Transfer learning in a biomaterial fibrosis model identifies in vivo senescence heterogeneity and contributions to vascularization and matrix production across species and diverse pathologies.

Cellular senescence is a state of permanent growth arrest that plays an important role in wound healing, tissue fibrosis, and tumor suppression. Despite senescent cells' (SnCs) pathological role and therapeutic interest, their phenotype in vivo remains poorly defined. Here, we developed an in vivo-derived senescence signature (SenSig) using a foreign body response-driven fibrosis model in a p16-CreERT2;Ai14 reporter mouse. We identified pericytes and "cartilage-like" fibroblasts as senescent and defined cell type-specific senescence-associated secretory phenotypes (SASPs). Transfer learning and senescence scoring identified these two SnC populations along with endothelial and epithelial SnCs in new and publicly available murine and human data single-cell RNA sequencing (scRNAseq) datasets from diverse pathologies. Signaling analysis uncovered crosstalk between SnCs and myeloid cells via an IL34-CSF1R-TGFßR signaling axis, contributing to tissue balance of vascularization and matrix production. Overall, our study provides a senescence signature and a computational approach that may be broadly applied to identify SnC transcriptional profiles and SASP factors in wound healing, aging, and other pathologies.

Charged hybrid block copolymer-lipid-cholesterol vesicles: pH, ionic environment, and composition dependence of phase transitions.

The impacts of pH, salt concentration (expressed as Debye length), and composition on the phase behavior of hybrid block copolymer-lipid-cholesterol bilayers incorporating carboxyl-terminated poly(butadiene)-block-poly(ethylene oxide) copolymer (PBdPEO1800(-)) or/and non-carboxyl-terminated PBdPEO (PBdPEO1800 or/and PBdPEO950), egg sphingomyelin (egg SM), and cholesterol were examined using fluorescence spectroscopy of laurdan. Laurdan emission spectra were decomposed into three lognormal curves as functions of energy. The ratio of the area of the mid-energy peak to the sum of the areas of all three peaks was evaluated as vesicles were cooled, yielding temperature breakpoint values (Tbreak) expected to be within the range of the phase transition temperature. Tbreak values displayed dependence on pH, Debye length, and vesicle composition consistent with an electrostatic repulsion contribution to vesicle phase behavior. Increased pH and Debye length, for which a greater dissociated fraction of PBdPEO1800(-) and a greater energy of electrostatic repulsion would be expected, resulted in Tbreak values as much as 10 °C less than at low pH or short Debye lengths. Additionally, at Debye lengths comparable to those at physiologically relevant ionic strength, Tbreak at pH 5.9 was observed to be slightly higher than at pH 7.0 for vesicles containing 50 mol% PBdPEO1800(-). Electrostatic effects observed for hybrid vesicles incorporating significant amounts of carboxyl-terminated polymer may have the ability to drive phase separation in response to pH drops-such as those observed after endocytosis-in physiologically relevant conditions, suggesting the utility of such materials for drug delivery.

Effects of Trehalose Solutions at Different Concentrations on High-Intensity Intermittent Exercise Performance.

Trehalose solution ingested during exercise induces gradual increases in blood glucose levels and the insulin response compared with glucose solution. Trehalose solution aids in the maintenance of performance in the later stages of prolonged exercise. The purpose of this study was to identify the lowest concentration at which the properties of trehalose could be exploited. Groups of 12 healthy men (21.3 ± 1.3 years) and 10 healthy men (21.1 ± 0.7 years) with recreational training were included in experiments 1 and 2, respectively. Both experiments followed the same protocol. After fasting for 12 h, the participants performed a 60 min constant-load exercise at 40% V˙O2 peak using a bicycle ergometer and ingested 500 mL of a trial drink (experiment 1: water, 8% glucose, and 6 or 8% trehalose; experiment 2: 4 or 6% trehalose). They performed four sets of the Wingate test combined with a 30 min constant-load exercise at 40% V˙O2 peak. The experiment was conducted using a randomized cross-over design; trial drink experiments were conducted over intervals of 7 to 12 days. The exercise performance was evaluated based on mean power in the Wingate test. Blood was collected from the fingertip at 12 points during each experiment to measure blood glucose levels. During the high-intensity 5 h intermittent exercise, no differences were found between the groups in exercise performance in the later stages with concentrations of 8, 6, and 4% trehalose solution. The results suggest that trehalose could be useful for making a new type of mixed carbohydrate solution. Further studies to determine the trehalose response of individual athletes during endurance exercise are needed.

Carbohydrate Mouth Rinse and Spray Improve Prolonged Exercise Performance in Recreationally Trained Male College Students.

Mouth rinsing with a carbohydrate (CHO) solution has emerged as a sports nutrition strategy to increase endurance performance. This study aimed to clarify the effects of two forms of CHO sensing in the mouth (i.e., CHO mouth rinse (CMR) and CHO mouth spray (CMS)) on exercise performance during prolonged exercise, including ultra-high intensity intermittent exercise over time. We conducted the following experimental trials: (1) 6% glucose solution (G), (2) 6% CMR, (3) 6% CMS, and (4) water (WAT). These trials were conducted at least 1 week apart in a randomized crossover design. Eight male college students performed constant-load exercise for 60 min (intensity 40% VO2peak), four sets of the Wingate test (three 30 s Wingate tests with a 4 min recovery between each test), and a constant-load exercise for 30 min (intensity 40% VO2peak). The mean exercise power output (Watt), ratings of perceived exertion, and blood glucose levels were measured. We found that the mean power values of the CMR and CMS in the third and fourth sets was significantly higher than that of WAT (p < 0.05), and that the G trial did not show a significant difference from any other trial. Thus, when compared to G or WAT, CMR and CMS can help improve endurance exercise performance.

Functionalizing silica sol-gel with entrapped plant virus-based immunosorbent nanoparticles.

Advancements in understanding and engineering of virus-based nanomaterials (VBNs) for biomedical applications motivate a need to explore the interfaces between VBNs and other biomedically-relevant chemistries and materials. While several strategies have been used to investigate some of these interfaces with promising initial results, including VBN-containing slow-release implants and VBN-activated bioceramic bone scaffolds, there remains a need to establish VBN-immobilized three dimensional materials that exhibit improved stability and diffusion characteristics for biosensing and other analyte-capture applications. Silica sol-gel chemistries have been researched for biomedical applications over several decades and are well understood; various cellular organisms and biomolecules (e.g., bacteria, algae, enzymes) have been immobilized in silica sol-gels to improve viability, activity, and form factor (i.e., ease of use). Here we present the immobilization of an antibody-binding VBN in silica sol-gel by pore confinement. We have shown that the resulting system is sufficiently diffuse to allow antibodies to migrate in and out of the matrix. We also show that the immobilized VBN is capable of antibody binding and elution functionality under different buffer conditions for multiple use cycles. The promising results of the VBN and silica sol-gel interface indicate a general applicability for VBN-based bioseparations and biosensing applications.

Characterization of phase separation phenomena in hybrid lipid/block copolymer/cholesterol bilayers using laurdan fluorescence with log-normal multipeak analysis.

Phase separation phenomena in hybrid lipid/block copolymer/cholesterol bilayers combining polybutadiene-block-polyethylene oxide (PBdPEO), egg sphingomyelin (egg SM), and cholesterol were studied with fluorescence spectroscopy and microscopy for comparison to lipid bilayers composed of palmitoyl oleoyl phosphatidylcholine (POPC), egg SM, and cholesterol. Laurdan emission spectra were decomposed into three lognormal curves. The temperature dependence of the ratios of the areas of the middle and lowest energy peaks revealed temperature break-point (Tbreak) values that were in better agreement, compared to generalized polarization inflection temperatures, with phase transition temperatures in giant unilamellar vesicles (GUVs). Agreement between GUV and spectroscopy results was further improved for hybrid vesicles by using the ratio of the area of the middle peak to the sum of the areas all three peaks to find the Tbreak values. For the hybrid vesicles, trends at Tbreak are hypothesized to be correlated with the mechanisms by which the phase transition takes place, supported by the compositional range as well as the morphologies of domains observed in GUVs. Low miscibility of PBdPEO and egg SM is suggested by the finding of relatively high Tbreak values at cholesterol contents greater than 30 mol%. Further, GUV phase behavior suggests stronger partitioning of cholesterol into PBdPEO than into POPC, and less miscibility of PBdPEO than POPC with egg SM. These results, summarized using a heat-map, contribute to the limited body of knowledge regarding the effect of cholesterol on hybrid membranes, with potential application toward the development of such materials for drug delivery or membrane protein reconstitution.

p21 produces a bioactive secretome that places stressed cells under immunosurveillance.

Immune cells identify and destroy damaged cells to prevent them from causing cancer or other pathologies by mechanisms that remain poorly understood. Here, we report that the cell-cycle inhibitor p21 places cells under immunosurveillance to establish a biological timer mechanism that controls cell fate. p21 activates retinoblastoma protein (Rb)­dependent transcription at select gene promoters to generate a complex bioactive secretome, termed p21-activated secretory phenotype (PASP). The PASP includes the chemokine CXCL14, which promptly attracts macrophages. These macrophages disengage if cells normalize p21 within 4 days, but if p21 induction persists, they polarize toward an M1 phenotype and lymphocytes mount a cytotoxic T cell response to eliminate target cells, including preneoplastic cells. Thus, p21 concurrently induces proliferative arrest and immunosurveillance of cells under duress.

Single Ingestion of Trehalose Enhances Prolonged Exercise Performance by Effective Use of Glucose and Lipid in Healthy Men.

Trehalose increases blood glucose levels slowly and induces a slight insulin response. The present study aimed to study the effect of trehalose on prolonged exercise performance. The participants were 12 healthy men (age: 21.3 ± 0.9 y). After an overnight fast (12 h), they first exercised with a constant load (intensity: 40% V˙O2peak) for 60 min using a bicycle ergometer. They continued to exercise with a constant load (40% V˙O2peak) for 30 min between four sets of the 30-s Wingate test. After the 1st set, each participant ingested 500 mL water (control), 8% glucose, or 8% trehalose in three trials. These three trials were at least one week apart and were conducted in a double-blind and randomized crossover manner. Blood was collected for seven biochemical parameters at 12 time points during the experiment. The area under the curve of adrenaline after ingestion of trehalose was significantly lower than that for water and tended to be lower than that for glucose in the later stage of the exercise. Lower secretion of adrenaline after a single dose of 8% trehalose during prolonged exercise reflected the preservation of carbohydrates in the body in the later stage of the exercise. In conclusion, a single ingestion of trehalose helped to maintain prolonged exercise performance.

Hybrid lipid/block copolymer vesicles display broad phase coexistence region.

The fluidity and polar environment of ~100 nm hybrid vesicles combining dipalmitoylphosphatidylcholine (DPPC) and poly(1,2-butadiene)-block-polyethylene oxide (PBd-PEO, average molecular weight 950 g/mol) were studied upon vesicle heating using the fluorescence spectroscopy techniques of DPH anisotropy and laurdan generalized polarization (GP). These techniques indicated PBd-PEO membranes are less ordered than solid DPPC, but slightly more ordered than fluid DPPC or dioleoylphosphatidylcholine (DOPC) membranes. We find the DPH anisotropy values are less than expected from additivity of the components' anisotropies in the fluid phase mixture of DPPC and PBd-PEO, inferring that DPPC strongly fluidizes the PBd-PEO. We use transitions in DPH anisotropy and laurdan GP to create a temperature/composition phase diagram for DPPC/PBd-PEO which we find displays a significantly broader solid/fluid phase coexistence region than DPPC/DOPC, showing that DPPC partitions less readily into fluid PBd-PEO than into fluid DOPC. The existence of a broad solid/fluid phase coexistence region in DPPC/PBd-PEO vesicles is verified by Förster resonance energy transfer results and the visualization of phase separation in giant unilamellar vesicles containing up to 95% PBd-PEO and a single phase in 100% PBd-PEO vesicles at room temperature. These results add to the limited knowledge of phase behavior and phase diagrams of hybrid vesicles, and should be useful in understanding and tailoring membrane surface architecture toward biomedical applications such as drug delivery or membrane protein reconstitution.

Injectable and Spatially Patterned Microporous Annealed Particle (MAP) Hydrogels for Tissue Repair Applications.

Spatially patterned hydrogels are becoming increasingly popular in the field of regenerative medicine and tissue repair because of their ability to guide cell infiltration and migration. However, postfabrication technologies are usually required to spatially pattern a hydrogel, making these hydrogels difficult to translate into the clinic. Here, an injectable spatially patterned hydrogel is reported using hyaluronic acid (HA)-based particle hydrogels. These particle hydrogels are sequentially loaded into a syringe to form a pattern and, once injected, they maintain the pattern. The applicability of this hydrogel in a wound healing skin model, a subcutaneous implant model, as well as a stroke brain model is examined and distinct patterning in all models tested is shown. This injectable and spatially patterned hydrogel can be used to create physical or biochemical gradients. Further, this design can better match the scaffold properties within the physical location of the tissue (e.g., wound border vs wound center). This allows for better design features within the material that promote repair and regeneration.

Cresyl Violet Stains Mast Cells Selectively: Its Application to Counterstaining in Immunohistochemistry.

The thiazine dye toluidine blue (TB) is well known to stain mast cells and hyaline cartilage metachromatically, and thus is mostly often used for their identification. However, TB is not suitable for counterstaining in immunohistochemistry, because of its high-background staining in the cytoplasm of other cell species and in extracellular structures. To expand the knowledge about dyestuffs staining mast cells in consideration with their usage in immunohistochemistry, we determined the stainability of several thiazines and oxazines, which are structurally related compounds to TB, using sections of mast cell-containing tissues. We found that all azine dyes used metachromatically stained mast cells and cartilage. Among these dyes, an oxazines cresyl violet (CV) stained mast cells with lower background, suggesting that those are useful for detecting mast cells and for counterstaining in immunohistochemistry. To ascertain its utility, CV was used in immunostaining of bHSDs in sections from adult rat ovary. Immunopositive signals reflected by DAB development in brown were clearly detected even after CV staining. We conclude that, similar to thiazines, oxazines stain mast cells metachromatically, and that of these, CV is more useful as a counterstain in immunohistochemistry than TB.

Cyclin A2 is an RNA binding protein that controls Mre11 mRNA translation.

Cyclin A2 activates the cyclin-dependent kinases Cdk1 and Cdk2 and is expressed at elevated levels from S phase until early mitosis. We found that mutant mice that cannot elevate cyclin A2 are chromosomally unstable and tumor-prone. Underlying the chromosomal instability is a failure to up-regulate the meiotic recombination 11 (Mre11) nuclease in S phase, which leads to impaired resolution of stalled replication forks, insufficient repair of double-stranded DNA breaks, and improper segregation of sister chromosomes. Unexpectedly, cyclin A2 controlled Mre11 abundance through a C-terminal RNA binding domain that selectively and directly binds Mre11 transcripts to mediate polysome loading and translation. These data reveal cyclin A2 as a mechanistically diverse regulator of DNA replication combining multifaceted kinase-dependent functions with a kinase-independent, RNA binding-dependent role that ensures adequate repair of common replication errors.

Acute toxic effects of 3,3'-iminodipropionitrile on hypothalamic-pituitary-gonadal axis in male rats.

Exposure to 3,3'-iminodipropionitrile (IDPN) causes persistent neurotoxicity, while its reproductive toxicity in female rats is transient, indicating that gonadotropin-releasing hormone (GnRH) neurons and gonadotrophs receive little or no damage from IDPN and that the transient gonadal toxicity may be also observed in males. To clarify these points, the acute toxic effects of IDPN on hypothalamic-pituitary-gonadal axis of male rats were examined histologically, biochemically and serologically. A single intraperitoneal injection of IDPN (1000 mg/kg body weight) induced signs of neurotoxicity within a day; nevertheless, GnRH neurons were not affected throughout the experimental period. Four days after IDPN treatment, the plasma level of testosterone but not gonadotropins decreased and active caspase 3-immunopositive spermatids increased; both parameters returned to normal levels afterwards. Data from our studies revealed that while IDPN had little or no toxic effect on GnRH neurons or gonadotrophs it was transiently toxic to gonads in both sexes.

Number of CGG repeats in the FMR1 gene of Japanese patients with primary ovarian insufficiency.

OBJECTIVE: To define the number of CGG repeats in the FMR1 gene of Japanese patients with primary ovarian insufficiency (POI) and normal controls. DESIGN: Retrospective, controlled cohort study. SETTING: Outpatient department of an academic tertiary center. PATIENT(S): One hundred twenty-eight consecutive Japanese patients with sporadic, nonsyndromic POI and 98 controls with normal menstruation. INTERVENTION(S): Deoxyribonucleic acid was obtained from the plasma of each subject. MAIN OUTCOME MEASURE(S): Differences in the distribution of CGG repeat numbers between patients with POI and controls. RESULT(S): Six alleles in the intermediate range and two in the premutation range were found in five and two patients with POI, respectively, but none were identified in normal controls. The prevalence of FMR1 premutation among Japanese POI patients was 1.56% (2 of 128). The prevalence of having >36 CGG repeats in the FMR1 gene was significantly higher in patients with POI than in controls, and age at the onset of amenorrhea was significantly lower in patients with >38 repeats. CONCLUSION(S): More than 36 CGG repeats in the FMR1 might intensify the etiology of POI, at least up to the premutation range.

International flow of Zambian nurses.

This commentary paper highlights changing patterns of outward migration of Zambian nurses. The aim is to discuss these pattern changes in the light of policy developments in Zambia and in receiving countries. Prior to 2000, South Africa was the most important destination for Zambian registered nurses. In 2000, new destination countries, such as the United Kingdom, became available, resulting in a substantial increase in migration from Zambia. This is attributable to the policy of active recruitment by the United Kingdom's National Health Service and Zambia's policy of offering Voluntary Separation Packages: early retirement lump-sum payments promoted by the government, which nurses used towards migration costs. The dramatic decline in migration to the United Kingdom since 2004 is likely to be due to increased difficulties in obtaining United Kingdom registration and work permits. Despite smaller numbers, enrolled nurses are also leaving Zambia for other destination countries, a significant new development. This paper stresses the need for nurse managers and policy-makers to pay more attention to these wider nurse migration trends in Zambia, and argues that the focus of any migration strategy should be on how to retain a motivated workforce through improving working conditions and policy initiatives to encourage nurses to stay within the public sector.

Melatonin binding sites in estrogen receptor-positive cells derived from human endometrial cancer.

Our previous work showed that melatonin (N-acetyl-5-methoxytryptamine) inhibits proliferation of the human endometrial cancer cell line, Ishikawa, which is estrogen receptor-positive. The aim of the present study was to determine whether Ishikawa cells possess membrane melatonin receptors. Binding of the radioligand 2-[125I]-iodomelatonin to membrane preparations obtained from Ishikawa cells was detectable, saturable and stable. Scatchard analysis revealed that the dissociation constant (Kd) of the binding sites was 179.0 pm (similar to that of the MT2 [Mel1b] melatonin receptor subtype), and that the concentration (Bmax) of the binding sites was 12.9 fmol/mg protein. Luzindole, a selective MT2 melatonin receptor antagonist, significantly suppressed binding of 2-[125I]-iodomelatonin at all concentrations tested (10(-8) to 10(-4) m). These results suggest that the MT2 melatonin receptor subtype is present in the membranes of Ishikawa cells, and that the antiproliferative effect of melatonin on Ishikawa cells is mediated via the MT2 receptor. This may have implications for the use of melatonin in endometrial cancer therapy.