Obesity Induces Temporally Regulated Alterations in the extracellular matrix that drive breast tumor invasion and metastasis.

Obesity is associated with increased incidence and metastasis of triple-negative breast cancer (TNBC), an aggressive breast cancer subtype. The extracellular matrix (ECM) is a major component of the tumor microenvironment that drives metastasis. To characterize the temporal effects of age and high-fat diet-driven weight gain on the ECM, we injected allograft tumor cells at 4-week intervals into mammary fat pads of mice fed a control or high-fat diet (HFD), assessing tumor growth and metastasis and evaluating the ECM composition of the mammary fat pads, lungs, and livers. Tumor growth was increased in obese mice after 12 weeks on the HFD. Liver metastasis increased in obese mice only at 4 weeks, and elevated body weight correlated with increased metastasis to the lungs but not the liver. Whole decellularized ECM coupled with proteomics indicated that early stages of obesity were sufficient to induce changes in the ECM composition. Obesity led to increased abundance of the pro-invasive ECM proteins collagen IV and collagen VI in the mammary glands and enhanced the invasive capacity of cancer cells. Cells of stromal vascular fraction and adipose stem and progenitor cells were primarily responsible for secreting collagen IV and VI, not adipocytes. Longer exposure to HFD increased the invasive potential of ECM isolated from lung and liver, with significant changes in ECM composition found in the liver with short-term HFD exposure. Together, this data suggests that changes in the breast, lung, and liver ECM underlie some of the effects of obesity on TNBC incidence and metastasis.

Impact of telehealth on patient-provider communication in prenatal care for pregnant women from underserved settings.

INTRODUCTION: Telehealth has emerged as a promising supplementary modality in prenatal care. However, its impact on patient-provider communication (PPC), especially among pregnant women from underserved settings, requires comprehensive evaluation. This study examined the factors associated with the quality of patient-provider communication during the COVID-19 pandemic among pregnant telehealth users and non-users. METHODS: Using a cross-sectional study design, 242 women were surveyed (response rate = 23%) regarding their experience with telehealth, quality of PPC, and experiences of discrimination during prenatal care. Multiple regression models were used to identify the factors associated with the quality of PPC during the COVID-19 pandemic. A sub-group analysis explored the factors associated with the quality of PPC separately among telehealth users and non-users. RESULTS: The majority of the participants were on Medicaid (95%) and self-identified as Black/African American (57.3%). Regression analyses revealed a negative relationship between telehealth use during pregnancy and the quality of PPC (ß = -1.13, P = 0.002). Irrespective of the telehealth use, the experience of discrimination was associated with poor quality of PPC among users (ß = -3.47, P = .02) and non-users (ß = -.78, P = .03), while adjusting for sociodemographic factors and social support during pregnancy. DISCUSSION: While telehealth offers advantages like convenience, increased accessibility, and continuity of care, challenges in establishing effective PPC in virtual settings have emerged that emphasize the necessity for comprehensive provider training extending beyond technical competencies. The persistent issue of perceived discrimination, impacting PPC across both groups, underscores the necessity to rethink existing strategies of mandatory training to increase providers' knowledge.

An optimized purification protocol for enzymatically synthesized S-adenosyl-L-methionine (SAM) for applications in solution state infrared spectroscopic studies.

S-adenosyl-L-methionine (SAM) is an abundant biomolecule used by methyltransferases to regulate a wide range of essential cellular processes such as gene expression, cell signaling, protein functions, and metabolism. Despite considerable effort, there remain many specificity challenges associated with designing small molecule inhibitors for methyltransferases, most of which exhibit off-target effects. Interestingly, NMR evidence suggests that SAM undergoes conformeric exchange between several states when free in solution. Infrared spectroscopy can detect different conformers of molecules if present in appreciable populations. When SAM is noncovalently bound within enzyme active sites, the nature and the number of different conformations of the molecule are likely to be altered from when it is free in solution. If there are unique structures or different numbers of conformers between different methyltransferase active sites, solution-state information may provide promising structural leads to increase inhibitor specificity for a particular methyltransferase. Toward this goal, frequencies measured in SAM's infrared spectra must be assigned to the motions of specific atoms via isotope incorporation at discrete positions. The incorporation of isotopes into SAM's structure can be accomplished via an established enzymatic synthesis using isotopically labeled precursors. However, published protocols produced an intense and highly variable IR signal which overlapped with many of the signals from SAM rendering comparison between isotopes challenging. We observed this intense absorption to be from co-purifying salts and the SAM counterion, producing a strong, broad signal at 1100 cm-1. Here, we report a revised SAM purification protocol that mitigates the contaminating salts and present the first IR spectra of isotopically labeled CD3-SAM. These results provide a foundation for isotopic labeling experiments of SAM that will define which atoms participate in individual molecular vibrations, as a means to detect specific molecular conformations.

Age and obesity-driven changes in the extracellular matrix of the primary tumor and metastatic site influence tumor invasion and metastatic outgrowth.

Younger age and obesity increase the incidence and metastasis of triple-negative breast cancer (TNBC), an aggressive subtype of breast cancer. The extracellular matrix (ECM) promotes tumor invasion and metastasis. We characterized the effect of age and obesity on the ECM of mammary fat pads, lungs, and liver using a diet-induced obesity (DIO) model. At 4 week intervals, we either injected the mammary fat pads with allograft tumor cells to characterize tumor growth and metastasis or isolated the mammary fat pads and livers to characterize the ECM. Age had no effect on tumor growth but increased lung and liver metastasis after 16 weeks. Obesity increased tumor growth starting at 12 weeks, increased liver metastasis only at 4 weeks, and weight gain correlated to increased lung but not liver metastasis. Utilizing whole decellularized ECM coupled with proteomics, we found that early stages of obesity were sufficient to induce changes in the ECM composition and invasive potential of mammary fat pads with increased abundance of pro-invasive ECM proteins Collagen IV and Collagen VI. We identified cells of stromal vascular fraction and adipose stem and progenitor cells as primarily responsible for secreting Collagen IV and VI, not adipocytes. We characterized the changes in ECM in the lungs and liver, and determined that older age decreases the metastatic potential of lung and liver ECM while later-stage obesity increases the metastatic potential. These data implicate ECM changes in the primary tumor and metastatic microenvironment as mechanisms by which age and obesity contribute to breast cancer progression.

Evidence for a Long-Lived, Cu-Coupled and Oxygen-Inert Disulfide Radical Anion in the Assembly of Metallothionein-3 Cu(I)4-Thiolate Cluster.

The human copper-binding protein metallothionein-3 (MT-3) can reduce Cu(II) to Cu(I) and form a polynuclear Cu(I)4-Cys5-6 cluster concomitant with intramolecular disulfide bonds formation, but the cluster is unusually inert toward O2 and redox-cycling. We utilized a combined array of rapid-mixing spectroscopic techniques to identify and characterize the transient radical intermediates formed in the reaction between Zn7MT-3 and Cu(II) to form Cu(I)4Zn(II)4MT-3. Stopped-flow electronic absorption spectroscopy reveals the rapid formation of transient species with absorption centered at 430-450 nm and consistent with the generation of disulfide radical anions (DRAs) upon reduction of Cu(II) by MT-3 cysteine thiolates. These DRAs are oxygen-stable and unusually long-lived, with lifetimes in the seconds regime. Subsequent DRAs reduction by Cu(II) leads to the formation of a redox-inert Cu(I)4-Cys5 cluster with short Cu-Cu distances (<2.8 Å), as revealed by low-temperature (77 K) luminescence spectroscopy. Rapid freeze-quench Raman and electron paramagnetic resonance (EPR) spectroscopy characterization of the intermediates confirmed the DRA nature of the sulfur-centered radicals and their subsequent oxidation to disulfide bonds upon Cu(II) reduction, generating the final Cu(I)4-thiolate cluster. EPR simulation analysis of the radical g- and A-values indicate that the DRAs are directly coupled to Cu(I), potentially explaining the observed DRA stability in the presence of O2. We thus provide evidence that the MT-3 Cu(I)4-Cys5 cluster assembly process involves the controlled formation of novel long-lived, copper-coupled, and oxygen-stable disulfide radical anion transient intermediates.

Reconstitution reveals two paths of force transmission through the kinetochore.

Partitioning duplicated chromosomes equally between daughter cells is a microtubule-mediated process essential to eukaryotic life. A multi-protein machine, the kinetochore, drives chromosome segregation by coupling the chromosomes to dynamic microtubule tips, even as the tips grow and shrink through the gain and loss of subunits. The kinetochore must harness, transmit, and sense mitotic forces, as a lack of tension signals incorrect chromosome-microtubule attachment and precipitates error correction mechanisms. But though the field has arrived at a 'parts list' of dozens of kinetochore proteins organized into subcomplexes, the path of force transmission through these components has remained unclear. Here we report reconstitution of functional Saccharomyces cerevisiae kinetochore assemblies from recombinantly expressed proteins. The reconstituted kinetochores are capable of self-assembling in vitro, coupling centromeric nucleosomes to dynamic microtubules, and withstanding mitotically relevant forces. They reveal two distinct pathways of force transmission and Ndc80c recruitment.

Seeing is believing: our evolving view of kinetochore structure, composition, and assembly.

This review highlights three recent trends in the field of kinetochore biology: the proliferation of structural data for kinetochore protein complexes (including CBF3, Dam1c, Mis12cMIND, and CENP-NLChl4/Iml3); the growing consensus that the kinetochore is a dynamic structure whose composition changes as the cell cycle progresses; and the mounting evidence of multiple pathways whereby the microtubule-binding elements of the outer kinetochore may be recruited by inner kinetochore proteins. Our focus is on the two best-studied systems in the field: human and budding yeast kinetochores. This review will demonstrate the remarkable similarity of these two systems, as well as their intriguing differences.

Babies in traffic: infant vocalizations and listener sex modulate auditory motion perception.

Infant vocalizations and "looming sounds" are classes of environmental stimuli that are critically important to survival but can have dramatically different emotional valences. Here, we simultaneously presented listeners with a stationary infant vocalization and a 3D virtual looming tone for which listeners made auditory time-to-arrival judgments. Negatively valenced infant cries produced more cautious (anticipatory) estimates of auditory arrival time of the tone over a no-vocalization control. Positively valenced laughs had the opposite effect, and across all conditions, men showed smaller anticipatory biases than women. In Experiment 2, vocalization-matched vocoded noise stimuli did not influence concurrent auditory time-to-arrival estimates compared with a control condition. In Experiment 3, listeners estimated the egocentric distance of a looming tone that stopped before arriving. For distant stopping points, women estimated the stopping point as closer when the tone was presented with an infant cry than when it was presented with a laugh. For near stopping points, women showed no differential effect of vocalization type. Men did not show differential effects of vocalization type at either distance. Our results support the idea that both the sex of the listener and the emotional valence of infant vocalizations can influence auditory motion perception and can modulate motor responses to other behaviorally relevant environmental sounds. We also find support for previous work that shows sex differences in emotion processing are diminished under conditions of higher stress.