Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy.

Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range of applications due to poor performance under two-photon microscopy, a method of choice for deep-tissue recording. To improve indicators, we developed a multiparameter high-throughput platform to optimize voltage indicators for two-photon microscopy. Using this system, we identified JEDI-2P, an indicator that is faster, brighter, and more sensitive and photostable than its predecessors. We demonstrate that JEDI-2P can report light-evoked responses in axonal termini of Drosophila interneurons and the dendrites and somata of amacrine cells of isolated mouse retina. JEDI-2P can also optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min using both resonant-scanning and ULoVE random-access microscopy. Finally, ULoVE recording of JEDI-2P can robustly detect spikes at depths exceeding 400 µm and report voltage correlations in pairs of neurons.

How Placenta Promotes the Successful Reproduction in High-Altitude Populations: A Transcriptome Comparison between Adaptation and Acclimatization.

As the best adapted high altitude population, Tibetans feature a relatively high offspring survival rate. Genome-wide studies have identified hundreds of candidate SNPs related to high altitude adaptation of Tibetans, although most of them have unknown functional relevance. To explore the mechanisms behind successful reproduction at high altitudes, we compared the placental transcriptomes of Tibetans, sea level Hans (SLHan), and Han immigrants (ImHan). Among the three populations, placentas from ImHan showed a hyperactive gene expression pattern. Their increased activation demonstrates a hypoxic stress response similar to sea level individuals experiencing hypoxic conditions. Unlike ImHan, Tibetan placentas were characterized by the significant up-regulation of placenta-specific genes, and the activation of autophagy and the tricarboxylic acid (TCA) cycle. Certain conserved hypoxia response functions, including the antioxidant system and angiogenesis, were activated in both ImHan and Tibetans, but mediated by different genes. The coherence of specific transcriptome features linked to possible genetic contribution was observed in Tibetans. Furthermore, we identified a novel Tibetan-specific EPAS1 isoform with a partial deletion at exon six, which may be involved in the adaption to hypoxia through the EPAS1-centred gene network in the placenta. Overall, our results show that the placenta grants successful pregnancies in Tibetans by strengthening the natural functions of the placenta itself. On the other hand, the placenta of ImHan was in an inhabiting time-dependent acclimatization process representing a common hypoxic stress response pattern.

Ultra-precise detection of mutations by droplet-based amplification of circularized DNA.

BACKGROUND: NGS (next generation sequencing) has been widely used in studies of biological processes, ranging from microbial evolution to cancer genomics. However, the error rate of NGS (0.1 % ~ 1 %) is still remaining a great challenge for comprehensively investigating the low frequency variations, and the current solution methods have suffered severe amplification bias or low efficiency. RESULTS: We creatively developed Droplet-CirSeq for relatively efficient, low-bias and ultra-sensitive identification of variations by combining millions of picoliter uniform-sized droplets with Cir-seq. Droplet-CirSeq is entitled with an incredibly low error rate of 3 ~ 5 X 10(-6). To systematically evaluate the performances of amplification uniformity and capability of mutation identification for Droplet-CirSeq, we took the mixtures of two E. coli strains as specific instances to simulate the circumstances of mutations with different frequencies. Compared with Cir-seq, the coefficient of variance of read depth for Droplet-CirSeq was 10 times less (p = 2.6 X 10(-3)), and the identified allele frequency presented more concentrated to the authentic frequency of mixtures (p = 4.8 X 10(-3)), illustrating a significant improvement of amplification bias and accuracy in allele frequency determination. Additionally, Droplet-CirSeq detected 2.5 times genuine SNPs (p < 0.001), achieved a 2.8 times lower false positive rate (p < 0.05) and a 1.5 times lower false negative rate (p < 0.001), in the case of a 3 pg DNA input. Intriguingly, the false positive sites predominantly represented in two types of base substitutions (G- > A, C- > T). Our findings indicated that 30 pg DNA input accommodated in 5 ~ 10 million droplets resulted in maximal detection of authentic mutations compared to 3 pg (p = 1.2 X 10(-8)) and 300 pg input (p = 2.2 X 10(-3)). CONCLUSIONS: We developed a method namely Droplet-CirSeq to significantly improve the amplification bias, which presents obvious superiority over the currently prevalent methods in exploitation of ultra-low frequency mutations. Droplet-CirSeq would be promisingly used in the identification of low frequency mutations initiated from extremely low input DNA, such as DNA of uncultured microorganisms, captured DNA of target region, circulation DNA of plasma et al, and its creative conception of rolling circle amplification in droplets would also be used in other low input DNA amplification fields.

Development of a two-stage feeding strategy based on the kind and level of feeding nutrients for improving fed-batch production of L-threonine by Escherichia coli.

Fed-batch fermentation is the predominant method for industrial production of amino acids. In this study, we comprehensively investigated the effects of four kinds of feeding nutrients and developed an accurate optimization strategy for fed-batch production of L-threonine. The production of L-threonine was severely inhibited when cell growth ceased in the bath culture. Similarly, L-threonine production was also associated with cell growth in the carbon-, phosphate-, and sulfate-limited fed-batch cultures, but the accumulation of L-threonine was markedly increased because of the extended production time in the growth stage. Interestingly, auxotrophic amino acid (L-isoleucine)-limited feeding promoted L-threonine production over the non-growth phase. Metabolite analysis indicates that substantial production of acetate and glutamate and the resulting accumulation of ammonium may lead to the inhibition of L-threonine production. During the growth phase, the levels of L-isoleucine were accurately optimized by balancing cell growth and production with Pontryagin's maximum principle, basing on the relationship between the specific growth rate µ and specific production rate ρ. Furthermore, the depletion of L-isoleucine and phosphate at the end of the growth phase favored the synthesis of L-threonine in the subsequent non-growth phase. Combining the two-stage feeding profiles, the final L-threonine concentration and conversion rate were increased by 5.9- and 2.1-fold, respectively, compared to batch processes without feeding control. The identification of efficient feeding nutrient and the development of accurate feeding strategies provide potential guidelines for microbial production of amino acids.

Development and application of an arabinose-inducible expression system by facilitating inducer uptake in Corynebacterium glutamicum.

Corynebacterium glutamicum is currently used for the industrial production of a variety of biological materials. Many available inducible expression systems in this species use lac-derived promoters from Escherichia coli that exhibit much lower levels of inducible expression and leaky basal expression. We developed an arabinose-inducible expression system that contains the L-arabinose regulator AraC, the P(BAD) promoter from the araBAD operon, and the L-arabinose transporter AraE, all of which are derived from E. coli. The level of inducible P(BAD)-based expression could be modulated over a wide concentration range from 0.001 to 0.4% L-arabinose. This system tightly controlled the expression of the uracil phosphoribosyltransferase without leaky expression. When the gene encoding green fluorescent protein (GFP) was under the control of P(BAD) promoter, flow cytometry analysis showed that GFP was expressed in a highly homogeneous profile throughout the cell population. In contrast to the case in E. coli, P(BAD) induction was not significantly affected in the presence of different carbon sources in C. glutamicum, which makes it useful in fermentation applications. We used this system to regulate the expression of the odhI gene from C. glutamicum, which encodes an inhibitor of α-oxoglutarate dehydrogenase, resulting in high levels of glutamate production (up to 13.7 mM) under biotin nonlimiting conditions. This system provides an efficient tool available for molecular biology and metabolic engineering of C. glutamicum.

Common Postzygotic Mutational Signatures in Healthy Adult Tissues Related to Embryonic Hypoxia.

Postzygotic mutations are acquired in normal tissues throughout an individual's lifetime and hold clues for identifying mutagenic factors. Here, we investigated postzygotic mutation spectra of healthy individuals using optimized ultra-deep exome sequencing of the time-series samples from the same volunteer as well as the samples from different individuals. In blood, sperm, and muscle cells, we resolved three common types of mutational signatures. Signatures A and B represent clock-like mutational processes, and the polymorphisms of epigenetic regulation genes influence the proportion of signature B in mutation profiles. Notably, signature C, characterized by C>T transitions at GpCpN sites, tends to be a feature of diverse normal tissues. Mutations of this type are likely to occur early during embryonic development, supported by their relatively high allelic frequencies, presence in multiple tissues, and decrease in occurrence with age. Almost none of the public datasets for tumors feature this signature, except for 19.6% of samples of clear cell renal cell carcinoma with increased activation of the hypoxia-inducible factor 1 (HIF-1) signaling pathway. Moreover, the accumulation of signature C in the mutation profile was accelerated in a human embryonic stem cell line with drug-induced activation of HIF-1α. Thus, embryonic hypoxia may explain this novel signature across multiple normal tissues. Our study suggests that hypoxic condition in an early stage of embryonic development is a crucial factor inducing C>T transitions at GpCpN sites; and individuals' genetic background may also influence their postzygotic mutation profiles.

A synthetic circuit for buffering gene dosage variation between individual mammalian cells.

Precise control of gene expression is critical for biological research and biotechnology. However, transient plasmid transfections in mammalian cells produce a wide distribution of copy numbers per cell, and consequently, high expression heterogeneity. Here, we report plasmid-based synthetic circuits - Equalizers - that buffer copy-number variation at the single-cell level. Equalizers couple a transcriptional negative feedback loop with post-transcriptional incoherent feedforward control. Computational modeling suggests that the combination of these two topologies enables Equalizers to operate over a wide range of plasmid copy numbers. We demonstrate experimentally that Equalizers outperform other gene dosage compensation topologies and produce as low cell-to-cell variation as chromosomally integrated genes. We also show that episome-encoded Equalizers enable the rapid generation of extrachromosomal cell lines with stable and uniform expression. Overall, Equalizers are simple and versatile devices for homogeneous gene expression and can facilitate the engineering of synthetic circuits that function reliably in every cell.

Versatile phenotype-activated cell sorting.

Unraveling the genetic and epigenetic determinants of phenotypes is critical for understanding and re-engineering biology and would benefit from improved methods to separate cells based on phenotypes. Here, we report SPOTlight, a versatile high-throughput technique to isolate individual yeast or human cells with unique spatiotemporal profiles from heterogeneous populations. SPOTlight relies on imaging visual phenotypes by microscopy, precise optical tagging of single target cells, and retrieval of tagged cells by fluorescence-activated cell sorting. To illustrate SPOTlight's ability to screen cells based on temporal properties, we chose to develop a photostable yellow fluorescent protein for extended imaging experiments. We screened 3 million cells expressing mutagenesis libraries and identified a bright new variant, mGold, that is the most photostable yellow fluorescent protein reported to date. We anticipate that the versatility of SPOTlight will facilitate its deployment to decipher the rules of life, understand diseases, and engineer new molecules and cells.

Noninvasive chimeric DNA profiling identifies tumor-originated HBV integrants contributing to viral antigen expression in liver cancer.

BACKGROUND: Host genome integration of HBV sequence is considered to be significant in HBV antigen expression and the development of hepatocellular carcinoma (HCC). METHOD: We developed a probe-based capture strategy to enrich integrated HBV DNA for deep-sequencing analysis of integration sites in paired patient samples derived from tumor, liver tissue adjacent to tumor, saliva and plasma, as a platform for exploring the correlation, significance and utility of detecting integrations in these sample types. RESULTS: Most significantly, alpha fetoprotein levels significantly correlated to the amounts of integrations detected in tumor. Viral-host chimeric DNA fragments were successfully detected at high sequencing coverage in plasma rather than saliva samples from HCC patients, and each fragment of this type was only seen once in plasma from chronic hepatitis B patients. Almost all plasma chimeric fragments were derived from integrations in tumor rather than in adjacent liver tissues. Over 50% of them may produce viral-host chimeric transcripts according to deep RNA sequencing in paired tissue samples. Particularly, in patients with low HBV DNA level (< 250 UI/ml), the seemingly normal HBsAg titers may be explained by larger amounts of integrations detected. Meanwhile, we developed a strategy to predict integrants by pairing breakpoints for each integration event. Among four resolved viral patterns, the majority of Pattern I events (81.2%) retained the complete opening reading frame for HBV surface proteins. CONCLUSION: We achieve the efficient enrichment of plasma cell-free chimeric DNA from integration site, and demonstrate that chimeric DNA profiling in plasma is a promising noninvasive approach to monitor HBV integration in liver cancer development and to determine the ability of integrated sequences to express viral proteins that can be targeted, e.g. by immunotherapies.

Reprogramming One-Carbon Metabolic Pathways To Decouple l-Serine Catabolism from Cell Growth in Corynebacterium glutamicum.

l-Serine, the principal one-carbon source for DNA biosynthesis, is difficult for microorganisms to accumulate due to the coupling of l-serine catabolism and microbial growth. Here, we reprogrammed the one-carbon unit metabolic pathways in Corynebacterium glutamicum to decouple l-serine catabolism from cell growth. In silico model-based simulation showed a negative influence on glyA-encoding serine hydroxymethyltransferase flux with l-serine productivity. Attenuation of glyA transcription resulted in increased l-serine accumulation, and a decrease in purine pools, poor growth and longer cell shapes. The gcvTHP-encoded glycine cleavage (Gcv) system from Escherichia coli was introduced into C. glutamicum, allowing glycine-derived 13CH2 to be assimilated into intracellular purine synthesis, which resulted in an increased amount of one-carbon units. Gcv introduction not only restored cell viability and morphology but also increased l-serine accumulation. Moreover, comparative proteomic analysis indicated that abundance changes of the enzymes involved in one-carbon unit cycles might be responsible for maintaining one-carbon unit homeostasis. Reprogramming of the one-carbon metabolic pathways allowed cells to reach a comparable growth rate to accumulate 13.21 g/L l-serine by fed-batch fermentation in minimal medium. This novel strategy provides new insights into the regulation of cellular properties and essential metabolite accumulation by introducing an extrinsic pathway.

Genetic variants of TREML2 are associated with HLA-B27-positive ankylosing spondylitis.

Although ankylosing spondylitis (AS) is a common, highly heritable arthropathy, the precise genetic mechanism underlying the disease remains elusive. Here, we investigate the disease-causing mutations in a large AS family with distinguished complexity, consisting of 23 patients covering four generations and exhibiting a mixed HLA-B27 (+) and (-) status. Linkage analysis with 32 members using three methods and whole-exome sequencing analysis with three HLA-B27 (+) patients, one HLA-B27 (-) patient, and one healthy individual did not identify a mutation common to all of the patients, strongly suggesting the existence of genetic heterogeneity in this large pedigree. However, if only B27-positive patients were analyzed, the linkage analysis located a 22-Mb region harboring the HLA gene cluster in chromosome 6 (LOD = 4.2), and the subsequent exome analysis identified two non-synonymous mutations in the TREML2 and IP6K3 genes. These genes were resequenced among 370 sporadic AS patients and 487 healthy individuals. A significantly higher mutation frequency of TREML2 was observed in AS patients (1.51% versus 0.21%). The results obtained for the AS pedigree and sporadic patients suggest that mutation of TREML2 is a major factor leading to AS for HLA-B27 (+) members in this large family and that TREML2 is also a susceptibility gene promoting the development of ankylosing spondylitis in HLA-B27 (+) individuals.

Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq.

Detection of de novo, low-frequency mutations is essential for characterizing cancer genomes and heterogeneous cell populations. However, the screening capacity of current ultrasensitive NGS methods is inadequate owing to either low-efficiency read utilization or severe amplification bias. Here, we present o2n-seq, an ultrasensitive and high-efficiency NGS library preparation method for discovering de novo, low-frequency mutations. O2n-seq reduces the error rate of NGS to 10-5-10-8. The efficiency of its data usage is about 10-30 times higher than that of barcode-based strategies. For detecting mutations with allele frequency (AF) 1% in 4.6 Mb-sized genome, the sensitivity and specificity of o2n-seq reach to 99% and 98.64%, respectively. For mutations with AF around 0.07% in phix174, o2n-seq detects all the mutations with 100% specificity. Moreover, we successfully apply o2n-seq to screen de novo, low-frequency mutations in human tumours. O2n-seq will aid to characterize the landscape of somatic mutations in research and clinical settings.

Conjunctival Microbiome Changes Associated With Soft Contact Lens and Orthokeratology Lens Wearing.

Purpose: Usage of different types of contact lenses is associated with increased risk of sight-threatening complications. Changes in the ocular microbiome caused by contact lens wear are suggested to affect infection development in those individuals. To address this question, this study compares conjunctival microbial communities in contact lens wearers with those in noncontact lens wearers. Methods: Paired-end sequencing of the V3 region of the 16S rRNA gene was used to characterize the bacterial communities on the conjunctival surfaces of contact lens wearers and nonwearers. Results: No differences in microbial diversity were detected between contact lens wearers and nonwearers. Nevertheless, some slight microbe variability was evident between these two different groups. Bacillus, Tatumella and Lactobacillus abundance was less in orthokeratology lens (OKL) wearers than in nonwearers. In soft contact lenses (SCL) wearers, Delftia abundance decreased whereas Elizabethkingia levels increased. The difference in the SCL and nonwearer group was smaller than that in the OKL group. Variations in the conjunctival taxonomic composition between SCL wearers were larger than those in other groups. Sex differences in the conjunctival microbiota makeup were only evident among nonwearers. Conclusions: Even though there were slight percentage changes between contact lens wearers and nonwearers in some microbes, there were no differences in their diversity. On the other hand, contact lens usage might cause relative abundance of some taxa to change. Our results will help assess whether or not conjunctival microbiome changes caused by contact lens wear affect infection risk.

An Old Story Retold: Loss of G1 Control Defines A Distinct Genomic Subtype of Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) has a high mortality rate. To determine the molecular basis of ESCC development, this study sought to identify characteristic genome-wide alterations in ESCC, including exonic mutations and structural alterations. The clinical implications of these genetic alterations were also analyzed. Exome sequencing and verification were performed for nine pairs of ESCC and the matched blood samples, followed by validation with additional samples using Sanger sequencing. Whole-genome SNP arrays were employed to detect copy number alteration (CNA) and loss of heterozygosity (LOH) in 55 cases, including the nine ESCC samples subjected to exome sequencing. A total of 108 non-synonymous somatic mutations (NSSMs) in 102 genes were verified in nine patients. The chromatin modification process was found to be enriched in our gene ontology (GO) analysis. Tumor genomes with TP53 mutations were significantly more unstable than those without TP53 mutations. In terms of the landscape of genomic alterations, deletion of 9p21.3 covering CDKN2A/2B (30.9%), amplification of 11q13.3 covering CCND1 (30.9%), and TP53 point mutation (50.9%) occurred in two-thirds of the cases. These results suggest that the deregulation of the G1 phase during the cell cycle is a key event in ESCC. Furthermore, six minimal common regions were found to be significantly altered in ESCC samples and three of them, 9p21.3, 7p11.2, and 3p12.1, were associated with lymph node metastasis. With the high correlation of TP53 mutation and genomic instability in ESCC, the amplification of CCND1, the deletion of CDKN2A/2B, and the somatic mutation of TP53 appear to play pivotal roles via G1 deregulation and therefore helps to classify this cancer into different genomic subtypes. These findings provide clinical significance that could be useful in future molecular diagnoses and therapeutic targeting.

Genetic and biochemical characterization of Corynebacterium glutamicum ATP phosphoribosyltransferase and its three mutants resistant to feedback inhibition by histidine.

ATP phosphoribosyltransferase (ATP-PRT) catalyzes the condensation of ATP and PRPP at the first step of histidine biosynthesis and is regulated by a feedback inhibition from product histidine. Here, we report the genetic and biochemical characterization of such an enzyme, HisG(Cg), from Corynebacterium glutamicum, including site-directed mutagenesis of the histidine-binding site for the first time. Gene disruption and complementation experiments showed that HisG(Cg) is essential for histidine biosynthesis. HisG(Cg) activity was noncompetitively inhibited by histidine and the α-amino group of histidine were found to play an important role for its binding to HisG(Cg). Homology-based modeling predicted that four residues (N215, L231, T235 and A270) in the C-terminal domain of HisG(Cg) may affect the histidine inhibition. Mutating these residues in HisG(Cg) did not cause significant change in the specific activities of the enzyme but resulted in the generation of mutant ones resistant to histidine inhibition. Our data identified that the mutant N215K/L231F/T235A resists to histidine inhibition the most with 37-fold increase in K(i) value. As expected, overexpressing a hisG(Cg) gene containing N215K/L231F/T235A mutations in vivo promoted histidine accumulation to a final concentration of 0.15 ± 0.01 mM. Our results demonstrated that the polarity change of electrostatic potential of mutant protein surface prevents histidine from binding to the C-terminal domain of HisG(Cg), resulting in the release of allosteric inhibition. Considering that these residues were highly conserved in ATP-PRTs from different genera of Gram-positive bacteria the mechanism by histidine inhibition as exhibited in Corynebacterium glutamicum probably represents a ubiquitously inhibitory mechanism of ATP-PRTs by histidine.

Metabolic engineering and flux analysis of Corynebacterium glutamicum for L-serine production.

L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and attenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase (PGDH), in combination with the co-expression of 3-phosphoglycerate kinase (PGK) and feedback-resistant PGDH (PGDH(r)). The resulting strain, SER-8, exhibited a lower specific growth rate and significant differences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached (14.22 ± 1.41) µmol g(CDM) (-1), which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway (PPP) towards the glycolysis pathway (EMP), and the expression of PGDH(r) improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR resulted in down-regulation of serine hydroxymethyltransferase (SHMT) expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C(1) units generation by introducing the glycine cleavage system (GCV) to degrade the excessive glycine is a promising target for L-serine production in C. glutamicum.