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Background: Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples., Results: In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories., Conclusions: The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products. Video Abstract.

Introduction: Multiple gallbladders represent a rare congenital disorder, and coexistence with carcinoma is extremely rare, leading to a high possibility of misdiagnosis and surgical complications. In this study, a case was reported and the literature was reviewed., Case Presentation: An 80-year-old woman was diagnosed with acute cholecystitis via ultrasonography and was successfully treated with antibiotics. After the patient's biliary colic relapsed, she was referred to our hospital. Multiple imaging modalities revealed duplication of her gallbladder (H-type) and suggested coexistence with carcinoma. According to preoperative evaluations, we assumed the patient had stage IIIA disease, and cholecystectomy, cholangiography using a near-infrared ray vision system, and sectionectomy of segments 4a and 5 were performed. Contrary to the high standardized uptake values obtained by 18 F-fluoro-2-deoxy-D-glucose positron emission tomography, gallbladder carcinoma was pathologically diagnosed as stage 0 mucosal cancer. Seven days after the operation, portal thrombosis of the posterior branch was revealed, and conservative therapy was indicated; satisfactory results were achieved. The patient was discharged 65 days after surgery. No recurrence was observed for 1 year after surgery., Conclusions: An extremely rare case of malignancy in a duplicated gallbladder was reported, and the literature was reviewed. Accurate estimations are feasible for diagnoses of multiple gallbladders, where correct evaluations are vital, especially in malignant cases. Because of the possibility of malignancy, resected accessory gallbladders should be scrutinized pathologically.

To prevent lifestyle diseases, wearable bio-signal monitoring systems for daily life monitoring have attracted attention. Wearable systems have strict size and weight constraints, which impose significant limitations of the battery capacity and the signal-to-noise ratio of bio-signals. This report describes an electrocardiograph (ECG) processor for use with a wearable healthcare system. It comprises an analog front end, a 12-bit ADC, a robust Instantaneous Heart Rate (IHR) monitor, a 32-bit Cortex-M0 core, and 64 Kbyte Ferroelectric Random Access Memory (FeRAM). The IHR monitor uses a short-term autocorrelation (STAC) algorithm to improve the heart-rate detection accuracy despite its use in noisy conditions. The ECG processor chip consumes 13.7 μA for heart rate logging application.

This paper describes an electrocardiograph (ECG) monitoring SoC using a non-volatile MCU (NVMCU) and a noise-tolerant instantaneous heartbeat detector. The novelty of this work is the combination of the non-volatile MCU for normally off computing and a noise-tolerant-QRS (heartbeat) detector to achieve both low-power and noise tolerance. To minimize the stand-by current of MCU, a non-volatile flip-flop and a 6T-4C NVRAM are used. Proposed plate-line charge-share and bit-line non-precharge techniques also contribute to mitigate the active power overhead of 6T-4C NVRAM. The proposed accurate heartbeat detector uses coarse-fine autocorrelation and a template matching technique. Accurate heartbeat detection also contributes system-level power reduction because the active ratio of ADC and digital block can be reduced using heartbeat prediction. Measurement results show that the fully integrated ECG-SoC consumes 6.14 μ A including 1.28- μA non-volatile MCU and 0.7- μA heartbeat detector.

The separation of the first two lineages - trophectoderm (TE) and inner cell mass (ICM) - is a crucial event in the development of the early embryo. The ICM, which constitutes the pluripotent founder cell population, develops into the embryo proper, whereas the TE, which comprises the surrounding outer layer, supports the development of the ICM before and after implantation. Cdx2, the first transcription factor expressed specifically in the developing TE, is crucial for the differentiation of cells into the TE, as lack of zygotic Cdx2 expression leads to a failure of embryos to hatch and implant into the uterus. However, speculation exists as to whether maternal Cdx2 is required for initiation of TE lineage separation. Here, we show that effective elimination of both maternal and zygotic Cdx2 transcripts by an RNA interference approach resulted in failure of embryo hatching and implantation, but the developing blastocysts exhibited normal gross morphology, indicating that TE differentiation had been initiated. Expression of keratin 8, a marker for differentiated TE, further confirmed the identity of the TE lineage in Cdx2-deficient embryos. However, these embryos exhibited low mitochondrial activity and abnormal ultrastructure, indicating that Cdx2 plays a key role in the regulation of TE function. Furthermore, we found that embryonic compaction does not act as a 'switch' regulator to turn on Cdx2 expression. Our results clearly demonstrate that neither maternal nor zygotic Cdx2 transcripts direct the initiation of ICM/TE lineage separation.

The large micromeres of the 32-cell stage of sea urchin embryos are autonomously specified and differentiate into primary mesenchyme cells (PMCs), giving rise to the skeletogenic cells. We previously demonstrated that HpEts, an ets-related transcription factor, plays an essential role in the specification of PMCs in sea urchin embryos. In order to clarify the function of HpEts in the gene regulatory network involved in PMC specification, we analyzed the zygotic expression pattern and the cis-regulatory region of HpEts, and examined the activity of the HpEts protein as a transcription factor. Intron-based PCR reveals that zygotic expression of HpEts starts at the cleavage stage, and that the rate of transcription reaches maximum at the unhatched blastula stage. A series of progressive deletions of the fragments from -4.2 kbp to +1206 bp of the HpEts, which directs PMC-specific expression, caused a gradual decrease in the specificity, implying that coordination of several cis-regulatory elements regulates the expression in PMCs. A minimum cis-element required for the temporal expression is located within a 10 bp from -243 bp to -234 bp. The HpEts protein remains in the cytoplasm of entire embryonic cells in the cleavage stage. At the unhatched blastula stage, the HpEts protein translocates into the nucleus in presumptive PMCs. Transactivation assays demonstrate that the HpEts protein activates a promoter of Spicule Matrix Protein 50 (SM50), which is a target of HpEts, which binds to the regulatory region of SM50.

Embryonic germ-line cells are unipotent cells that give rise to either sperm or oocytes. However, pluripotent stem cells can be derived from primordial germ cells (PGCs) or spermatogonia, suggesting that germ-line cells retain a capacity for pluripotency. Here, we made genome-wide comparisons of the gene expression profiles of freshly isolated PGCs, in vitro-formed PGCs (iPGCs), and other stem cell lines, including embryonic stem cells (ESCs), embryonic germ cells (EGCs) and germ-line stem (GS) cells. Comparing PGC with ESC, 382 genes/transcripts were significantly up-regulated in ESC, while 188 were elevated in PGC. This suggests that PGCs possess transcription program distinct from that of ESC, although both share expression of many pluripotency-associated genes. Our micro-array analysis showed that the analyzed samples could be classified into two groups: one consisting of all the ESCs and most of EGCs, and the other containing PGC samples, iPGC, one type of female EGC and GS cells. We then identified "signature" genes for the two groups, and used them to characterize GS cells, EGC, and iPGCs, and revealed developmental status of each cell type. The relationships between PGCs and stem cells derived from embryos or germ cells are discussed in light of these findings.

Quantification of DNA methyltransferases Dnmt3a and Dnmt3a2, and Dnmt3L in isolated male gonocytes in day 16.5 embryos confirmed that not Dnmt3a but Dnmt3a2 and Dnmt3L were the major Dnmt3s. The expression level of Dnmt3L constituted 5- to 10-fold molar excess compared to that of Dnmt3a2. The stimulation property of the DNA methylation activity of Dnmt3a2 with Dnmt3L towards substrate DNA in naked or nucleosomes was similar to that of Dnmt3a. However, the DNA methylation activity of not Dnmt3a but Dnmt3a2 was severely inhibited at the physiological salt concentration. Interestingly, the activity of Dnmt3a2 was significantly detected in the presence of Dnmt3L even at the physiological salt concentration. This indicates that Dnmt3a2 functions only in the presence of Dnmt3L in male gonocytes, and may explain why Dnmt3L is required specifically in mouse gonocytes for DNA methylation.

Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation.

Signals from micromere descendants play a crucial role in sea urchin development. In this study, we demonstrate that these micromere descendants express HpTb, a T-brain homolog of Hemicentrotus pulcherrimus. HpTb is expressed transiently from the hatched blastula stage through the mesenchyme blastula stage to the gastrula stage. By a combination of embryo microsurgery and antisense morpholino experiments, we show that HpTb is involved in the production of archenteron induction signals. However, HpTb is not involved in the production of signals responsible for the specification of secondary mesenchyme cells, the initial specification of primary mesenchyme cells, or the specification of endoderm. HpTb expression is controlled by nuclear localization of beta-catenin, suggesting that HpTb is in a downstream component of the Wnt signaling cascade. We also propose the possibility that HpTb is involved in the cascade responsible for the production of signals required for the spicule formation as well as signals from the vegetal hemisphere required for the differentiation of aboral ectoderm.

The preparation, ESR spectra, isolation, and X-ray crystallographic structure of N-(arylthio)-2-tert-butyl-4,6-diarylphenylaminyls (1) and N-(arylthio)-4-tert-butyl-2,6-diarylphenylaminyls (2) are described. The aminyls are generated by PbO(2) oxidation of N-(arylthio)-2-tert-butyl-4,6-diarylanilines and N-(arylthio)-4-tert-butyl-2,6-diarylanilines. The kinetic ESR study shows that the aminyls are quite persistent, even in the presence of oxygen, and exist in the individual radical forms. Among the seventeen aminyls prepared, N-[(4-nitrophenyl)thio]-2-tert-butyl-4,6-diphenylphenylaminyl (1b), N-[(4-nitrophenyl)thio]-2-tert-butyl-4,6-bis(4-chlorophenyl)phenylaminyl (1f), N-[(4-nitrophenyl)thio]-4-tert-butyl-2,6-diphenylphenylaminyl (2b), N-[(4-nitrophenyl)thio]-4-tert-butyl-2,6-bis(4-chlorophenyl)phenylaminyl (2h), and N-[(3,5-dichlorophenyl)thio]-4-tert-butyl-2,6-bis(4-chlorophenyl)phenylaminyl (2j) are isolated as radical crystals. The crystallographic structures of 1b and 2b are determined by the X-ray crystallographic analyses. Aminyls 1 and 2 give similar ESR spectra consisting of 1:1:1 triplets with the a(N) values of 0.921-0.948 mT. Deuteration of the phenyl groups on the anilino benzene ring gives rise to a further splitting of the nitrogen 1:1:1 triplet by the anilino meta (0.126-0.138) and phenylthiyl ortho and para protons (0.077-0.096 mT). Upon recording at high gain, one of the partly deuterated aminyls gives satellite lines due to (33)S isotopes at natural abundance from which a(33)(S) is determined to be 0.51 mT. The ESR parameters for 1 and 2 are compared with those for structurally close N-(arylthio)-2,4,6-triarylphenylaminyl and N-(arylthio)-2,4,6-tri-tert-butylphenylaminyl.