Biology subjects

Oh, J.

Publications and source records attributed to Oh, J..

90 records · Page 5Linked to original sources

bioRxiv2020

Deficiency in DNA mismatch repair of methylation damage is a major mutational process in cancer

DNA mismatch repair (MMR) is essential for maintaining genome integrity with its deficiency predisposing to cancer1. MMR is well known for its role in the post-replicative repair of mismatched base pairs that escape proofreading by DNA polymerases following cell division2. Yet, cancer genome sequencing has revealed that MMR deficient cancers not only have high mutation burden but also harbour multiple mutational signatures3, suggesting that MMR has pleotropic effects on DNA repair. The mechanisms underlying these mutational signatures have remained unclear despite studies using a range of in vitro4,5 and in vivo6 models of MMR deficiency. Here, using mutation data from cancer genomes, we identify a previously unknown function of MMR, showing that the loss of non-canonical replication-independent MMR activity is a major mutational process in human cancers. MMR is comprised of the MutS (MSH2/MSH6) and MutL (MLH1/PMS2) complexes7. Cancers with deficiency of MutS exhibit mutational signature contributions distinct from those deficient of MutL. This disparity is attributed to mutations arising from the unrepaired deamination of 5-methylcytosine (5mC), i.e. methylation damage, as opposed to replicative errors by DNA polymerases induced mismatches. Repair of methylation damage is strongly associated with H3K36me3 chromatin but independent of binding of MBD4, a DNA glycosylase that recognise 5mC and can repair methylation damage. As H3K36me3 recruits MutS, our results suggest that MutS is the essential factor in mediating the repair of methylation damage. Cell line models of MMR deficiency display little evidence of 5mC deamination-induced mutations as their rapid rate of proliferation limits for the opportunity for methylation damage. We thus uncover a non-canonical role of MMR in the protection against methylation damage in non-dividing cells.

genomics↗

bioRxiv2020

Thymic rejuvenation via induced thymic epithelial cells (iTECs) from FOXN1-overexpressing fibroblasts to counteract inflammaging

Age-associated systemic, chronic, sterile inflammatory condition (inflammaging) is partially attributed to increased self (auto)-reactivity, resulting from disruption of central tolerance in the aged, involuted thymus. Age-related thymic involution causally results from gradually declined expression of the transcription factor forkhead box N1 (FOXN1) in thymic epithelial cells (TECs), while exogenous FOXN1 in TECs can significantly rescue age-related thymic involution. Given the findings that induced TECs (iTECs) from FOXN1-overexpressing embryonic fibroblasts can generate an ectopic de novo thymus under the kidney capsule and intra-thymically injected natural young TECs can lead to middle-aged thymus regrowth, we sought to expand upon these two findings by applying them as a novel thymic rejuvenation strategy with two types of promoter-driven (Rosa26CreERT and FoxN1Cre) Cre-mediated iTECs. We engrafted iTECs, rather than natural young TECs, directly into the aged thymus and/or peri-thymus and found a significantly rejuvenated architecture and function in the native aged murine thymus. The engrafted iTECs drove regrowth of the aged thymus in both male and female mice, showing not only increased thymopoiesis, but also reinforcement of thymocyte negative selection, thereby, reducing senescent T cells and auto-reactive T cell-mediated inflammaging phenotypes in old mice. Therefore, this is a promising thymic rejuvenation strategy with preclinical significance, which can potentially rescue declined thymopoiesis and impaired negative selection to significantly, albeit partially, restore the defective central tolerance and reduce subclinical chronic inflammatory symptoms in the elderly. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=165 HEIGHT=200 SRC="FIGDIR/small/995357v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@d48c22org.highwire.dtl.DTLVardef@3c3ac4org.highwire.dtl.DTLVardef@3636b3org.highwire.dtl.DTLVardef@1416073_HPS_FORMAT_FIGEXP M_FIG C_FIG A novel rejuvenation strategy via the FOXN1-TEC axis using induced two types of FOXN1-overexpressing embryonic fibroblasts (termed iTECs) by intrathymic injection is able to counteract age-related thymic involution, which rescued negative selection, thereby, reducing peripheral T cell-associated inflammaging conditions.

immunology↗

bioRxiv2020

Controlling the Growth of the Skin Commensal Staphylococcus epidermidis Using D-Alanine Auxotrophy

Using live microbes as therapeutic candidates is a strategy that has gained traction across multiple therapeutic areas. In the skin, commensal microorganisms play a crucial role in maintaining skin barrier function, homeostasis, and cutaneous immunity. Alterations of the homeostatic skin microbiome are associated with a number of skin diseases. Here, we present the design of an engineered commensal organism, Staphylococcus epidermidis, for use as a live biotherapeutic product (LBP) candidate for skin diseases. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics, or genetic elements conferring antibiotic resistance, enables modulation of therapeutic exposure and improves safety. We therefore constructed an auxotrophic strain of S. epidermidis that requires exogenously supplied D-alanine. The S. epidermidis strain, NRRL B-4268 {Delta}alr1{Delta}alr2{Delta}dat (SE{Delta}{Delta}{Delta}) contains deletions of three biosynthetic genes: two alanine racemase genes, alr1 and alr2 (SE1674 and SE1079), and the D-alanine aminotransferase gene, dat (SE1423). These three deletions restricted growth in D-alanine deficient media, pooled human blood, and skin. In the presence of D-alanine, SE{Delta}{Delta}{Delta} colonized and increased expression of human {beta}-defensin 2 in cultured human skin models in vitro. SE{Delta}{Delta}{Delta}, showed a low propensity to revert to D-alanine prototrophy, and did not form biofilms on plastic in vitro. These studies support the potential safety and utility of SE{Delta}{Delta}{Delta} as a live biotherapeutic strain whose growth can be controlled by D-alanine.

microbiology↗

bioRxiv2020

An enhanced characterization of the human skin microbiome: a new biodiversity of microbial interactions

The healthy human skin microbiome is shaped by skin site physiology, individual-specific factors, and is largely stable over time despite significant environmental perturbation. Studies identifying these characteristics used shotgun metagenomic sequencing for high resolution reconstruction of the bacteria, fungi, and viruses in the community. However, these conclusions were drawn from a relatively small proportion of the total sequence reads analyzable by mapping to known reference genomes. Reference-free approaches, based on de novo assembly of reads into genome fragments, are also limited in their ability to capture low abundance species, small genomes, and to discriminate between more similar genomes. To account for the large fraction of non-human unmapped reads on the skin--referred to as microbial dark matter--we used a hybrid de novo and reference-based approach to annotate a metagenomic dataset of 698 healthy human skin samples. This approach reduced the overall proportion of uncharacterized reads from 42% to 17%. With our refined characterization, we revisited assumptions about the skin microbiome, and demonstrated higher biodiversity and lower stability, particularly in dry and moist skin sites. To investigate hypotheses underlying stability, we examined growth dynamics and interspecies interactions in these communities. Surprisingly, even though most skin sites were relatively stable, many dominant skin microbes, including Cutibacterium acnes and staphylococci, were actively growing in the skin, with poor or no relationship between growth rate and relative abundance, suggesting that host selection or interspecies competition may be important factors maintaining community homeostasis. To investigate other mechanisms facilitating adaptation to a specific skin site, we identified Staphylococcus epidermidis genes that are likely involved in stress response and provide mechanisms essential for growth in oily sites. Finally, horizontal gene transfer--another mechanism of competition by which strains may swap antagonistic or virulent coding regions--was relatively limited in healthy skin, but suggested exchange of different metabolic and environmental tolerance pathways. Altogether, our findings underscore the value of a combined reference-based and de novo approach to provide significant new insights into microbial composition, physiology, and interspecies interactions to maintain community homeostasis in the healthy human skin microbiome.

microbiology↗

bioRxiv2019

Measurements of 3-D morphology of individual red blood cells upon localized photothermal effects using gold nanorods

We investigated the in-situ photothermal response of human red blood cells (RBCs) by combining photothermal heat generation and 3D quantitative phase imaging techniques. Gold-nanorod-coated substrates were excited using near-infrared light to generate local heat to RBCs, and response was measured by imaging 3-D refractive index tomograms of cells under various near infrared (NIR) excitation conditions. On photothermal treatment, cell morphology changed from discoid to crescent shapes, cell volume and dry mass decreased, and hemoglobin concentration increased. We also investigated the irreversible deformation of RBCs when multiple intense excitation shocks are applied. These results provide a new understanding of thermodynamic aspects of cell biology and hematology. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biophysics↗

bioRxiv2019

RNA Sequencing by Direct Tagmentation of RNA/DNA Hybrids

Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status but it relies on second strand cDNA synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol, and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods. Significance StatementRNA sequencing is widely used to measure gene expression in biomedical research; therefore, improvements in the simplicity and accuracy of the technology are desirable. All existing RNA sequencing methods rely on the conversion of RNA into double-stranded DNA through reverse transcription followed by second strand synthesis. The latter step requires additional enzymes and purification, and introduces sequence-dependent bias. Here, we show that Tn5 transposase, which randomly binds and cuts double-stranded DNA, can directly fragment and prime the RNA/DNA heteroduplexes generated by reverse transcription. The primed fragments are then subject to PCR amplification. This provides a new approach for simple and accurate RNA characterization and quantification.

molecular biology↗

bioRxiv2019

A conveyor feeder for animal experiments

Several different types of open source feeders have been used in animal experiments in cognitive biology, neuroscience, psychology and related fields. These feeders use either dry pellets, which have hard surface and simple shape, or liquid food types such as sucrose solution. These food types can be rather easily manipulated due to its physical attributes. Although it is beneficial in terms of controllability, animal subjects often lose motivation to interact with operant conditioning devices offering such food items. Using natural food items such as fruits, insects, worms and pieces of meat will be helpful to keep the subjects motivation high, however, those food items are not very well suited for currently available open source feeders due to its physical attributes, including its complex shape, sticky and delicate texture. We made a feeder to deliver such natural food items to animal subjects for operant conditioning, using relatively cheap and easily obtainable parts. For a validation, we built a full operant conditioning device for wolves and dogs, containing two of these feeders, a pressure-sensitive monitor and a speaker.\n\nSpecifications table\n\nO_TBL View this table:\norg.highwire.dtl.DTLVardef@f65995org.highwire.dtl.DTLVardef@1736cacorg.highwire.dtl.DTLVardef@e3b1c6org.highwire.dtl.DTLVardef@ac3e9eorg.highwire.dtl.DTLVardef@432ab4_HPS_FORMAT_FIGEXP M_TBL C_TBL

animal behavior and cognition↗

bioRxiv2019

Convolutional neural network model to predict causal risk factors that share complex regulatory features

Major progress in disease genetics has been made through genome-wide association studies (GWASs). One of the key tasks for post-GWAS analyses is to identify causal noncoding variants with regulatory function. Here, on the basis of > 2,000 functional features, we developed a convolutional neural network framework for combinatorial, nonlinear modeling of complex patterns shared by risk variants scattered among multiple associated loci. When applied for major psychiatric disorders and autoimmune diseases, neural and immune features, respectively, exhibited high explanatory power while reflecting the pathophysiology of the relevant disease. The predicted causal variants were concentrated in active regulatory regions of relevant cell types and tended to be in physical contact with transcription factors while residing in evolutionarily conserved regions and resulting in expression changes of genes related to the given disease. We demonstrate some examples of novel candidate causal variants and associated genes. Our method is expected to contribute to the identification and functional interpretation of causal noncoding variants in post-GWAS analyses.

genetics↗

bioRxiv2019

3.1A structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids

Despite significant advances in all aspects of single particle cryo-electron microscopy (cryo-EM), specimen preparation still remains a challenge. During sample preparation, macromolecules interact with the air-water interface, which often leads to detrimental effects such as denaturation or adoption of preferred orientations, ultimately hindering structure determination. Randomly biotinylating the protein of interest and then tethering it to a cryo-EM grid coated with two-dimensional crystals of streptavidin (acting as an affinity surface) can prevent the protein from interacting with the air-water interface. Recently, this approach was successfully used to solve a high-resolution structure of a test sample, a bacterial ribosome. However, the general applicability of this method to samples where interaction with the air-water interface is problematic remains to be determined. Here we report a 3.1[A] structure of a RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion (Pol II EC(CPD)) solved using streptavidin grids. Our previous attempt to solve this structure using conventional sample preparation methods resulted in a poor quality cryo-EM map due to Pol II EC(CPD)s adopting a strong preferred orientation. Imaging the same sample on streptavidin grids led to a high-resolution structure with little anisotropy, showing that streptavidin affinity grids could be used as a general strategy to address challenges posed by interaction with the air-water interface.

molecular biology↗

bioRxiv2019

Sarcopenia is an independent predictor of hospitalization in chronic kidney disease outpatients

BackgroundPatients with chronic kidney disease (CKD) experience much more marked and earlier muscle wasting than subjects who do not have chronic illnesses. However, a few studies that have examined sarcopenia have been reported in CKD patients. We investigated the prevalence of sarcopenia in predialysis and dialysis outpatients with CKD and explored its relationship with the clinical outcomes.\n\nMeasurementsSarcopenia was defined as reduced muscle strength accompanied by decreased adjusted appendicular skeletal muscle (ASM), while those patients who exhibited only one of these characteristics were categorized as presarcopenic patients. ASM was measured by bioimpedence analysis, and muscle strength was evaluated by handgrips. ASM was adjusted by weight (ASM/wt). Patients were prospectively followed for up to 2 years.\n\nResultsOne hundred seventy-nine patients were recruited (114 male and 65 female patients who were classified into 103 predialysis patients and 76 dialysis patients, with 44.7% having diabetes). Their mean age was 60.6 {+/-} 13.5 years old. The prevalence of sarcopenia was 9.5%, while 55.9% of the patients were categorized as presarcopenic. The ASM/wt index showed significant correlations with age, handgrip strength, HOMA-IR and frailty scores. Multivariate Cox proportional hazards models demonstrated that the risk of hospitalization was significantly higher for patients with presarcopenia [hazard ratio (HR), 2.48; 95% confidence interval (CI), 1.180-5.230], and the risk of hospitalization was much higher for patients with sarcopenia than for patients in the nonsarcopenic group (HR, 9.11; 95% CI, 2.295-25.182)\n\nConclusionsSarcopenia and presarcopenia, which were defined using the ASM/wt index and handgrip strength, predicted a poorer, hospitalization-free survival in CKD patients

scientific communication and education↗

bioRxiv2018

Xrs2 and Tel1 independently contribute to MR-mediated DNA tethering and replisome stability

The yeast Mre11-Rad50-Xrs2 (MRX) complex has structural, signaling and catalytic functions in the cellular response to DNA damage. Xrs2, the eukaryotic-specific component of the complex, is required for nuclear import of Mre11 and Rad50, and to recruit the Tel1 kinase to damage sites. We show that nuclear-localized MR complex (Mre11-NLS) catalyzes homology-dependent repair without Xrs2, but MR cannot activate Tel1 and it fails to tether DSBs resulting in sensitivity to genotoxins, replisome instability and increased gross chromosome rearrangements (GCRs). Fusing the Tel1 interaction domain from Xrs2 to Mre11-NLS is sufficient to restore telomere elongation and Tel1 signaling to Xrs2-deficient cells. Furthermore, Tel1 stabilizes Mre11-DNA association, and this stabilization function becomes important for DNA damage resistance in the absence of Xrs2. Enforcing Tel1 recruitment to the nuclear MR complex fully rescues end tethering, stalled replication fork stability and suppresses GCRs, highlighting important roles for Xrs2 and Tel1 to ensure optimal MR activity.\n\nHighlightsO_LIXrs2 is required for recruitment but not for activation of Tel1 kinase\nC_LIO_LITel1 and Xrs2 function independently to optimize MR activity at DSBs and stalled replication forks\nC_LIO_LIStable association of Mre11 at DSBs is required to maintain end-to-end tethering\nC_LIO_LIMR-mediated DNA tethering promotes replisome stability and genome integrity\nC_LI

genetics↗

bioRxiv2018

F-actin dynamics transform filopodial bridges into intercellular nanotubes capable of distant cell communication

A novel actin-based bridge connecting cells has been recognized as a new pathway for the distant transport of cytoplasmic components, viruses, or pathogenic substances between cells. However, it is not yet known how such a fine structure extends over several hundred micrometres and remains robust for several hours. Using optical fluorescence imaging methods, we found that random contact promotes the formation of filopodial bridges through N-cadherin interactions between filopodia, which are slender actin-rich plasma membrane protrusions. These filopodial bridges eventually evolve into a single actin-based bridge (intercellular nanotube) that connects two cells via an intermediate state that involves a helical structure. Surprisingly, the twisting of two filopodia is likely to result from the rotational motion of actin filaments inside the filopodia by myosin V. The accumulated torsion of the filopodia triggers the release of one of the paired filopodia, whose end is attached to the other cell body by an N-cadherin cluster. The resulting retraction of the filopodium by retrograde F-actin flow leaves a single bridge. The N-cadherin/catenin cluster is likely to form a synapse between the intercellular nanotube and the cell body. This study sheds light on the formation mechanism of the filopodial bridge-based intercellular nanotubes for long-distance communication between cells.

biophysics↗

bioRxiv2018

Microbiome-TP53 Gene Interaction in Human Lung Cancer

BackgroundLung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier integrity and increases susceptibility to infections. Herein, we hypothesized that somatic mutations together with cigarette smoke generate a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from controls (n=33) and cancer cases (n=143), we conducted 16S rRNA bacterial gene sequencing, with RNA-seq data from lung cancer cases in The Cancer Genome Atlas (n=1112) serving as the validation cohort.\n\nResultsOverall, we demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue. In squamous cell carcinoma (SCC) specifically, a separate group of taxa were identified, in which Acidovorax was enriched in smokers (P =0.0013). Acidovorax temporans was identified by fluorescent in situ hybridization within tumor sections, and confirmed by two separate 16S rRNA strategies. Further, these taxa, including Acidovorax, exhibited higher abundance among the subset of SCC cases with TP53 mutations, an association not seen in adenocarcinomas (AD).\n\nConclusionsThe results of this comprehensive study show both a microbiome-gene and microbiome-exposure interactions in SCC lung cancer tissue. Specifically, tumors harboring TP53 mutations, which can damage epithelial function, have a unique bacterial consortia which is higher in relative abundance in smoking-associated SCC. Given the significant need for clinical diagnostic tools in lung cancer, this study may provide novel biomarkers for early detection.

cancer biology↗

bioRxiv2017

Population genomic analysis reveals contrasting demographic changes of two closely related dolphin species in the last glacial

Population genomic data can be used to infer historical effective population sizes (Ne), which help study the impact of past climate changes on biodiversity. Previous genome sequencing of one individual of the common bottlenose dolphin Tursiops truncatus revealed an unusual, sharp rise in Ne during the last glacial, raising questions about the reliability, generality, underlying cause, and biological implication of this finding. Here we first verify this result by additional sampling of T. truncatus. We then sequence and analyze the genomes of its close relative, the Indo-Pacific bottlenose dolphin T. aduncus. The two species exhibit contrasting demographic changes in the last glacial, likely through actual changes in population size and/or alterations in the level of gene flow among populations. Our findings demonstrate that even closely related species can have drastically different responses to climatic changes, making predicting the fate of individual species in the ongoing global warming a serious challenge.

evolutionary biology↗

bioRxiv2017

ReprDB and panDB: minimalist databases with maximal microbial representation

BackgroundProfiling of shotgun metagenomic samples is hindered by a lack of unified microbial reference genome databases that i) assemble genomic information from all open access microbial genomes, ii) have relatively small sizes, and iii) are compatible to various metagenomic read mapping tools. Moreover, computational tools to rapidly compile and update such databases to accommodate the rapid increase in new reference genomes do not exist. As a result, database-guided analyses often fail to profile a substantial fraction of metagenomic shotgun sequencing reads from complex microbiomes.\n\nResultsWe report pipelines that efficiently traverse all open access microbial genomes and assemble non-redundant genomic information. The pipelines result in two species-resolution microbial reference databases of relatively small sizes: reprDB, which assembles microbial representative or reference genomes, and panDB, for which we developed a novel iterative alignment algorithm to identify and assemble non-redundant genomic regions in multiple sequenced strains. With the databases, we managed to assign taxonomic labels and genome positions to the majority of metagenomic reads from human skin and gut microbiomes, demonstrating a significant improvement over a previous database-guided analysis on the same datasets.\n\nConclusionsreprDB and panDB leverage the rapid increases in the number of open access microbial genomes to more fully profile metagenomic samples. Additionally, the databases exclude redundant sequence information to avoid inflated storage or memory space and indexing or analyses time. Finally, the novel iterative alignment algorithm significantly increases efficiency in pan-genome identification and can be useful in comparative genomic analyses.

bioinformatics↗

bioRxiv2017

An automated statistical technique for counting distinct multiple sclerosis lesions can recover aspects of lesion history and provide relevant disease information

BackgroundLesion load is a common biomarker in multiple sclerosis, yet it has historically shown modest associations with clinical outcomes. Lesion count, which encapsulates the natural history of lesion formation and is thought to provide complementary information, is difficult to assess in patients with confluent (i.e. spatially overlapping) lesions. We introduce a statistical technique for cross-sectionally counting pathologically distinct lesions.\n\nMethodsMRI is used to assess the probability of lesion at each location. The texture of this map is quantified using a novel technique, and clusters resembling the center of a lesion are counted.\n\nResultsValidity was demonstrated by comparing the proposed count to a gold-standard count in 60 subjects observed longitudinally. The counts were highly correlated (r = .97, p < .001) and not significantly different (t59 = -0.83, p > .40). Reliability was determined using 14 scans of a clinically stable subject acquired at 7 sites, and variability of lesion count was equivalent to that of lesion load. Accounting for lesion load and age, lesion count was negatively associated (t58 = -2.73, p < .01) with the Expanded Disability Status Scale (EDSS). Average lesion size had a higher association with EDSS (r =.35, p < .01) than lesion load (r = .10, p > .40) or lesion count (r = -.12, p > .30) alone.\n\nConclusionThese findings demonstrate that it is possible to recover important aspects of the natural history of lesion formation without longitudinal data, and suggest that lesion size provides complementary information about disease.\n\nGrant SupportThe project described was supported in part by the NIH grants R01 NS085211, R21 NS093349, and R01 NS094456 from the National Institute of Neurological Disorders and Stroke (NINDS). The study was also supported by the Intramural Research Program of NINDS and the Race to Erase MS Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

neuroscience↗

bioRxiv2017

A universal, genome-wide guide finder for CRISPR/Cas9 targeting in microbial genomes

BackgroundThe CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression or activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, no pan-bacterial, genome-wide tools exist for guide discovery. We have created Guide Finder: a customizable, user-friendly program that can design guides for any annotated bacterial genome.\n\nResultsGuide Finder designs guides from NGG PAM sites for any number of genes using an annotated genome and fasta file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, a feature unique to Guide Finder. Guide Finder has been tested on a variety of diverse bacterial genomes, on average finding guides for 95% of genes. Moreover, guides designed by the program are functionally useful--focusing on CRISPRi as a potential application--as demonstrated by essential gene knockdown in two staphylococcal species.\n\nConclusionsThrough the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies for a variety of bacterial species.

microbiology↗

bioRxiv2017

Refractive index as an intrinsic imaging contrast for 3-D label-free live cell imaging

Live cell imaging provides essential information in the investigation of cell biology and related pathophysiology. Refractive index (RI) can serve as intrinsic optical imaging contrast for 3-D label-free and quantitative live cell imaging, and provide invaluable information to understand various dynamics of cells and tissues for the study of numerous fields. Recently significant advances have been made in imaging methods and analysis approaches utilizing RI, which are now being transferred to biological and medical research fields, providing novel approaches to investigate the pathophysiology of cells. To provide insight how RI can be used as an imaging contrast for imaging of biological specimens, here we provide the basic principle of RI-based imaging techniques and summarize recent progress on applications, ranging from microbiology, hematology, infectious diseases, hematology, and histopathology.

cell biology↗