toward a cure for CF provides a roadmap for other monogenic diseases. Over the past 30 years, the advances that built upon knowledge of the gene and the CFTR protein to develop effective therapeutics have been remarkable, and yet, the setbacks have also been challenging. Technological progress in other fields has often circumvented the barriers. This review focuses on key aspects of CF diagnostics and current approaches to develop new therapies for all CFTR mutations. It also highlights the major research advances that underpinned progress toward treatments and considers the remaining obstacles.