Impaired intestinal free fatty acid transport followed by chylomicron malformation, not pancreatic insufficiency, cause metabolic defects in cystic fibrosis.

Intestinal disease is one of the earliest manifestations of cystic fibrosis (CF) in children and is closely tied to deficits in growth and nutrition, both of which are directly linked to future mortality. Patients are treated aggressively with pancreatic enzyme replacement therapy and a high-fat diet to circumvent fat malabsorption, but this does not reverse growth and nutritional defects. We hypothesized that defects in chylomicron production could explain why CF body weights and nutrition are so resistant to clinical treatments. We used gold standard intestinal lipid absorption and metabolism approaches, including mouse mesenteric lymph cannulation, in vivo chylomicron secretion kinetics, transmission electron microscopy, small intestinal organoids, and chylomicron metabolism assays to test this hypothesis. In mice expressing the G542X mutation in cystic fibrosis transmembrane conductance regulator (CFTR-/- mice), we find that defective FFA trafficking across the epithelium into enterocytes drives a chylomicron formation defect. Furthermore, G542X mice secrete small, triglyceride-poor chylomicrons into the lymph and blood. These defective chylomicrons are cleared into extraintestinal tissues at ∼10-fold faster than WT chylomicrons. This defect in FFA absorption resulting in dysfunctional chylomicrons cannot be explained by steatorrhea or pancreatic insufficiency and is maintained in primary small intestinal organoids treated with micellar lipids. These studies suggest that the ultrahigh-fat diet that most people with CF are counselled to follow may instead make steatorrhea and malabsorption defects worse by overloading the absorptive capacity of the CF small intestine.

Bone Marrow Transplantation Rescues Monocyte Recruitment Defect and Improves Cystic Fibrosis in Mice.

Cystic fibrosis (CF) is an inherited life-threatening disease accompanied by repeated lung infections and multiorgan inflammation that affects tens of thousands of people worldwide. The causative gene, cystic fibrosis transmembrane conductance regulator (CFTR), is mutated in CF patients. CFTR functions in epithelial cells have traditionally been thought to cause the disease symptoms. Recent work has shown an additional defect: monocytes from CF patients show a deficiency in integrin activation and adhesion. Because monocytes play critical roles in controlling infections, defective monocyte function may contribute to CF progression. In this study, we demonstrate that monocytes from CFTRΔF508 mice (CF mice) show defective adhesion under flow. Transplanting CF mice with wild-type (WT) bone marrow after sublethal irradiation replaced most (60-80%) CF monocytes with WT monocytes, significantly improved survival, and reduced inflammation. WT/CF mixed bone marrow chimeras directly demonstrated defective CF monocyte recruitment to the bronchoalveolar lavage and the intestinal lamina propria in vivo. WT mice reconstituted with CF bone marrow also show lethality, suggesting that the CF defect in monocytes is not only necessary but also sufficient to cause disease. We also show that monocyte-specific knockout of CFTR retards weight gains and exacerbates dextran sulfate sodium-induced colitis. Our findings show that providing WT monocytes by bone marrow transfer rescues mortality in CF mice, suggesting that similar approaches may mitigate disease in CF patients.

Bronchopulmonary Dysplasia and Pulmonary Hypertension. The Role of Smooth Muscle adh5.

Bronchopulmonary dysplasia (BPD) is characterized by alveolar simplification, airway hyperreactivity, and pulmonary hypertension. In our BPD model, we have investigated the metabolism of the bronchodilator and pulmonary vasodilator GSNO (S-nitrosoglutathione). We have shown the GSNO catabolic enzyme encoded by adh5 (alcohol dehydrogenase-5), GSNO reductase, is epigenetically upregulated in hyperoxia. Here, we investigated the distribution of GSNO reductase expression in human BPD and created an animal model that recapitulates the human data. Blinded comparisons of GSNO reductase protein expression were performed in human lung tissues from infants and children with and without BPD. BPD phenotypes were evaluated in global (adh5-/-) and conditional smooth muscle (smooth muscle/adh5-/-) adh5 knockout mice. GSNO reductase was prominently expressed in the airways and vessels of human BPD subjects. Compared with controls, expression was greater in BPD smooth muscle, particularly in vascular smooth muscle (2.4-fold; P = 0.003). The BPD mouse model of neonatal hyperoxia caused significant alveolar simplification, airway hyperreactivity, and right ventricular and vessel hypertrophy. Global adh5-/- mice were protected from all three aspects of BPD, whereas smooth muscle/adh5-/- mice were only protected from pulmonary hypertensive changes. These data suggest adh5 is required for the development of BPD. Expression in the pulmonary vasculature is relevant to the pathophysiology of BPD-associated pulmonary hypertension. GSNO-mimetic agents or GSNO reductase inhibitors, both of which are currently in clinical trials for other conditions, could be considered for further study in BPD.

Lysophosphatidic Acid Increases Maturation of Brush Borders and SGLT1 Activity in MYO5B-deficient Mice, a Model of Microvillus Inclusion Disease.

BACKGROUND & AIM: Myosin VB (MYO5B) is an essential trafficking protein for membrane recycling in gastrointestinal epithelial cells. The inactivating mutations of MYO5B cause the congenital diarrheal disease, microvillus inclusion disease (MVID). MYO5B deficiency in mice causes mislocalization of SGLT1 and NHE3, but retained apical function of CFTR, resulting in malabsorption and secretory diarrhea. Activation of lysophosphatidic acid (LPA) receptors can improve diarrhea, but the effect of LPA on MVID symptoms is unclear. We investigated whether LPA administration can reduce the epithelial deficits in MYO5B-knockout mice. METHODS: Studies were conducted with tamoxifen-induced, intestine-specific knockout of MYO5B (VilCreERT2;Myo5bflox/flox) and littermate controls. Mice were given LPA, an LPAR2 agonist (GRI977143), or vehicle for 4 days after a single injection of tamoxifen. Apical SGLT1 and CFTR activities were measured in Üssing chambers. Intestinal tissues were collected, and localization of membrane transporters was evaluated by immunofluorescence analysis in tissue sections and enteroids. RNA sequencing and enrichment analysis were performed with isolated jejunal epithelial cells. RESULTS: Daily administration of LPA reduced villus blunting, frequency of multivesicular bodies, and levels of cathepsins in intestinal tissues of MYO5B-knockout mice compared with vehicle administration. LPA partially restored the brush border height and the localization of SGLT1 and NHE3 in small intestine of MYO5B-knockout mice and enteroids. The SGLT1-dependent short-circuit current was increased and abnormal CFTR activities were decreased in jejunum from MYO5B-knockout mice given LPA compared with vehicle. CONCLUSIONS: LPA may regulate a MYO5B-independent trafficking mechanism and brush border maturation, and therefore be developed for treatment of MVID.

Mutations in the mitochondrial ribosomal protein MRPS22 lead to primary ovarian insufficiency.

Primary ovarian insufficiency (POI) is characterized by amenorrhea and loss or dysfunction of ovarian follicles prior to the age of 40. POI has been associated with autosomal recessive mutations in genes involving hormonal signaling and folliculogenesis, however, the genetic etiology of POI most often remains unknown. Here we report MRPS22 homozygous missense variants c.404G>A (p.R135Q) and c.605G>A (p.R202H) identified in four females from two independent consanguineous families as a novel genetic cause of POI in adolescents. Both missense mutations identified in MRPS22 are rare, occurred in highly evolutionarily conserved residues, and are predicted to be deleterious to protein function. In contrast to prior reports of mutations in MRPS22 associated with severe mitochondrial disease, the POI phenotype is far less severe. Consistent with this genotype-phenotype correlation, mitochondrial defects in oxidative phosphorylation or rRNA levels were not detected in fibroblasts derived from the POI patients, suggesting a non-bioenergetic or tissue-specific mitochondrial defect. Furthermore, we demonstrate in a Drosophila model that mRpS22 deficiency specifically in somatic cells of the ovary had no effect on fertility, whereas flies with mRpS22 deficiency specifically in germ cells were infertile and agametic, demonstrating a cell autonomous requirement for mRpS22 in germ cell development. These findings collectively identify that MRPS22, a component of the small mitochondrial ribosome subunit, is critical for ovarian development and may therefore provide insight into the pathophysiology and treatment of ovarian dysfunction.

Synergy between Readthrough and Nonsense Mediated Decay Inhibition in a Murine Model of Cystic Fibrosis Nonsense Mutations.

Many heritable genetic disorders arise from nonsense mutations, which generate premature termination codons (PTCs) in transcribed mRNA. PTCs ablate protein synthesis by prematurely terminating the translation of mutant mRNA, as well as reducing mutant mRNA quantity through targeted degradation by nonsense-mediated decay (NMD) mechanisms. Therapeutic strategies for nonsense mutations include facilitating ribosomal readthrough of the PTC and/or inhibiting NMD to restore protein function. However, the efficacy of combining readthrough agents and NMD inhibitors has not been thoroughly explored. In this study, we examined combinations of known NMD inhibitors and readthrough agents using functional analysis of the CFTR protein in primary cells from a mouse model carrying a G542X nonsense mutation in Cftr. We observed synergy between an inhibitor of the NMD component SMG-1 (SMG1i) and the readthrough agents G418, gentamicin, and paromomycin, but did not observe synergy with readthrough caused by amikacin, tobramycin, PTC124, escin, or amlexanox. These results indicate that treatment with NMD inhibitors can increase the quantity of functional protein following readthrough, and that combining NMD inhibitors and readthrough agents represents a potential therapeutic option for treating nonsense mutations.

Linaclotide improves gastrointestinal transit in cystic fibrosis mice by inhibiting sodium/hydrogen exchanger 3.

Gastrointestinal dysfunction in cystic fibrosis (CF) is a prominent source of pain among patients with CF. Linaclotide, a guanylate cyclase C (GCC) receptor agonist, is a US Food and Drug Administration-approved drug prescribed for chronic constipation but has not been widely used in CF, as the cystic fibrosis transmembrane conductance regulator (CFTR) is the main mechanism of action. However, anecdotal clinical evidence suggests that linaclotide may be effective for treating some gastrointestinal symptoms in CF. The goal of this study was to determine the effectiveness and mechanism of linaclotide in treating CF gastrointestinal disorders using CF mouse models. Intestinal transit, chloride secretion, and intestinal lumen fluidity were assessed in wild-type and CF mouse models in response to linaclotide. CFTR and sodium/hydrogen exchanger 3 (NHE3) response to linaclotide was also evaluated. Linaclotide treatment improved intestinal transit in mice carrying either F508del or null Cftr mutations but did not induce detectable Cl- secretion. Linaclotide increased fluid retention and fluidity of CF intestinal contents, suggesting inhibition of fluid absorption. Targeted inhibition of sodium absorption by the NHE3 inhibitor tenapanor produced improvements in gastrointestinal transit similar to those produced by linaclotide treatment, suggesting that inhibition of fluid absorption by linaclotide contributes to improved gastrointestinal transit in CF. Our results demonstrate that linaclotide improves gastrointestinal transit in CF mouse models by increasing luminal fluidity through inhibiting NHE3-mediated sodium absorption. Further studies are necessary to assess whether linaclotide could improve CF intestinal pathologies in patients. GCC signaling and NHE3 inhibition may be therapeutic targets for CF intestinal manifestations. NEW & NOTEWORTHY Linaclotide's primary mechanism of action in alleviating chronic constipation is through cystic fibrosis transmembrane conductance regulator (CFTR), negating its use in patients with cystic fibrosis (CF). For the first time, our findings suggest that in the absence of CFTR, linaclotide can improve fluidity of the intestinal lumen through the inhibition of sodium/hydrogen exchanger 3. These findings suggest that linaclotide could improve CF intestinal pathologies in patients.

Dynamic expression of long noncoding RNAs reveals their potential roles in spermatogenesis and fertility.

Mammalian reproduction requires that males and females produce functional haploid germ cells through complex cellular differentiation processes known as spermatogenesis and oogenesis, respectively. While numerous studies have functionally characterized protein-coding genes and small noncoding RNAs (microRNAs and piRNAs) that are essential for gametogenesis, the roles of regulatory long noncoding RNAs (lncRNAs) are yet to be fully characterized. Previously, we and others have demonstrated that intergenic regions of the mammalian genome encode thousands of long noncoding RNAs, and many studies have now demonstrated their critical roles in key biological processes. Thus, we postulated that some lncRNAs may also impact mammalian spermatogenesis and fertility. In this study, we identified a dynamic expression pattern of lncRNAs during murine spermatogenesis. Importantly, we identified a subset of lncRNAs and very few mRNAs that appear to escape meiotic sex chromosome inactivation, an epigenetic process that leads to the silencing of the X- and Y-chromosomes at the pachytene stage of meiosis. Further, some of these lncRNAs and mRNAs show a strong testis expression pattern suggesting that they may play key roles in spermatogenesis. Lastly, we generated a mouse knockout of one X-linked lncRNA, Tslrn1 (testis-specific long noncoding RNA 1), and found that males carrying a Tslrn1 deletion displayed normal fertility but a significant reduction in spermatozoa. Our findings demonstrate that dysregulation of specific mammalian lncRNAs is a novel mechanism of low sperm count or infertility, thus potentially providing new biomarkers and therapeutic strategies.

Moving beyond genetics: is FAM13A a major biological contributor in lung physiology and chronic lung diseases?

Variants in FAM13A have been found in genome-wide association studies (GWAS) to associate with lung function in the general population as well as in several common chronic lung diseases (CLD) such as chronic obstructive pulmonary disease (COPD), asthma, as well as in idiopathic interstitial pneumonias (IIP). The gene was cloned in 2004, yet the encoded protein has not been characterised and its function is unknown. The redundancy of its genetic contribution in CLD suggests a major function of this gene both in lung physiology and CLD. This review provides a brief summary of the current knowledge of FAM13A, and demonstrates the necessity to resolve its biological function besides its well accepted genetic contribution. Further interpretations of FAM13A variants may help in the understanding of CLD mechanisms and reveal opportunity for intervention.

SMC1beta-deficient female mice provide evidence that cohesins are a missing link in age-related nondisjunction.

Mitotic chromosome segregation is facilitated by the cohesin complex, which maintains physical connections between sister chromatids until anaphase. Meiotic cell division is considerably more complex, as cohesion must be released sequentially to facilitate orderly segregation of chromosomes at both meiosis I and meiosis II. This necessitates meiosis-specific cohesin components; recent studies in rodents suggest that these influence chromosome behavior during both cell division and meiotic prophase. To elucidate the role of the meiosis-specific cohesin SMC1beta (encoded by Smc1l2) in oogenesis, we carried out meiotic studies of female SMC1beta-deficient mice. Our results provide the first direct evidence that SMC1beta acts as a chiasma binder in mammals, stabilizing sites of exchange until anaphase. Additionally, our observations support the hypothesis that deficient cohesion is an underlying cause of human age-related aneuploidy.

In vivo editing of lung stem cells for durable gene correction in mice.

In vivo genome correction holds promise for generating durable disease cures; yet, effective stem cell editing remains challenging. In this work, we demonstrate that optimized lung-targeting lipid nanoparticles (LNPs) enable high levels of genome editing in stem cells, yielding durable responses. Intravenously administered gene-editing LNPs in activatable tdTomato mice achieved >70% lung stem cell editing, sustaining tdTomato expression in >80% of lung epithelial cells for 660 days. Addressing cystic fibrosis (CF), NG-ABE8e messenger RNA (mRNA)-sgR553X LNPs mediated >95% cystic fibrosis transmembrane conductance regulator (CFTR) DNA correction, restored CFTR function in primary patient-derived bronchial epithelial cells equivalent to Trikafta for F508del, corrected intestinal organoids and corrected R553X nonsense mutations in 50% of lung stem cells in CF mice. These findings introduce LNP-enabled tissue stem cell editing for disease-modifying genome correction.

A minimally invasive bronchoscopic approach for direct delivery to murine airways and application to models of pulmonary infection.

The laboratory mouse is used extensively for human disease modeling and preclinical therapeutic testing for efficacy, biodistribution, and toxicity. The variety of murine models available, and the ability to create new ones, eclipses all other species, but the size of mice and their organs create challenges for many in vivo studies. For pulmonary research, improved methods to access murine airways and lungs, and track substances administered to them, would be desirable. A nonsurgical endoscopic system with a camera, effectively a bronchoscope, coupled with a cryoimaging fluorescence microscopy technique to view the lungs in 3D, is described here that allows visualization of the procedure, including the anatomical location at which substances are instilled and fluorescence detection of those substances. We have applied it to bacterial infection studies to characterize better and optimize a chronic lung infection murine model in which we instill bacteria-laden agarose beads into the airways and lungs to extend the duration of the infection and inflammation. The use of the endoscope as guidance for placing a catheter into the airways is simple and quick, requiring only momentary sedation, and reduces post-procedural mortality compared with our previous instillation method that includes a trans-tracheal surgery. The endoscopic method improves speed and precision of delivery while reducing the stress on animals and the number of animals generated and used for experiments.

Lung SORT LNPs enable precise homology-directed repair mediated CRISPR/Cas genome correction in cystic fibrosis models.

Approximately 10% of Cystic Fibrosis (CF) patients, particularly those with CF transmembrane conductance regulator (CFTR) gene nonsense mutations, lack effective treatments. The potential of gene correction therapy through delivery of the CRISPR/Cas system to CF-relevant organs/cells is hindered by the lack of efficient genome editor delivery carriers. Herein, we report improved Lung Selective Organ Targeting Lipid Nanoparticles (SORT LNPs) for efficient delivery of Cas9 mRNA, sgRNA, and donor ssDNA templates, enabling precise homology-directed repair-mediated gene correction in CF models. Optimized Lung SORT LNPs deliver mRNA to lung basal cells in Ai9 reporter mice. SORT LNP treatment successfully corrected the CFTR mutations in homozygous G542X mice and in patient-derived human bronchial epithelial cells with homozygous F508del mutations, leading to the restoration of CFTR protein expression and chloride transport function. This proof-of-concept study will contribute to accelerating the clinical development of mRNA LNPs for CF treatment through CRISPR/Cas gene correction.

Cystic fibrosis growth retardation is not correlated with loss of Cftr in the intestinal epithelium.

Maldigestion due to exocrine pancreatic insufficiency leads to intestinal malabsorption and consequent malnutrition, a mechanism proposed to cause growth retardation associated with cystic fibrosis (CF). However, although enzyme replacement therapy combined with increased caloric intake improves weight gain, the effect on stature is not significant, suggesting that growth retardation has a more complex etiology. Mouse models of CF support this, since these animals do not experience exocrine pancreatic insufficiency yet are growth impaired. Cftr absence from the intestinal epithelium has been suggested as a primary source of growth retardation in CF mice, a concept we directly tested by generating mouse models with Cftr selectively inactivated or restored in intestinal epithelium. The relationship between growth and functional characteristics of the intestines, including transepithelial electrophysiology, incidence of intestinal obstruction, and histopathology, were assessed. Absence of Cftr exclusively from intestinal epithelium resulted in loss of cAMP-stimulated short-circuit current, goblet cell hyperplasia, and occurrence of intestinal obstructions but only slight and transient impaired growth. In contrast, specifically restoring Cftr to the intestinal epithelium resulted in restoration of ion transport and completely protected against obstruction and histopathological anomalies, but growth was indistinguishable from CF mice. These results indicate that absence of Cftr in the intestinal epithelium is an important contributor to the intestinal obstruction phenotype in CF but does not correlate with the observed growth reduction in CF.

Cohesin SMC1 beta is required for meiotic chromosome dynamics, sister chromatid cohesion and DNA recombination.

Sister chromatid cohesion ensures the faithful segregation of chromosomes in mitosis and in both meiotic divisions. Meiosis-specific components of the cohesin complex, including the recently described SMC1 isoform SMC1 beta, were suggested to be required for meiotic sister chromatid cohesion and DNA recombination. Here we show that SMC1 beta-deficient mice of both sexes are sterile. Male meiosis is blocked in pachytene; female meiosis is highly error-prone but continues until metaphase II. Prophase axial elements (AEs) are markedly shortened, chromatin extends further from the AEs, chromosome synapsis is incomplete, and sister chromatid cohesion in chromosome arms and at centromeres is lost prematurely. In addition, crossover-associated recombination foci are absent or reduced, and meiosis-specific perinuclear telomere arrangements are impaired. Thus, SMC1 beta has a key role in meiotic cohesion, the assembly of AEs, synapsis, recombination, and chromosome movements.

Enhancing Cystic Fibrosis Immune Regulation.

In cystic fibrosis (CF), sustained infection and exuberant inflammation results in debilitating and often fatal lung disease. Advancement in CF therapeutics has provided successful treatment regimens for a variety of clinical consequences in CF; however effective means to treat the pulmonary infection and inflammation continues to be problematic. Even with the successful development of small molecule cystic fibrosis transmembrane conductance regulator (CFTR) correctors and potentiators, there is only a modest effect on established infection and inflammation in CF patients. In the pursuit of therapeutics to treat inflammation, the conundrum to address is how to overcome the inflammatory response without jeopardizing the required immunity to manage pathogens and prevent infection. The key therapeutic would have the capacity to dull the inflammatory response, while sustaining the ability to manage infections. Advances in cell-based therapy have opened up the avenue for dynamic and versatile immune interventions that may support this requirement. Cell based therapy has the capacity to augment the patient's own ability to manage their inflammatory status while at the same time sustaining anti-pathogen immunity. The studies highlighted in this manuscript outline the potential use of cell-based therapy for CF. The data demonstrate that 1) total bone marrow aspirates containing Cftr sufficient hematopoietic and mesenchymal stem cells (hMSCs) provide Cftr deficient mice >50% improvement in survival and improved management of infection and inflammation; 2) myeloid cells can provide sufficient Cftr to provide pre-clinical anti-inflammatory and antimicrobial benefit; 3) hMSCs provide significant improvement in survival and management of infection and inflammation in CF; 4) the combined interaction between macrophages and hMSCs can potentially enhance anti-inflammatory and antimicrobial support through manipulating PPARγ. These data support the development of optimized cell-based therapeutics to enhance CF patient's own immune repertoire and capacity to maintain the balance between inflammation and pathogen management.

The Burkholderia cenocepacia Type VI Secretion System Effector TecA Is a Virulence Factor in Mouse Models of Lung Infection.

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex (Bcc), a group of bacteria with members responsible for causing lung infections in cystic fibrosis (CF) patients. The most severe outcome of Bcc infection in CF patients is cepacia syndrome, a disease characterized by necrotizing pneumonia with bacteremia and sepsis. B. cenocepacia is strongly associated with cepacia syndrome, making it one of the most virulent members of the Bcc. Mechanisms underlying the pathogenesis of B. cenocepacia in lung infections and cepacia syndrome remain to be uncovered. B. cenocepacia is primarily an intracellular pathogen and encodes the type VI secretion system (T6SS) effector TecA, which is translocated into host phagocytes. TecA is a deamidase that inactivates multiple Rho GTPases, including RhoA. Inactivation of RhoA by TecA triggers assembly of the pyrin inflammasome, leading to secretion of proinflammatory cytokines, such as interleukin-1ß, from macrophages. Previous work with the B. cenocepacia clinical isolate J2315 showed that TecA increases immunopathology during acute lung infection in C57BL/6 mice and suggested that this effector acts as a virulence factor by triggering assembly of the pyrin inflammasome. Here, we extend these results using a second B. cenocepacia clinical isolate, AU1054, to demonstrate that TecA exacerbates weight loss and lethality during lung infection in C57BL/6 mice and mice engineered to have a CF genotype. Unexpectedly, pyrin was dispensable for TecA virulence activity in both mouse infection models. Our findings establish that TecA is a B. cenocepacia virulence factor that exacerbates lung inflammation, weight loss, and lethality in mouse infection models. IMPORTANCE B. cenocepacia is often considered the most virulent species in the Bcc because of its close association with cepacia syndrome in addition to its capacity to cause chronic lung infections in CF patients (1). Prior to the current study, virulence factors of B. cenocepacia important for causing lethal disease had not been identified in a CF animal model of lung infection. Results of this study describe a CF mouse model and its use in demonstrating that the T6SS effector TecA of B. cenocepacia exacerbates inflammatory cell recruitment and weight loss and is required for lethality and, thus, acts as a key virulence factor during lung infection. This model will be important in further studies to better understand TecA's role as a virulence factor and in investigating ways to prevent or treat B. cenocepacia infections in CF patients. Additionally, TecA may be the founding member of a family of virulence factors in opportunistic pathogens.

Analyzing complex traits with congenic strains.

Congenic strains continue to be a fundamental resource for dissecting the genetic basis of complex traits. Traditionally, genetic variants (QTLs) that account for phenotypic variation in a panel of congenic strains are sought first by comparing phenotypes for each strain to the host (reference) strain, and then by examining the results to identify a common chromosome segment that provides the best match between genotype and phenotype across the panel. However, this "common-segment" method has significant limitations, including the subjective nature of the genetic model and an inability to deal formally with strain phenotypes that do not fit the model. We propose an alternative that we call "sequential" analysis and that is based on a unique principle of QTL analysis where each strain, corresponding to a single genotype, is tested individually for QTL effects rather than testing the congenic panel collectively for common effects across heterogeneous backgrounds. A minimum spanning tree, based on principles of graph theory, is used to determine the optimal sequence of strain comparisons. For two traits in two panels of congenic strains in mice, we compared results for the sequential method with the common-segment method as well as with two standard methods of QTL analysis, namely, interval mapping and multiple linear regression. The general utility of the sequential method was demonstrated with analysis of five additional traits in congenic panels from mice and rats. Sequential analysis rigorously resolved phenotypic heterogeneity among strains in the congenic panels and found QTLs that other methods failed to detect.

Voltage-gated potassium channel proteins and stereoselective S-nitroso-l-cysteine signaling.

S-nitroso-l-cysteine (L-CSNO) behaves as a ligand. Its soluble guanylate cyclase-independent (sGC-independent) effects are stereoselective - that is, not recapitulated by S-nitroso-d-cysteine (D-CSNO) - and are inhibited by chemical congeners. However, candidate L-CSNO receptors have not been identified. Here, we have used 2 complementary affinity chromatography assays - followed by unbiased proteomic analysis - to identify voltage-gated K+ channel (Kv) proteins as binding partners for L-CSNO. Stereoselective L-CSNO-Kv interaction was confirmed structurally and functionally using surface plasmon resonance spectroscopy; hydrogen deuterium exchange; and, in Kv1.1/Kv1.2/Kvß2-overexpressing cells, patch clamp assays. Remarkably, these sGC-independent L-CSNO effects did not involve S-nitrosylation of Kv proteins. In isolated rat and mouse respiratory control (petrosyl) ganglia, L-CSNO stereoselectively inhibited Kv channel function. Genetic ablation of Kv1.1 prevented this effect. In intact animals, L-CSNO injection at the level of the carotid body dramatically and stereoselectively increased minute ventilation while having no effect on blood pressure; this effect was inhibited by the L-CSNO congener S-methyl-l-cysteine. Kv proteins are physiologically relevant targets of endogenous L-CSNO. This may be a signaling pathway of broad relevance.

Delivering on the promise of gene editing for cystic fibrosis.

In this review, we describe a path for translation of gene editing into therapy for cystic fibrosis (CF). Cystic fibrosis results from mutations in the CFTR gene, with one allele predominant in patient populations. This simple, genetic etiology makes gene editing appealing for treatment of this disease. There already have been success in applying this approach to cystic fibrosis in cell and animal models, although these advances have been modest in comparison to advances for other disease. Less than six years after its first demonstration in animals, CRISPR/Cas gene editing is in early clinical trials for several disorders. Most clinical trials, thus far, attempt to edit genes in cells of the blood lineages. The advantage of the blood is that the stem cells are known, can be isolated, edited, selected, expanded, and returned to the body. The likely next trials will be in the liver, which is accessible to many delivery methods. For cystic fibrosis, the biggest hurdle is to deliver editors to other, less accessible organs. We outline a path by which delivery can be improved. The translation of new therapies doesn't occur in isolation, and the development of gene editors is occurring as advances in gene therapy and small molecule therapeutics are being made. The advances made in gene therapy may help develop delivery vehicles for gene editing, although major improvements are needed. Conversely, the approval of effective small molecule therapies for many patients with cystic fibrosis will raise the bar for translation of gene editing.