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Comparison of the inhibitory effects of Lactobacillus supernatant and coculture on Gardnerella vaginalis
No full textObjective: Vaginal lactobacilli play a crucial role in inhibiting bacteria such as Gardnerella vaginalis (G. vaginalis), a key contributor to dysbiosis and bacterial vaginosis. We aimed to compare the inhibitory effects of Lactobacillus cell-free supernatants on G. vaginalis growth with those observed in Lactobacillus-G. vaginalis coculture, an experimental setup that more closely mimics in vivo conditions.
Results: To identify the optimal medium for coculture experiments, MRS broth and NYC-III broth were compared. NYC-III significantly enhanced the growth of G. vaginalis and four of the five tested Lactobacillus strains. We then developed a direct quantitative PCR (qPCR) method that allowed us to specifically measure G. vaginalis genome concentrations in cocultures with Lactobacillus. This direct qPCR did not require DNA extraction and had a 4,096-fold dynamic range. We then assessed the inhibition of G. vaginalis growth by Lactobacillus cell-free supernatants in G. vaginalis cultures by measuring optical density and the Lactobacillus-mediated inhibition in cocultures by the G. vaginalis specific direct qPCR. The two measurement methods showed different levels of inhibitory activity for two of the five Lactobacillus strains tested. These findings suggest that coculture experiments should be conducted in place of, or in addition to, supernatant-based inhibitory assays
Cryo‐EM structures of amyloid β filaments from cotton wool plaques in Alzheimer disease caused by the PSEN1 V261I and A431E mutations
No full textBackground:
One of the neuropathologic phenotypes of Alzheimer disease (AD), associated with missense and deletion mutations in PSEN1, is the presence of cotton wool plaques (CWPs). CWPs are round amyloid β (Aβ) immunopositive structures, lacking an amyloid core; they are well recognizable in hematoxilin ‐eosin stained preparations and are not fluorescent using Thioflavin S (ThS), thus being morphologically different from Aβ cored plaques and Aβ diffuse deposits. Amino‐terminally truncated and post‐translationally modified Aβ peptide species are the main component of CWPs. Tau immunopositive neurites may be seen in CWPs and neurofibrillary tangles may be numerous amidst CWPs
Method:
We used cryo‐electron microscopy to study the structure of Aβ filaments extracted from the brains of two individuals affected by dominantly inherited AD caused by the PSEN1 V261I and A431E mutations, respectively.
Result:
CWPs consist predominantly of two novel arrangements of type I Aβ filaments, and we have designated them as type Ic and type Id Aβ filaments. The latter filaments exhibit unique protofilament packing arrangements. While the type Ic Aβ filaments share a largely similar S‐shaped monomeric configuration with type I Aβ filaments, type Id Aβ filaments display an antiparallel and asymmetric packing, in contrast to the twofold parallel arrangement characteristic of type Ib and Ic Aβ filaments.
Conclusion:
The formation of type Ic and type Id Aβ filaments in CWPs may be linked to a distinct array of Aβ peptide species present in CWPs, differing from those found in core plaques in sporadic AD. This work provides novel insights into the role that different types of Aβ filaments may play in the composition of plaque morphology. Aggregation of Aβ as type Ic and type Id Aβ filaments may be the basis for the phenotype of CWPs. The characterization of Aβ filaments from CWPs, is important for the understanding of the role that the structure and assembly of amyloid peptides may play in the morphological diversity of Aβdeposits as well as for developing new diagnostic and therapeutic strategies for AD
Acute endothelial stresses identify microRNA let-7b-5p and non-coding SLC11A2 (NRAMP2/DMT1) exon as biomarkers that overlap with those detected in malignant and non-malignant diseases
No full textBackground: Disease biomarkers are often identified long after initiating pathologies, hampering mechanistic understanding and the development of preventative strategies. We hypothesized that aberrant cellular responses to normally-encountered stresses may be relevant to later disease states.
Aim: To model two under-explored acute cellular stresses for blood-exposed cells, and cross-reference to known biomarkers of disease.
Methods: Normal primary human endothelial cells (ECs) were treated for 1-6 h with cycloheximide (CHX) 100 μg/ml to inhibit protein translation and nonsense-mediated decay (modelling the integrated stress response), or 10 μmol/l ferric citrate (modelling diurnal variation in serum iron that can be augmented by treatments prescribed 8 million times/year in England). Directional whole transcriptome RNA-seq identified differentially expressed genes and micro(mi)RNAs. Customized novel scripts examined the expression of 517 225 exons to predict 1 h CHX-stabilized exons. Validations were by cel-miR-39-spiked qRT-PCR and RNA-seq in other EC types, peripheral blood mononuclear cells (PBMCs) and plasma.
Results: miRNAs fell transiently at 1 h after 10 μmol/l ferric citrate (P < 0.01), specifically in let-7 family member pre-miRNAs ('let-7', P < 0.05), where there was an accompanying differential 6 h increased expression of 570 let-7-target mRNAs identified through TargetScan (P < 0.0001). qRT-PCR and RNA-seq validations in other normal ECs, plasma and PBMCs confirmed up to 80% falls in pre-let-7b/let-7b-5p after 1 h iron, and exon 3B of the SLC11A2 (NRAMP2/DMT1)-encoded iron/copper transporter as a novel exon most consistently stabilized following 1 h treatment with CHX. Overlaps with disease biomarkers for cancer, growth retardation and multiple organ-specific diseases were identified.
Conclusions: Biomarkers for normal, acute cellular responses overlap with disease-state biomarkers, warranting further study
Communication Work When Care is at Stake: One Family’s Journey with a Health Insurance Denial
No full textDenials of health insurance claims in the U.S. have been increasing rapidly in recent years, taking a toll on patients, families, and the health care system. Yet health communication scholars know little about the storied experiences of those affected by these denials. In this essay, I reflect on my own family’s efforts to secure insurance coverage for an orthopedic surgery for my son after our insurer repeatedly refused coverage. In doing so, I draw attention to the ways that coping with a health insurance denial involves a distinct type of communication labor and contributes to advancing research using the Integrative Theory of Communication Work. I contend that health communication scholars are uniquely positioned to contribute to our understanding of health insurance denials and to suggest improvements to this complex bureaucratic process
Frontal Theta Event‐Related Oscillations During a Continuous Performance Test: The Influence of Trauma Type and Fluid Intelligence Polygenic Score
No full textBackground: Trauma exposure during adolescence can lead to impaired executive function and altered neural development in related cognitive control networks. Studies have shown that adolescents with a family history of alcohol use disorders have a disproportionately high rate of trauma exposure, as well as impaired response inhibition, making them particularly vulnerable to cognitive impairment and poor mental health outcomes in adulthood. While studies have suggested that this may be due partly to genetic influences, no study to our knowledge has investigated the influences of trauma exposure and polygenic scores (PGS) for cognitive function on later cognitive function.
Methods: This study used data from trauma-exposed individuals in the Collaborative Study on the Genetics of Alcoholism prospective cohort (N = 912), comprising offspring from alcohol-dependent high-risk and comparison families, to investigate main and interaction effects of PGS for cognitive function (fluid intelligence score, UK Biobank study) and trauma exposure (nonsexual assaultive, nonassaultive, sexual assaultive) on performance measures and frontal theta event-related oscillations (EROs) during a continuous performance test (CPT).
Results: A significant interaction between fluid intelligence PGS and nonsexual assaultive trauma was observed for CPT ERO power (B = 0.094, p < 0.01), such that individuals with a lower PGS who experienced a nonsexual assaultive traumatic exposure had lower frontal theta ERO power during the cued no-go condition of the CPT.
Conclusion: These findings suggest that a polygenic predisposition for higher fluid intelligence may be associated with differences in neural response inhibition depending on trauma type
Disulfide bonds are critical for stabilizing cell division, cell envelope biogenesis, and antibiotic resistance proteins in mycobacteria
No full textMycobacteria, including Mycobacterium tuberculosis-the etiological agent of tuberculosis-possess a unique and impermeable cell envelope that is critical for survival and antibiotic resistance. The assembly and maintenance of this envelope depend on properly folded proteins, yet the role of disulfide bond formation in these processes remains poorly understood. Mycobacteria rely on two membrane enzymes, disulfide bond formation protein A (DsbA) and vitamin K epoxide reductase (VKOR), for introducing disulfide bonds into exported proteins. In silico studies predict that ~64% of exported proteins contain even numbers of cysteine residues and thence disulfide bonding; nevertheless, substrates of the DsbA-VKOR pathway remain largely unknown. Here, we demonstrate that DsbA and VKOR introduce disulfide bonds into substrate proteins and identify several essential proteins that depend on oxidative folding in the mycobacterial cell envelope. Using bioinformatics and cysteine profiling proteomics, we uncover numerous exported proteins that require disulfide bonds for stability. Cysteine derivatization in whole cells confirms that key proteins, including LamA (MmpS3), PstP, LpqW, and EmbB, rely on disulfide bonds for proper function. Furthermore, chemical inhibition of VKOR phenocopies vkor deletion, thus highlighting its essential role in maintaining mycomembrane integrity. These findings address a critical gap in understanding mycobacterial cell envelope biogenesis and underscore the DsbA-VKOR system as a promising target for disrupting cell envelope homeostasis in drug-resistant Mycobacteria.
IMPORTANCE: This work addresses a major deficiency in understanding mycobacterial cell envelope processes and highlights the biological and clinical implications of oxidative protein folding in mycobacteria. This process, marked by the formation of disulfide bonds, is essential for the stability of exported proteins. While disulfide bond formation studies in Gram-negative bacteria suggested a similar role in mycobacteria, the underlying consequences of disulfide bonds remained unclear. Thus, we began investigating the diverse physiological functions dependent on disulfide bonds in Mycobacteria using a combination of bioinformatics, proteomics, and genetic and biochemical approaches. We identified hundreds of proteins affected by oxidative protein folding and validated essential substrates of this process. We show that disulfide bonds are not only crucial for the stability and function of key mycobacterial proteins but also represent a novel therapeutic target against antimicrobial resistance. Our findings underscore the potential of targeting disulfide bond formation to disrupt mycomembrane assembly, opening new avenues for antimycobacterial drug development
Challenges and opportunities in state-of-the-art proteomics analysis for biomarker development from plasma extracellular vesicles
No full textExtracellular vesicles (EVs) are membrane-bound particles secreted by cells, playing crucial roles in intercellular communication. The composition of EVs can undergo changes in response to stress and disease conditions, making them excellent biomarker candidates. However, extracting protein information from EVs can be challenging due to their low abundance in complex biofluids and copurification with contaminant proteins and particles. Techniques to enrich EVs have their strengths and limitations, without one being able to purify EVs to complete homogeneity. This can lead to compromised recovery rates and increased complexity, making data interpretation difficult. In this viewpoint article, we explore the concept that better characterization of EV composition, followed by quantification of EV proteins in complex samples, might be a more viable route for biomarker development. Mass spectrometers can provide reproducible deep coverage of the EV proteome, despite sample impurities. This paradigm shift presents opportunities to integrate advanced bioinformatics tools to refine the EV proteome landscape, identify novel biomarkers, and streamline validation processes in biomarker development. By focusing on leveraging technology rather than achieving absolute purity, this approach can transform current practices and open opportunities for robust biomarker discovery. Herein, we highlight not only such opportunities but also challenges to implement this concept. SUMMARY: Extracellular vesicles (EVs) have enormous potential as biomarkers of diseases, as they can carry signatures of the cells they are derived from and the pathogenesis process. Biofluids, such as blood plasma, are highly complex and contain many components with physicochemical properties similar to those of EVs, making it challenging to obtain pure EV fractions. Challenges in obtaining pure preparations represent a main hurdle for studying EVs, and their components are potential biomarkers. This article explores the concept of studying EV proteins within complex samples, discussing opportunities and needs to move this field forward
Recurrent Melanoma In Situ at the Skin Graft Site of a Prior Malignant Melanoma of the Left Upper Extremity: A Case Report
No full textWe present the case of a 43-year-old Caucasian, fair-skinned male with a history of multiple malignant melanomas, who presented in February 2024 for excision of a new melanoma arising within a prior skin graft. The graft had been harvested from his thigh to close an excision of a primary malignant melanoma with a depth of 0.73 mm in 2018. Extensive pathologic interrogation, as well as systemic work-up - including positron emission tomography and computed tomography scans - was performed to determine whether this lesion represented local recurrent disease or a new primary melanoma. This workup was consistent with a primary melanoma in situ, which led to the decision to proceed with skin graft resection and full-thickness skin grafting from his lower left abdomen. The entire previous skin graft was excised. Subsequent surgical pathologic examination of the excised skin graft demonstrated no residual malignant melanoma. This interesting case demonstrates a unique site of primary melanoma occurrence in a split-thickness skin graft. While this may be the result of incomplete tumor resection at the primary operation, the initial excision had wide margins of 2 cm, and pathological examination demonstrated negative margins. Additionally, the skin graft harvest site was carefully selected, as it came from a relatively sun-protected site. This presentation highlights the importance of continued surveillance of melanoma excision sites, warning patients of possible tumor recurrence or new primary occurrence, and setting expectations that incidental findings may occur
A comparison of neighborhood socioeconomic deprivation measures and the association with survival among Black and White women with endometrial cancer
No full textBackground: Black women with endometrial cancer have twice the mortality compared with White. Survival disparities remain after accounting for individual-level socioeconomic and cancer-related factors. We investigated associations between area-based deprivation and survival and explored whether area-based deprivation attenuates the association between race and survival, among a cohort of Black and White women.
Methods: Data from endometrial cancers diagnosed between 2013 and 2022 were collected from a comprehensive cancer registry covering Metropolitan Detroit. Addresses at diagnosis were linked to the area deprivation (ADI) and social vulnerability (SVI) indices. Adjusted Fine and Gray models and Cox proportional hazard models were run investigating associations between area-based deprivation measures and survival; analyses were conducted estimating the proportion of the association between race and survival that was attenuated by area-based measures.
Results: Higher deprivation was associated with poorer survival, adjusted for race, insurance status, and tumor characteristics. Compared with the least disadvantaged quartile, the quartile with the highest disadvantage using ADI and SVI had 1.18 [95% confidence interval (CI), 0.99-1.43] and 1.40 (1.14-1.71) times the hazard of endometrial cancer-specific mortality, respectively. ADI and SVI attenuated 18% (3%-38%) and 27% (10%-48%) of associations between race and mortality overall and 24% (95% CI, 3%-61%) and 40% (95% CI, 16%-78%) among those with high-grade histology.
Conclusions: This study demonstrates a clear association between neighborhood-level disadvantage and survival among women with endometrial cancer living in Metropolitan Detroit. Neighborhood disadvantage attenuates the relationship between race and survival, particularly among those with high-grade histology.
Impact: These findings serve as motivation to understand how neighborhood affects cancer outcomes