A Global Analysis of the Adaptations Required for Sterol Catabolism in Mycobacterium Tuberculosis: A Dissertation

Systems biology approaches have allowed for comprehensive understanding of complicated biological processes. Here, we’ve developed a global phenotypic profiling method by improving upon transposon mutagenesis methods for identifying genes required for bacterial growth in various conditions. By using the massively parallel power of Illumina sequencing, we precisely redefined the genes required for the growth of Mycobacterium Tuberculosis (Mtb) in vitro. This adapted technique provided more informative data with both increased dynamic range and resolution. As a result, we quantitatively assessed the fitness of individual mutants, as well as identified sub-genic essentiality. Mtb is well adapted to its nutrient-limiting intracellular niche. One important and novel adaptation is its ability to consume cholesterol for both energy and carbon. A combination of this genome-wide phenotypic analysis and global metabolite profiling was used to define the dedicated cholesterol catabolic pathway, as well as important transcriptional and metabolic adaptations required for the consumption of this carbon source. We identified the methylcitrate cycle (MCC) and an unexpected gluconeogenic route as essential pathways. Furthermore, we found that the cholesterol-dependent transcriptional induction of these metabolic enzymes was also essential for growth on this substrate, a function mediated by the Rv1129c regulatory protein. Using a combination of genetic and chemical methods to inhibit these pathways, we show that cholesterol represents a significant source of carbon during intracellular growth in macrophages. Finally, we have begun to define the mechanism by which lipids, such as cholesterol, are imported into the cell by investigating the assembly of the ABC-like lipid transporter, Mce1. The subunits of this system are localized to the cell wall and data is provided to support a novel mechanism for Mce-dependent import of lipids, such as cholesterol. In sum, this global analysis of host cholesterol utilization during infection provides insight into each step of this complicated process; import into the bacterial cell, the degradation of the molecule into primary metabolites, and the transformation of these metabolites into carbon and energy

The effect of additional characters on choice of referring expression: Everyone counts☆

Two story-telling experiments examine the process of choosing between pronouns and proper names in speaking. Such choices are traditionally attributed to speakers striving to make referring expressions maximally interpretable to addressees. The experiments revealed a novel effect: even when a pronoun would not be ambiguous, the presence of another character in the discourse decreased pronoun use and increased latencies to refer to the most prominent character in the discourse. In other words, speakers were more likely to call Minnie Minnie than shewhen Donald was also present. Even when the referent character appeared alone in the stimulus picture, the presence of another character in the preceding discourse reduced pronouns. Furthermore, pronoun use varied with features associated with the speaker’s degree of focus on the preceding discourse (e.g., narrative style and disfluency). We attribute this effect to competition for attentional resources in the speaker’s representation of the discourse

Mutations in HPV18 E1^E4 Impact Virus Capsid Assembly, Infectivity Competence, and Maturation.

The most highly expressed protein during the productive phase of the human papillomavirus (HPV) life cycle is E1^E4. Its full role during infection remains to be established. HPV E1^E4 is expressed during both the early and late stages of the virus life cycle and contributes to viral genome amplification. In an attempt to further outline the functions of E1^E4, and determine whether it plays a role in viral capsid assembly and viral infectivity, we examined wild-type E1^E4 as well as four E1^E4 truncation mutants. Our study revealed that HPV18 genomes containing the shortest truncated form of E1^E4, the 17/18 mutant, produced viral titers that were similar to wild-type virus and significantly higher compared to virions containing the three longer E1^E4 mutants. Additionally, the infectivity of virus containing the shortest E1^E4 mutation was equivalent to wild-type and significantly higher than the other three mutants. In contrast, infectivity was completely abrogated for virus containing the longer E1^E4 mutants, regardless of virion maturity. Taken together, our results indicate for the first time that HPV18 E1^E4 impacts capsid assembly and viral infectivity as well as virus maturation

Serum Iron Level is Associated with Time to Antibiotics in Cystic Fibrosis

Background: Serum levels of hepcidin‐25, a peptide hormone that reduces blood iron content, are elevated when patients with cystic fibrosis (CF) develop pulmonary exacerbation (PEx). Because hepcidin‐25 is unavailable as a clinical laboratory test, we questioned whether a one‐time serum iron level was associated with the subsequent number of days until PEx, as defined by the need to receive systemic antibiotics (ABX) for health deterioration. Methods: Clinical, biochemical, and microbiological parameters were simultaneously checked in 54 adults with CF. Charts were reviewed to determine when they first experienced a PEx after these parameters were assessed. Time to ABX was compared in subgroups with and without specific attributes. Multivariate linear regression was used to identify parameters that significantly explained variation in time to ABX. Results: In univariate analyses, time to ABX was significantly shorter in subjects with Aspergillus‐positive sputum cultures and CF‐related diabetes. Multivariate linear regression models demonstrated that shorter time to ABX was associated with younger age, lower serum iron level, and Aspergillus sputum culture positivity. Conclusions: Serum iron, age, and Aspergillus sputum culture positivity are factors associated with shorter time to subsequent PEx in CF adults

Patient Perspective on the Value of Genetic Counselling for Familial Pancreas Cancer

<p>Abstract</p> <p>Purpose</p> <p>To assess patient views regarding the value of genetic counselling for familial pancreas cancer in the absence of predictive genetic testing.</p> <p>Patients and methods</p> <p>At-risk adults with three or more relatives with pancreas cancer received genetic counselling prior to research screening via endoscopic ultrasound. Questionnaires were mailed after the visit to assess perceived value of the counselling session.</p> <p>Results</p> <p>Ninety-three percent of respondents felt genetic counselling for pancreas cancer was helpful despite the lack of a causative gene, while only 7% felt that it should not be offered until such a gene is discovered. Over half of respondents believed the pancreas cancer in their family was caused by a gene mutation, and 42% thought they had inherited the mutation. The average perceived lifetime risk of developing pancreas cancer was 51%, and 87% of respondents would ultimately seek predictive genetic testing. When more information is gained, 89% would be interested in another genetic counselling session, and 82% would recommend current genetic counselling for pancreas cancer to a friend or relative with a family history of the disease.</p> <p>Conclusion</p> <p>Despite the lack of an identified major causative gene for pancreas cancer, respondents found genetic counselling for this malignancy to be helpful. These patients perceive their personal cancer risk to be high, and would seek predictive genetic testing if it were available. Referral for genetic counselling should be offered to appropriate individuals.</p

Label-free Quantitative Proteomics Reveals a Role for the Mycobacterium tuberculosis SecA2 Pathway in Exporting Solute Binding Proteins and Mce Transporters to the Cell Wall

Mycobacterium tuberculosis is an example of a bacterial pathogen with a specialized SecA2-dependent protein export system that contributes to its virulence. Our understanding of the mechanistic basis of SecA2-dependent export and the role(s) of the SecA2 pathway in M. tuberculosis pathogenesis has been hindered by our limited knowledge of the proteins exported by the pathway. Here, we set out to identify M. tuberculosis proteins that use the SecA2 pathway for their export from the bacterial cytoplasm to the cell wall. Using label-free quantitative proteomics involving spectral counting, we compared the cell wall and cytoplasmic proteomes of wild type M. tuberculosis to that of a ΔsecA2 mutant. This work revealed a role for the M. tuberculosis SecA2 pathway in the cell wall localization of solute binding proteins that work with ABC transporters to import solutes. Another discovery was a profound effect of SecA2 on the cell wall localization of the Mce1 and Mce4 lipid transporters, which contribute to M. tuberculosis virulence. In addition to the effects on solute binding proteins and Mce transporter export, our label-free quantitative analysis revealed an unexpected relationship between SecA2 and the hypoxia-induced DosR regulon, which is associated with M. tuberculosis latency. Nearly half of the transcriptionally controlled DosR regulon of cytoplasmic proteins were detected at higher levels in the ΔsecA2 mutant versus wild type M. tuberculosis. By increasing the list of M. tuberculosis proteins known to be affected by the SecA2 pathway, this study expands our appreciation of the types of proteins exported by this pathway and guides our understanding of the mechanism of SecA2-dependent protein export in mycobacteria. At the same time, the newly identified SecA2-dependent proteins are helpful for understanding the significance of this pathway to M. tuberculosis virulence and physiology

JWST Imaging of the Cartwheel Galaxy Reveals Dust Associated with SN 2021afdx

We present near- and mid-infrared (0.9-18 $\mu$m) photometry of supernova (SN) 2021afdx, which was imaged serendipitously with the James Webb Space Telescope (JWST) as part of its Early Release Observations of the Cartwheel Galaxy. Our ground-based optical observations show it is likely to be a Type IIb SN, the explosion of a yellow supergiant, and its infrared spectral energy distribution (SED) $\approx$200 days after explosion shows two distinct components, which we attribute to hot ejecta and warm dust. By fitting models of dust emission to the SED, we derive a dust mass of $(3.8_{-0.3}^{+0.5}) \times 10^{-3}\ M_\odot$, which is the highest yet observed in a Type IIb SN but consistent with other Type II SNe observed by the Spitzer Space Telescope. We also find that the radius of the dust is significantly larger than the radius of the ejecta, as derived from spectroscopic velocities during the photospheric phase, which implies that we are seeing an infrared echo off of preexisting dust in the progenitor environment, rather than dust newly formed by the SN. Our results show the power of JWST to address questions of dust formation in SNe, and therefore the presence of dust in the early universe, with much larger samples than have been previously possible.Comment: updated to match accepted versio

Forgiveness-Reconciliation and Communication-Conflict-Resolution Interventions Versus Retested Controls in Early Married Couples

The first 6 months of marriage are optimal for marriage enrichment interventions. The Hope-Focused Approach to couple enrichment was presented as two 9-hr interventions--(a) Handling Our Problems Effectively (HOPE), which emphasized communication and conflict resolution, and (b) Forgiveness and Reconciliation through Experiencing Empathy (FREE). HOPE and FREE were compared with repeated assessment controls. Couples were randomly assigned and were assessed at pretreatment (t1); 1 month posttreatment (t2) and at 3- (t3), 6- (t4), and 12-month (t5) follow-ups using self-reports. In addition to self-report measures, couples were assessed at t1, t2, and t5 using salivary cortisol, and behavioral coding of decision making. Of 179 couples who began the study, 145 cases were analyzed. Both FREE and HOPE produced lasting positive changes on self-reports. For cortisol reactivity, HOPE and FREE reduced reactivity at t2, but only HOPE at t5. For coded behaviors, control couples deteriorated; FREE and HOPE did not change. Enrichment training was effective regardless of the focus of the training

Multifocal demyelinating motor neuropathy and hamartoma syndrome associated with a de novo PTEN mutation.

OBJECTIVE: To describe a patient with a multifocal demyelinating motor neuropathy with onset in childhood and a mutation in phosphatase and tensin homolog (PTEN), a tumor suppressor gene associated with inherited tumor susceptibility conditions, macrocephaly, autism, ataxia, tremor, and epilepsy. Functional implications of this protein have been investigated in Parkinson and Alzheimer diseases. METHODS: We performed whole-exome sequencing in the patient's genomic DNA validated by Sanger sequencing. Immunoblotting, in vitro enzymatic assay, and label-free shotgun proteomic profiling were performed in the patient's fibroblasts. RESULTS: The predominant clinical presentation of the patient was a childhood onset, asymmetric progressive multifocal motor neuropathy. In addition, he presented with macrocephaly, autism spectrum disorder, and skin hamartomas, considered as clinical criteria for PTEN-related hamartoma tumor syndrome. Extensive tumor screening did not detect any malignancies. We detected a novel de novo heterozygous c.269T>C, p.(Phe90Ser) PTEN variant, which was absent in both parents. The pathogenicity of the variant is supported by altered expression of several PTEN-associated proteins involved in tumorigenesis. Moreover, fibroblasts showed a defect in catalytic activity of PTEN against the secondary substrate, phosphatidylinositol 3,4-trisphosphate. In support of our findings, focal hypermyelination leading to peripheral neuropathy has been reported in PTEN-deficient mice. CONCLUSION: We describe a novel phenotype, PTEN-associated multifocal demyelinating motor neuropathy with a skin hamartoma syndrome. A similar mechanism may potentially underlie other forms of Charcot-Marie-Tooth disease with involvement of the phosphatidylinositol pathway

Identifying the SN 2022acko progenitor with JWST

We report on analysis using the James Webb Space Telescope (JWST) to identify a candidate progenitor star of the Type II-plateau supernova SN 2022acko in the nearby, barred spiral galaxy NGC 1300. To our knowledge, our discovery represents the first time JWST has been used to localize a progenitor system in pre-explosion archival Hubble Space Telescope (HST) images. We astrometrically registered a JWST NIRCam image from 2023 January, in which the SN was serendipitously captured, to pre-SN HST F160W and F814W images from 2017 and 2004, respectively. An object corresponding precisely to the SN position has been isolated with reasonable confidence. That object has a spectral energy distribution (SED) and overall luminosity consistent with a single-star model having an initial mass possibly somewhat less than the canonical 8 Msun theoretical threshold for core collapse (although masses as high as 9 Msun for the star are also possible); however, the star's SED and luminosity are inconsistent with that of a super-asymptotic giant branch star which might be a forerunner of an electron-capture SN. The properties of the progenitor alone imply that SN 2022acko is a relatively normal SN II-P, albeit most likely a low-luminosity one. The progenitor candidate should be confirmed with follow-up HST imaging at late times, when the SN has sufficiently faded. This potential use of JWST opens a new era of identifying SN progenitor candidates at high spatial resolution.Comment: 8 pages, substantial changes from v1, to appear in MNRA