Search

The sterility testing methods described in pharmacopoeias require an incubation period of 14 days to obtain analysis results. An alternative method that can significantly shorten the detection time and improve the accuracy is in urgent need to meet the sterility testing requirements of regenerative medicine products with a short shelf life. In this study, we developed the next-generation sequencing-based sterility test (NGSST) based on sequencing and multiple displacement amplification. The NGSST can be finished within 48 hours with five steps including whole genome amplification, sequencing, alignment, sterility testing report, and microorganism identification. We use RPKM ratio to minorize the influence of environmental bacteria and determine its cutoff based AUC curve. The NGSST showed high sensitivity in reporting contaminates at 0.1 CFU in supernatant of biological product or 1 CFU in cell suspension. Furthermore, we identified microorganisms in 5 primary umbilical cord mesenchymal stem cell samples that were tested positive by BacT/ALERTR 3D. Overall, the NGSST can serve as a promising alternative for sterility testing of biological products.

Strontium, a popular osteogenic component, has been incorporated into various types of orthopaedic biomaterials to enhance bone regeneration. Strontium performs dual effects in promoting bone formation and inhibiting bone resorption. Previous studies have focused on the effects of strontium ions (Sr2+) in regulating stem cell behavior to initiate regenerative capacity. However, its mechanisms for regulating the fate and homeostasis of stem cells have not been fully elucidated. In this study, the promotive effect of Sr2+ on the osteogenic differentiation of mesenchymal stem cells was confirmed both in vitro and in vivo. Interestingly, in response to Sr2+ treatment, stem cells performed asymmetric cell division to balance stemness maintenance and osteogenic differentiation. In initiating osteogenic differentiation, Sr2+ maintained more cells in the cell cycle by upregulating the population of S and G2/M phase cells, and this increase in the cell population contributed to enhanced osteogenic differentiation. The divided cells with different cell fates were observed, with one daughter cell maintained stemness, while the other committed to osteogenic lineage. Further investigation revealed that Sr2+ activated noncanonical Wnt signaling to regulate the expression and distribution of the Par complex, thus regulating cell division. As a result, the daughter cells committed to different cell fates due to the discriminately activation of osteogenic transcription factors caused by asymmetrically distributed Par3 and aPKC. The results of this study could facilitate the design of biomaterials for bone regeneration by providing a better understanding of cell fate determination regulated by strontium.

The study of heterogeneity in CRC single cell derived organoid using WES, WGS and ScRNA sequencing.

Additional file 1: Figure S1. Overview of the experimental procedure and data quality. Figure S2. Cell cycle phase assigned for cells throughout hESCs-derived LSCs differentiation. Figure S3. Pseudotime analysis characterizes expression changes throughout hESCs-derived LSCs differentiation. Figure S4. Transcriptional difference of subpopulations in hESCs-derived LSCs.

Additional file 1: Fig. S1. Workflow for data search and selection. Fig. S2. Overview of data collected for transcriptome variation analysis. (A) Boxplot showing correlation coefficient of transcriptome expression for each sample to others in the dataset before data selection step 3. 0.9 was selected as a cutoff for filtering out samples with lower median Pearson’s r.(B) Barplot showing number of samples in each study. (C) Histograms showing number of reads (up) and mapping results (down) across samples. Some MSCs derived from different anatomical parts of placenta, AM CM, CP, and CV. AD: Adipose tissue; AM: Amniotic membrane; BM: Bone marrow; CM: Chorionic membrane; CP: Chorionic plate; CV: Chorionic villi; ED: Endometrial; DP: Dental pulp; PL: Placenta; UC: Umbilical cord. Fig. S3. Transcriptome variation across MSC samples. (A) Scatter plot showing DM value and mean expression (CPM) for each expressed gene. (B) Scatter plot showing DM value and gene length for each expressed gene. (C) GESA positive results showing enrichment in GO cellular component (left) and molecular function (right) gene sets database based on ranked genes list in descending order by the DM value. (D) GESA negative results showing enrichment in GO gene sets database based on ranked genes list in descending order by the DM value. Only the top 20 terms with highest NES were presented (p

The sterility testing methods described in pharmacopoeias require an incubation period of 14 days to obtain analysis results. An alternative method that can significantly shorten the detection time and improve the accuracy is in urgent need to meet the sterility testing requirements of regenerative medicine products with a short shelf life. In this study, we developed the next-generation sequencing-based sterility test (NGSST) based on sequencing and multiple displacement amplification. The NGSST can be finished within 48 hours with five steps including whole genome amplification, sequencing, alignment, sterility testing report, and microorganism identification. We use RPKM ratio to minorize the influence of environmental bacteria and determine its cutoff based AUC curve. The NGSST showed high sensitivity in reporting contaminates at 0.1 CFU in supernatant of biological product or 1 CFU in cell suspension. Furthermore, we identified microorganisms in 5 primary umbilical cord mesenchymal stem cell samples that were tested positive by BacT/ALERTR 3D. Overall, the NGSST can serve as a promising alternative for sterility testing of biological products.

Background Mesenchymal stromal cell (MSC)-based therapies are being actively investigated in various inflammatory disorders. However, functional variability among MSCs cultured in vitro will lead to distinct therapeutic efficacies. Until now, the mechanisms behind immunomodulatory functional variability in MSCs are still unclear. Methods We systemically investigated transcriptomic variations among MSC samples derived from multiple tissues to reveal their effects on immunomodulatory functions of MSCs. We then analyzed transcriptomic changes of MSCs licensed with INFγ to identify potential molecular mechanisms that result in distinct MSC samples with different immunomodulatory potency. Results MSCs were clustered into distinct groups showing different functional enrichment according to transcriptomic patterns. Differential expression analysis indicated that different groups of MSCs deploy common regulation networks in response to inflammatory stimulation, while expression variation of genes in the networks could lead to different immunosuppressive capability. These different responsive genes also showed high expression variability among unlicensed MSC samples. Finally, a gene panel was derived from these different responsive genes and was able to regroup unlicensed MSCs with different immunosuppressive potencies. Conclusion This study revealed genes with expression variation that contribute to immunomodulatory functional variability of MSCs and provided us a strategy to identify candidate markers for functional variability assessment of MSCs.

Background Human pluripotent stem cell-derived limbal stem cells (hPSC-derived LSCs) provide a promising cell source for corneal transplants and ocular surface reconstruction. Although recent efforts in the identification of LSC markers have increased our understanding of the biology of LSCs, much more remains to be characterized in the developmental origin, cell fate determination, and identity of human LSCs. The lack of knowledge hindered the establishment of efficient differentiation protocols for generating hPSC-derived LSCs and held back their clinical application. Results Here, we performed a time-course single-cell RNA-seq to investigate transcriptional heterogeneity and expression changes of LSCs derived from human embryonic stem cells (hESCs). Based on current protocol, expression heterogeneity of reported LSC markers were identified in subpopulations of differentiated cells. EMT has been shown to occur during differentiation process, which could possibly result in generation of untargeted cells. Pseudotime trajectory analysis revealed transcriptional changes and signatures of commitment of hESCs-derived LSCs and their progeny—the transit amplifying cells. Conclusion Single-cell RNA-seq revealed time-course expression changes and significant transcriptional heterogeneity during hESC-derived LSC differentiation in vitro. Our results demonstrated candidate developmental trajectory and several new candidate markers for LSCs, which could facilitate elucidating the identity and developmental origin of human LSCs in vivo.

Non- Volatile Memory (NVM) systems require logging to support crash consistency. However, log operations introduce severe performance overhead. Recently, LAD was proposed to eliminate log operations for some transactions in which the total amount of updated cachelines is smaller than the Asynchronous DRAM Refresh (ADR) buffer, without affecting crash consistency. Nevertheless, on multicore, concurrent transactions tend to exhaust the ADR resource and hence log operations have to be conducted in LAD. In this study, we observe that a significant number of log operations could be avoided if each transaction run alone. To eliminate these unnecessary log operations, this paper proposes virtual ADR buffers to decouple buffering from the ADR's reliable writing data. Specifically, only logless operations are allowed to access ADR resources. Additionally, this paper proposes to adopt redo log with DRAM cache to speed up the transaction commit speed. The evaluation results demonstrate that our proposed scheme can efficiently reduce log operation up to 94.9 % and improve the transaction throughput up to 78.6 %.

The persistent memory (PM) requires maintaining the crash consistency and encrypting data, to ensure data recoverability and data confidentiality. The enforcement of these two goals does not only put more burden on programmers but also degrades performance. To address this issue, we propose a hardware-assisted encrypted persistent memory system. Specifically, logging and data encryption are assisted by hardware. Furthermore, we apply the counter-based encryption and the cipher feedback (CFB) mode encryption to data and log respectively, reducing the encryption overhead. Our primary experimental results show that the transaction throughput of the proposed design outperforms the baseline design by up to 34.4%.

Strontium as a popular osteogenic component has been incorporated into various types of orthopaedic biomaterials to enhance bone regeneration. It performs dual effects in promoting bone formation and inhibiting bone resorption. Studies have focused on the effects of strontium in regulating stem cells behaviors to initiate regenerative capacity. However, the mechanism of strontium on regulating stem cells fates and homeostasis has not been fully elucidated. In this study, the promoted effect of strontium on osteogenic differentiation of mesenchymal stem cells was confirmed both in vitro and in vivo. Interestingly, in responding to strontium treatment, stem cells performed asymmetric cell division to balance stemness maintenance and osteogenic differentiation. Asymmetric distribution of Par complex in daughter stem cells induced different cell fates by discriminately activating of osteogenic transcription factors. Strontium activated noncanonical Wnt signaling to regulate Par complex distribution. Understanding the mechanism of strontium on regulating stem cell fate could facilitate biomaterials designing to initiate endogenous bone regenerative capacity.

Cache pollution, by which weak-locality data unduly replaces strong-locality data, may notably degrade application performance in a shared-cache multicore machine. This paper presents NightWatch, a cache management subsystem that provides general, transparent and low-overhead pollution control to applications. NightWatch is based on the observation that data within the same memory chunk or chunks within the same allocation context often share similar locality property. NightWatch embodies this observation by online monitoring current cache locality to predict future behavior and restricting potential cache polluters proactively. We have integrated NightWatch into two popular allocators, tcmalloc and ptmalloc2 . Experiments with SPEC CPU2006 show that NightWatch improves application performance by up to 45 percent (18 percent on average), with an average monitoring overhead of 0.57 percent (up to 3.02 percent).

Based on Tunable Diode Laser Absorption Spectroscopy (TDLAS) technique and the feature of Vertical Cavity Surface Emitting Laser (VCSEL) wavelength scanning range up to dozens of nanometer, methane detection technique based on wavelength adaptive VCSEL without Thermoelectric Cooler (TEC), is proposed, then the laser methane sensor system design is followed. The reliability experiments study under variety of environments in laboratory, such as temperature impact test, damp heat, and cyclic and dusty impact test, were carried out. And then the test data and some analysis were given. These data verified the feasibility of the wavelength adaptive technique, and these data showed the laser methane sensor can provide long working time without any calibration, and they have the advantages of high accuracy and stability.