Stressors and Stress Responses in Cystic Fibrosis

Ageing is driven by the inexorable and stochastic accumulation of damage in biomolecules vital for proper cellular function. Although this process is fundamentally haphazard and uncontrollable, senescent decline and ageing is broadly influenced by genetic and extrinsic factors. Numerous gene mutations and treatments have been shown to extend the lifespan of diverse organisms ranging from the unicellular Saccharomyces cerevisiae to primates. It is becoming increasingly apparent that most such interventions ultimately interface with cellular stress response mechanisms, suggesting that longevity is intimately related to the ability of the organism to effectively cope with both intrinsic and extrinsic stress. Here, we survey the molecular mechanisms that link ageing to main stress response pathways, and mediate age-related changes in the effectiveness of the response to stress. We also discuss how each pathway contributes to modulate the ageing process. A better understanding of the dynamics and reciprocal interplay between stress responses and ageing is critical for the development of novel therapeutic strategies that exploit endogenous stress combat pathways against age-associated pathologies.

Etiology of azoospermia in 100 consecutive nonvasectomized men

Outcome of intracytoplasmic sperm injection for a couple in which the man is carrier of CFTR p.[R74W;V201M;D1270N] and p.P841R mutations and his spouse a heterozygous carrier of p.F508del mutation of the cystic fibrosis transmembrane conductance regulator gene

Cellular stress is something that a cell deals with on a regular basis. Fortunately, there are several different mechanisms that are set in place in order to deal with them. Depending on the stress present in the cell there will be different stress response pathways that are activated in order to mitigate the stress or initiate cellular death mechanisms. Transcription factors (TFs) are key components within these pathways, and this will be the focus of this thesis. TFs within mammalian stress response pathways are tasked with the essential regulation of multiple genes within these pathways and are oftentimes linked with diseases when mutated. ATF4 and P53 are two central TFs that work within the Integrated Stress Response and DNA damage response pathways respectively. This thesis will highlight important aspects of gene regulation within cell stress pathways and highlight these two central TFs and their roles in stress response pathways.

Cystic fibrosis (CF), a common lethal pulmonary disorder in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) that disturbs fluid homeostasis and host defense in target organs. The effects of CFTR and delta508-CFTR were assessed in transgenic mice that 1) lack CFTR expression (Cftr-/-); 2) express the human delta508 CFTR (CFTR(delta508)); 3) overexpress the normal human CFTR (CFTR(tg)) in respiratory epithelial cells. Genes were selected from Affymetrix Murine Gene-Chips analysis and subjected to functional classification, k-means clustering, promoter cis-elements/modules searching, literature mining, and pathway exploring. Genomic responses to Cftr-/- were not corrected by expression of CFTR(delta508). Genes regulating host defense, inflammation, fluid and electrolyte transport were similarly altered in Cftr-/- and CFTR(delta508) mice. CFTR(delta508) induced a primary disturbance in expression of genes regulating redox and antioxidant systems. Genomic responses to CFTR(tg) were modest and were not associated with lung pathology. CFTR(tg) and CFTR(delta508) induced genes encoding heat shock proteins and other chaperones but did not activate the endoplasmic reticulum-associated degradation pathway. RNAs encoding proteins that directly interact with CFTR were identified in each of the CFTR mouse models, supporting the hypothesis that CFTR functions within a multiprotein complex whose members interact at the level of protein-protein interactions and gene expression. Promoters of genes influenced by CFTR shared common regulatory elements, suggesting that their co-expression may be mediated by shared regulatory mechanisms. Genes and pathways involved in the response to CFTR may be of interest as modifiers of CF.

In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium

ObjectivesCystic fibrosis (CF) is a rare multi-systemic recessive disorder.1 The spectrum and frequencies of CFTR mutations causing CF varies among different populations in Europe and the Middle East, with varying...

Stress response pathways are emerging as conserved modulators of lifespan. The prevailing hypothesis is that activation of stress-responsive pathways, including the amino acid deprivation arm of the integrated stress response (ISR; the GCN2-ATF4 pathway), is prolongevity. Activation of ATF4 orthologs extends lifespan in Saccharomyces cerevisiae and Caenorhabditis elegans, but its role in other longer-lived organisms remains unclear. We comprehensively tested the role of the GCN2-ATF4 pathway in longevity in the fly (Drosophila melanogaster) for the first time. We used conditional genetic manipulation of dGCN2 and its downstream effector Drosophila ATF4 (crc; dATF4). In contrast to previous studies, we show that overexpression of dGCN2 and dATF4 significantly reduces lifespan, while knockdown (in vivo RNAi) of dATF4 extends lifespan. We confirmed that dATF4 activity was successfully modulated using a fluorescent dATF4 activation reporter. Borrelidin, a tRNA synthetase inhibitor, significantly reduced lifespan in a both dATF4 and diet-dependent manner, independent of microbial load, showing our modulation of dATF4 altered nutrient to ISR signaling. We further conducted long-read RNA sequencing and found that our manipulation of dATF4 changed global transcription in opposite directions, including known ATF4 target genes. Enrichment analysis revealed that dATF4 overexpression may drive metabolic stress, while dATF4 knockdown may upregulate proteostasis and DNA repair pathways. Our work reveals that ATF4 may exhibit a dual, dose-, and context-dependent role in aging. Chronic dATF4 activation is detrimental in flies, while chronic suppression is prolongevity. The GCN2-ATF4 pathway thus qualifies as a modifiable control of lifespan with cross-species relevance.

The relationship between cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations and congenital absence of the uterus and vagina (CAUV) was examined. CFTR mutations have previously been associated with congenital bilateral absence of the vas deferens (CBAVD). CBAVD is caused by a disruption in the vas deferens, a Wolffian duct derivative. Because the embryologic development of the Müllerian ducts directly depends on the prior normal development of the Wolffian ducts, the same gene products may be necessary for normal embryologic development of both ductal systems. This study evaluated the role of CFTR mutations in the development of CAUV. DNA samples from 25 patients with CAUV were tested for the presence of 33 of the most common CFTR mutations. Protein-coding DNA fragments from the CFTR gene were amplified in vitro by the polymerase chain reaction (PCR) and analyzed for mutations using allele-specific oligonucleotide (ASO) probes. Two patients were heterozygous for CFTR mutations. One was heterozygous for the W1282X mutation and the other was heterozygous for the DeltaF508 mutation. The incidence of the 33 CFTR mutations found in the patients with CAUV (8%) was twice that found in the general population (4%), but much less than the incidence of CFTR mutations in men with CBAVD (80%). This data suggests that it is unlikely for CFTR mutations to cause CAUV in females as they cause CBAVD in some males. Furthermore, the data suggest that CAUV in females may be the same disorder as CBAVD in males who do not have CFTR mutations.

Molecular characterization of the cystic fibrosis transmembrane conductance regulator gene in congenital absence of the vas deferens

Regulation of Chemokine Expression by NaCl Occurs Independently of Cystic Fibrosis Transmembrane Conductance Regulator in Macrophages

Microsphere Bead Arrays and Sequence Validation of 5/7/9T Genotypes for Multiplex Screening of Cystic Fibrosis Polymorphisms

The cystic fibrosis transmembrane conductance regulator (CFTR) gene, the gene responsible for the lethal hereditary disorder cystic fibrosis, codes for a membrane protein functioning as a cAMP-regulated Cl- channel. Evaluation of human CFTR mRNA transcripts from epithelial and nonepithelial cells demonstrated a CFTR cDNA containing a 260-base pair (bp) insertion between the known CFTR exons 23 and 24, introducing a premature stop codon that would result in a CFTR protein shortened by 61 amino acids at the carboxyl terminus compared to that expected from the normal reported human CFTR coding sequences. Sequence analysis of intron 23 of the CFTR gene demonstrated that the 260-bp insertion (named exon 24a), a part of the reported intron 23 and located consecutive to exon 24, is likely generated by an alternative splice acceptor site. The exon 24a+ CFTR mRNA transcripts represented 3-16% of the total CFTR transcripts in epithelial and nonepithelial cells. These observations suggest an unexpected plasticity of expression of the CFTR gene, where alternative splicing of precursor CFTR mRNA transcripts permits the use of an alternative exon derived from a genomic segment previously believed to function as an intron.

Tuberculosis (TB) is one of the most devastating infectious diseases caused by Mycobacterium tuberculosis (MTB). A high percentage of mortality and morbidity associated with TB has been reported globally with the highest number of cases reported in Asia. MTB is known to enter a state of dormancy and phenotypic drug resistance, when it is exposed to multiple stress conditions in the host microenvironment, and thereby it survives asymptomatically in latent phase in the host for decades and even for a lifetime. This raises the need for improved vaccine, drugs, and therapeutics, which could be achieved by a better understanding of the host-microbe interactions as well as immune responses during the infection. Recent studies have highlighted the importance of host cellular stress response pathways, such as unfolded protein response (UPR), oxidative stress response, integrated stress response (ISR), and autophagy during various infections. However, the role of these host stress response pathways during an MTB infection in the modulation of the immune response against the microbe is poorly understood. Therefore, through this chapter, we will highlight the cellular stress response pathways and various molecular mechanisms through which MTB influences the host innate as well as the adaptive immune response during infection, which might aid toward better design and development of therapeutics and vaccine candidates against TB.

In cystic fibrosis (CF), epithelial cells are unable to normally up-regulate apical membrane Cl- secretion in response to agents which increase cyclic AMP, but they do increase Cl- secretion in response to increases in intracellular Ca2+. Since intracellular divalent cations regulate the expression of many genes, we hypothesized that mobilization of intracellular Ca2+ and/or other divalent cations might modulate not only Ca(2+)-dependent Cl- channels but also cystic fibrosis transmembrane conductance regulator (CFTR) gene expression. To evaluate this concept, HT-29 human colon carcinoma cells were cultured under various conditions designed to manipulate intracellular divalent cation concentrations and CFTR gene expression was quantified at the levels of transcription, mRNA accumulation, mRNA half-life, and protein. Exposure to the divalent cation ionophores A23187 and ionomycin (agents which increase intracellular divalent cation concentrations) caused dose- and time-dependent reductions of CFTR mRNA levels, which could be blocked by the use of Ca(2+)- and Mg(2+)-free media. Ionophore-induced CFTR gene modulation was also observed with T84 human colon carcinoma cells and freshly isolated normal human bronchial epithelial cells. Incubation of HT-29 cells with thapsigargin, an agent that releases Ca2+ from intracellular stores, or in medium containing increased extracellular concentrations of Ca2+ or Mg2+ also caused down-regulation of CFTR mRNA levels. Transcription run-on analysis showed that, parallel with the decrease in CFTR mRNA levels, A23187 reduced the rate of transcription of the CFTR gene, while CFTR mRNA transcript half-life was unaffected. Consistent with the down-regulation of CFTR gene expression, CFTR protein levels also decreased after exposure to A23187. Thus, despite the independence of Ca(2+)-dependent Cl- channels and cyclic AMP-dependent CFTR-related Cl- channels in epithelial cells, increases in intracellular divalent cation concentrations down-regulate the expression of the CFTR gene at the transcriptional level, with consequent decreases in CFTR mRNA and protein.