NAC and Zuotin/Hsp70 chaperone systems coexist at the ribosome tunnel exit in vivo.

The area surrounding the tunnel exit of the 60S ribosomal subunit is a hub for proteins involved in maturation and folding of emerging nascent polypeptide chains. How different factors vie for positioning at the tunnel exit in the complex cellular environment is not well understood. We used in vivo site-specific cross-linking to approach this question, focusing on two abundant factors-the nascent chain-associated complex (NAC) and the Hsp70 chaperone system that includes the J-domain protein co-chaperone Zuotin. We found that NAC and Zuotin can cross-link to each other at the ribosome, even when translation initiation is inhibited. Positions yielding NAC-Zuotin cross-links indicate that when both are present the central globular domain of NAC is modestly shifted from the mutually exclusive position observed in cryogenic electron microscopy analysis. Cross-linking results also suggest that, even in NAC's presence, Hsp70 can situate in a manner conducive for productive nascent chain interaction-with the peptide binding site at the tunnel exit and the J-domain of Zuotin appropriately positioned to drive stabilization of nascent chain binding. Overall, our results are consistent with the idea that, in vivo, the NAC and Hsp70 systems can productively position on the ribosome simultaneously.

The Effect of Interlayer Delay on the Heat Accumulation, Microstructures, and Properties in Laser Hot Wire Directed Energy Deposition of Ti-6Al-4V Single-Wall.

Laser hot wire directed energy deposition (LHW-DED) is a layer-by-layer additive manufacturing technique that permits the fabrication of large-scale Ti-6Al-4V (Ti64) components with a high deposition rate and has gained traction in the aerospace sector in recent years. However, one of the major challenges in LHW-DED Ti64 is heat accumulation, which affects the part quality, microstructure, and properties of as-built specimens. These issues require a comprehensive understanding of the layerwise heat-accumulation-driven process-structure-property relationship in as-deposited samples. In this study, a systematic investigation was performed by fabricating three Ti-6Al-4V single-wall specimens with distinct interlayer delays, i.e., 0, 120, and 300 s. The real-time acquisition of high-fidelity thermal data and high-resolution melt pool images were utilized to demonstrate a direct correlation between layerwise heat accumulation and melt pool dimensions. The results revealed that the maximum heat buildup temperature of the topmost layer decreased from 660 °C to 263 °C with an increase to a 300 s interlayer delay, allowing for better control of the melt pool dimensions, which then resulted in improved part accuracy. Furthermore, the investigation of the location-specific composition, microstructure, and mechanical properties demonstrated that heat buildup resulted in the coarsening of microstructures and, consequently, the reduction of micro-hardness with increasing height. Extending the delay by 120 s resulted in a 5% improvement in the mechanical properties, including an increase in the yield strength from 817 MPa to 859 MPa and the ultimate tensile strength from 914 MPa to 959 MPa. Cooling rates estimated at 900 °C using a one-dimensional thermal model based on a numerical method allowed us to establish the process-structure-property relationship for the wall specimens. The study provides deeper insight into the effect of heat buildup in LHW-DED and serves as a guide for tailoring the properties of as-deposited specimens by regulating interlayer delay.

Thrombopoietin mimetic stimulates bone marrow vascular and stromal niches to mitigate acute radiation syndrome.

Background: Acute radiation syndrome (ARS) manifests after exposure to high doses of radiation in the instances of radiologic accidents or incidents. Facilitating the regeneration of the bone marrow (BM), namely the hematopoietic stem and progenitor cells (HSPCs), is a key in mitigating ARS and multi-organ failure. JNJ-26366821, a PEGylated thrombopoietin mimetic (TPOm) peptide, has been shown as an effective medical countermeasure (MCM) to treat hematopoietic-ARS (H-ARS) in mice. However, the activity of TPOm on regulating BM vascular and stromal niches to support HSPC regeneration has not yet been elucidated. Methods: C57BL/6J mice (9-14 weeks old) received sublethal or lethal total body irradiation (TBI), a model for H-ARS, by 137Cs or X-rays. At 24 hours post-irradiation, mice were subcutaneously injected with a single dose of TPOm (0.3 mg/kg or 1.0 mg/kg) or PBS (vehicle). At homeostasis and on days 4, 7, 10, 14, 18, and 21 post-TBI with and without TPOm treatment, BM was harvested for histology, BM flow cytometry of HSPCs, endothelial (EC) and mesenchymal stromal cells (MSC), and whole-mount confocal microscopy. For survival, irradiated mice were monitored and weighed for 30 days. Lastly, BM triple negative cells (TNC; CD45-, TER-119-, CD31-) were sorted for single-cell RNA-sequencing to examine transcriptomics after TBI with or without TPOm treatment. Results: At homeostasis, TPOm expanded the number of circulating platelets and HSPCs, ECs, and MSCs in the BM. Following sublethal TBI, TPOm improved BM architecture and promoted recovery of HSPCs, ECs, and MSCs. Furthermore, TPOm elevated VEGF-C levels in normal and irradiated mice. Following lethal irradiation, mice improved body weight recovery and 30-day survival when treated with TPOm after 137Cs and X-ray exposure. Additionally, TPOm reduced vascular dilation and permeability. Finally, single-cell RNA-seq analysis indicated that TPOm increased the expression of collagens in MSCs to enhance their interaction with other progenitors in BM and upregulated the regeneration pathway in MSCs. Conclusions: TPOm interacts with BM vascular and stromal niches to locally support hematopoietic reconstitution and systemically improve survival in mice after TBI. Therefore, this work warrants the development of TPOm as a potent radiation MCM for the treatment of ARS.

Thrombopoietin mimetic stimulates bone marrow vascular and stromal niches to mitigate acute radiation syndrome.

BACKGROUND: Acute radiation syndrome (ARS) manifests after exposure to high doses of radiation in the instances of radiologic accidents or incidents. Facilitating regeneration of the bone marrow (BM), namely the hematopoietic stem and progenitor cells (HSPCs), is key in mitigating ARS and multi-organ failure. JNJ-26366821, a PEGylated thrombopoietin mimetic (TPOm) peptide, has been shown as an effective medical countermeasure (MCM) to treat hematopoietic-ARS (H-ARS) in mice. However, the activity of TPOm on regulating BM vascular and stromal niches to support HSPC regeneration has yet to be elucidated. METHODS: C57BL/6J mice (9-14 weeks old) received sublethal or lethal total body irradiation (TBI), a model for H-ARS, by 137Cs or X-rays. At 24 h post-irradiation, mice were subcutaneously injected with a single dose of TPOm (0.3 mg/kg or 1.0 mg/kg) or PBS (vehicle). At homeostasis and on days 4, 7, 10, 14, 18, and 21 post-TBI with and without TPOm treatment, BM was harvested for histology, BM flow cytometry of HSPCs, endothelial (EC) and mesenchymal stromal cells (MSC), and whole-mount confocal microscopy. For survival, irradiated mice were monitored and weighed for 30 days. Lastly, BM triple negative cells (TNC; CD45-, TER-119-, CD31-) were sorted for single-cell RNA-sequencing to examine transcriptomics after TBI with or without TPOm treatment. RESULTS: At homeostasis, TPOm expanded the number of circulating platelets and HSPCs, ECs, and MSCs in the BM. Following sublethal TBI, TPOm improved BM architecture and promoted recovery of HSPCs, ECs, and MSCs. Furthermore, TPOm elevated VEGF-C levels in normal and irradiated mice. Following lethal irradiation, mice improved body weight recovery and 30-day survival when treated with TPOm after 137Cs and X-ray exposure. Additionally, TPOm reduced vascular dilation and permeability. Finally, single-cell RNA-seq analysis indicated that TPOm increased the expression of collagens in MSCs to enhance their interaction with other progenitors in BM and upregulated the regeneration pathway in MSCs. CONCLUSIONS: TPOm interacts with BM vascular and stromal niches to locally support hematopoietic reconstitution and systemically improve survival in mice after TBI. Therefore, this work warrants the development of TPOm as a potent radiation MCM for the treatment of ARS.

Cell-free SLC30A10 messenger ribonucleic acid (mRNA) expression and their association with vitamin-D level among non-small cell lung cancer (NSCLC) patients.

BACKGROUND: Lung cancer has been major cause of cancer related death and day by day Non-small cell lung cancer (NSCLC) cases are increasing globally. Present study explored the link between SLC30A10 mRNA expression with vitamin-D level among the NSCLC patients. METHODS: Present study included newly diagnosed 100 NSCLC patients and 100 healthy controls. Quantitative real time PCR was performed to check the SLC30A10 mRNA expression after cDNA synthesis from extracted total RNA from serum sample. Vitamin-D level was also analyzed in all the NSCLC patients by electrochemiluminscence based immunoassay method. RESULTS: Present research work observed decreased SLC30A10 mRNA expression (0.16 fold) among the NSCLC patients, decreased SLC30A10 mRNA expression was linked with advanced stage (0.15 fold, P < 00001) of disease and distant organ metastases (0.11 fold, P < 00001) compared to its contrast. Decreased level of vitamin-D was also observed with advanced stage (17.98 ng/ml, P < 00001) of disease and distant organ metastases (16.23 ng/ml, P < 00001) compared to its contrast. Positive correlation was observed between SLC30A10 mRNA expression with vitamin-D level among the NSCLC patients suggesting decrease or increase in SLC30A10 mRNA expression mau decreases or increase the vitamin-D level. NSCLC patients with vitamin-D deficiency had 0.14 reduced SCL30A10 mRNA expression while insufficient (P = 0 .06) and sufficient (P = 0.03) showed comparatively high SCL30A10 mRNA expression. CONCLUSION: Study concluded that down regulation of SLC30A10 mRNA and vitamin-D deficiency may involve in advancement of disease and distant organ metastases. It was also suggested that the decrease of increase in SLC30A10 expression may cause the decrease of increase in vitamin-D level among the NSCLC patients may be involved in disease severity and worseness of NSCLC disease.