Archival single-cell genomics reveals persistent subclones during DCIS progression.

Ductal carcinoma in situ (DCIS) is a common precursor of invasive breast cancer. Our understanding of its genomic progression to recurrent disease remains poor, partly due to challenges associated with the genomic profiling of formalin-fixed paraffin-embedded (FFPE) materials. Here, we developed Arc-well, a high-throughput single-cell DNA-sequencing method that is compatible with FFPE materials. We validated our method by profiling 40,330 single cells from cell lines, a frozen tissue, and 27 FFPE samples from breast, lung, and prostate tumors stored for 3-31 years. Analysis of 10 patients with matched DCIS and cancers that recurred 2-16 years later show that many primary DCIS had already undergone whole-genome doubling and clonal diversification and that they shared genomic lineages with persistent subclones in the recurrences. Evolutionary analysis suggests that most DCIS cases in our cohort underwent an evolutionary bottleneck, and further identified chromosome aberrations in the persistent subclones that were associated with recurrence.

A spontaneous complex structural variant in rcan-1 increases exploratory behavior and laboratory fitness of Caenorhabditis elegans.

Over long evolutionary timescales, major changes to the copy number, function, and genomic organization of genes occur, however, our understanding of the individual mutational events responsible for these changes is lacking. In this report, we study the genetic basis of adaptation of two strains of C. elegans to laboratory food sources using competition experiments on a panel of 89 recombinant inbred lines (RIL). Unexpectedly, we identified a single RIL with higher relative fitness than either of the parental strains. This strain also displayed a novel behavioral phenotype, resulting in higher propensity to explore bacterial lawns. Using bulk-segregant analysis and short-read resequencing of this RIL, we mapped the change in exploration behavior to a spontaneous, complex rearrangement of the rcan-1 gene that occurred during construction of the RIL panel. We resolved this rearrangement into five unique tandem inversion/duplications using Oxford Nanopore long-read sequencing. rcan-1 encodes an ortholog to human RCAN1/DSCR1 calcipressin gene, which has been implicated as a causal gene for Down syndrome. The genomic rearrangement in rcan-1 creates two complete and two truncated versions of the rcan-1 coding region, with a variety of modified 5' and 3' non-coding regions. While most copy-number variations (CNVs) are thought to act by increasing expression of duplicated genes, these changes to rcan-1 ultimately result in the reduction of its whole-body expression due to changes in the upstream regions. By backcrossing this rearrangement into a common genetic background to create a near isogenic line (NIL), we demonstrate that both the competitive advantage and exploration behavioral changes are linked to this complex genetic variant. This NIL strain does not phenocopy a strain containing an rcan-1 loss-of-function allele, which suggests that the residual expression of rcan-1 is necessary for its fitness effects. Our results demonstrate how colonization of new environments, such as those encountered in the laboratory, can create evolutionary pressure to modify gene function. This evolutionary mismatch can be resolved by an unexpectedly complex genetic change that simultaneously duplicates and diversifies a gene into two uniquely regulated genes. Our work shows how complex rearrangements can act to modify gene expression in ways besides increased gene dosage.

Extreme allelic heterogeneity at a Caenorhabditis elegans beta-tubulin locus explains natural resistance to benzimidazoles.

Benzimidazoles (BZ) are essential components of the limited chemotherapeutic arsenal available to control the global burden of parasitic nematodes. The emerging threat of BZ resistance among multiple nematode species necessitates the development of novel strategies to identify genetic and molecular mechanisms underlying this resistance. All detection of parasitic helminth resistance to BZ is focused on the genotyping of three variant sites in the orthologs of the ß-tubulin gene found to confer resistance in the free-living nematode Caenorhabditis elegans. Because of the limitations of laboratory and field experiments in parasitic nematodes, it is difficult to look beyond these three sites to identify additional mechanisms that might contribute to BZ resistance in the field. Here, we took an unbiased genome-wide mapping approach in the free-living nematode species C. elegans to identify the genetic underpinnings of natural resistance to the commonly used BZ, albendazole (ABZ). We found a wide range of natural variation in ABZ resistance in natural C. elegans populations. In agreement with known mechanisms of BZ resistance in parasites, we found that a majority of the variation in ABZ resistance among wild C. elegans strains is caused by variation in the ß-tubulin gene ben-1. This result shows empirically that resistance to ABZ naturally exists and segregates within the C. elegans population, suggesting that selection in natural niches could enrich for resistant alleles. We identified 25 distinct ben-1 alleles that are segregating at low frequencies within the C. elegans population, including many novel molecular variants. Population genetic analyses indicate that ben-1 variation arose multiple times during the evolutionary history of C. elegans and provide evidence that these alleles likely occurred recently because of local selective pressures. Additionally, we find purifying selection at all five ß-tubulin genes, despite predicted loss-of-function variants in ben-1, indicating that BZ resistance in natural niches is a stronger selective pressure than loss of one ß-tubulin gene. Furthermore, we used genome-editing to show that the most common parasitic nematode ß-tubulin allele that confers BZ resistance, F200Y, confers resistance in C. elegans. Importantly, we identified a novel genomic region that is correlated with ABZ resistance in the C. elegans population but independent of ben-1 and the other ß-tubulin loci, suggesting that there are multiple mechanisms underlying BZ resistance. Taken together, our results establish a population-level resource of nematode natural diversity as an important model for the study of mechanisms that give rise to BZ resistance.

Dicyanoisophorone-Based Near-Infrared-Emission Fluorescent Probe for Detecting NAD(P)H in Living Cells and in Vivo.

NADH and NADPH are ubiquitous coenzymes in all living cells that play vital roles in numerous redox reactions in cellular energy metabolism. To accurately detect the distribution and dynamic changes of NAD(P)H under physiological conditions is essential for understanding their biological functions and pathological roles. In this work, we developed a near-infrared (NIR)-emission fluorescent small-molecule probe (DCI-MQ) composed of a dicyanoisophorone chromophore conjugated to a quinolinium moiety for in vivo NAD(P)H detection. DCI-MQ has the advantages of high water solubility, a rapid response, extraordinary selectivity, great sensitivity (a detection limit of 12 nM), low cytotoxicity, and NIR emission (660 nm) in response to NAD(P)H. Moreover, the probe DCI-MQ was successfully applied for the detection and imaging of endogenous NAD(P)H in both living cells and tumor-bearing mice, which provides an effective tool for the study of NAD(P)H-related physiological and pathological processes.

Selection on a Subunit of the NURF Chromatin Remodeler Modifies Life History Traits in a Domesticated Strain of Caenorhabditis elegans.

Evolutionary life history theory seeks to explain how reproductive and survival traits are shaped by selection through allocations of an individual's resources to competing life functions. Although life-history traits evolve rapidly, little is known about the genetic and cellular mechanisms that control and couple these tradeoffs. Here, we find that two laboratory-adapted strains of C. elegans descended from a single common ancestor that lived in the 1950s have differences in a number of life-history traits, including reproductive timing, lifespan, dauer formation, growth rate, and offspring number. We identified a quantitative trait locus (QTL) of large effect that controls 24%-75% of the total trait variance in reproductive timing at various timepoints. Using CRISPR/Cas9-induced genome editing, we show this QTL is due in part to a 60 bp deletion in the 3' end of the nurf-1 gene, which is orthologous to the human gene encoding the BPTF component of the NURF chromatin remodeling complex. Besides reproduction, nurf-1 also regulates growth rate, lifespan, and dauer formation. The fitness consequences of this deletion are environment specific-it increases fitness in the growth conditions where it was fixed but decreases fitness in alternative laboratory growth conditions. We propose that chromatin remodeling, acting through nurf-1, is a pleiotropic regulator of life history trade-offs underlying the evolution of multiple traits across different species.

Highly Selective Fluorescent Probe for Imaging H2Se in Living Cells and in Vivo Based on the Disulfide Bond.

Hydrogen selenide (H2Se) is an important metabolite of dietary Se compounds and has been implicated in various pathological and physiological processes. The development of highly sensitive and selective methods for the sensing of H2Se is therefore very important. Herein, we developed a fluorescent probe (hemicyanine (Hcy)-H2Se) for detecting H2Se based on a new H2Se-specific receptor unit, 1,2-dithiane-4,5-diol. Hcy-H2Se showed high selectivity toward H2Se over thiols (RSH), hydrogen sulfide (H2S), and selenocysteine (Sec) and was further exploited for the fluorescence imaging of H2Se both in living cells and in vivo. Furthermore, with the aid of Hcy-H2Se, we demonstrated that H2Se can be generated and gradually accumulated in HepG2 cells under hypoxic conditions and in the solid tumor after treatment with Na2SeO3.

A prototypic lysine methyltransferase 4 from archaea with degenerate sequence specificity methylates chromatin proteins Sul7d and Cren7 in different patterns.

BACKGROUND: The origin of eukaryotic histone modification enzymes still remains obscure. RESULTS: Prototypic KMT4/Dot1 from Archaea targets chromatin proteins (Sul7d and Cren7) and shows increased activity on Sul7d, but not Cren7, in the presence of DNA. CONCLUSION: Promiscuous aKMT4 could be regulated by chromatin environment. SIGNIFICANCE: This study supports the prokaryotic origin model of eukaryotic histone methyltransferases and sheds light on chromatin dynamics in Archaea. Histone methylation is one of the major epigenetic modifications even in early diverging unicellular eukaryotes. We show that a widespread lysine methyltransferase from Archaea (aKMT4), bears striking structural and functional resemblance to the core of distantly related eukaryotic KMT4/Dot1. aKMT4 methylates a set of various proteins, including the chromatin proteins Sul7d and Cren7, and RNA exosome components. Csl4- and Rrp4-exosome complexes are methylated in different patterns. aKMT4 can self-methylate intramolecularly and compete with other proteins for the methyl group. Automethylation is inhibited by suitable substrates or DNA in a concentration-dependent manner. The automethylated enzyme shows relatively compromised activity. aKMT4-8A mutant with abrogated automethylation shows a more than 150% increase in methylation of substrates, suggesting a possible mechanism to regulate methyltransferase activity. More interestingly, methylation of Sul7d, but not Cren7, by aKMT4 is significantly enhanced by DNA. MS/MS and kinetic analysis further suggest that aKMT4 methylates Sul7d in the chromatin context. These data provide a clue to the possible regulation of aKMT4 activity by the local chromatin environment, albeit as a promiscuous enzyme required for extensive and variegated lysine methylation in Sulfolobus. This study supports the prokaryotic origin model of eukaryotic histone modification enzymes and sheds light on regulation of archaeal chromatin.

Knockdown of Smox protects the integrity of the blood-brain barrier through antioxidant effect and Nrf2 pathway activation in stroke.

Once an ischemic stroke occurs, reactive oxygen species (ROS) and oxidative stress degrade the tight connections between cerebral endothelial cells resulting in their damage. The expression of antioxidant genes may be enhanced, and ROS formation may be reduced following Nrf2 activation, which is associated with protection against ischemic stroke. Overexpression of spermine oxidase (Smox) in the neocortex led to increased H2O2 production. However, how Smox impacts the regulation of the blood-brain barrier (BBB) through antioxidants has not been examined yet. We conducted experiments both in the cell level and in the transient middle cerebral artery occlusion (tMCAO) model to evaluate the effect of Smox siRNA lentivirus (si-Smox) knockdown on BBB protection against ischemic stroke. Mice treated with si-Smox showed remarkably decreased BBB breakdown and reduced endothelial inflammation following stroke. The treatment with si-Smox significantly elevated the Bcl-2 to Bax ratio and decreased the production of cleaved caspase-3 in the tMCAO model. Further investigation revealed that the neuroprotective effect was the result of the antioxidant properties of si-Smox, which reduced oxidative stress and enhanced CD31+ cells in the peri-infarct cortical areas. Of significance, si-Smox activated Nrf2 in both bEnd.3 cells and tMCAO animals, and blocking Nrf2 with brusatol diminished the protective effects of si-Smox. The study findings suggest that si-Smox exerts neuroprotective effects and promotes angiogenesis by activating the Nrf2 pathway, thus decreasing oxidative stress and apoptosis caused by tMCAO. As a result, si-Smox may hold potential as a therapeutic candidate for preserving BBB integrity while treating ischemic stroke.

Multiplexed transcriptomic profiling of the fate of human CAR T cells in vivo via genetic barcoding with shielded small nucleotides.

The design of chimeric antigen receptor (CAR) T cells would benefit from knowledge of the fate of the cells in vivo. This requires the permanent labelling of CAR T cell products and their pooling in the same microenvironment. Here, we report a cell-barcoding method for the multiplexed longitudinal profiling of cells in vivo using single-cell RNA sequencing (scRNA-seq). The method, which we named shielded-small-nucleotide-based scRNA-seq (SSN-seq), is compatible with both 3' and 5' single-cell profiling, and enables the recording of cell identity, from cell infusion to isolation, by leveraging the ubiquitous Pol III U6 promoters to robustly express small-RNA barcodes modified with direct-capture sequences. By using SSN-seq to track the dynamics of the states of CAR T cells in a tumour-rechallenge mouse model of leukaemia, we found that a combination of cytokines and small-molecule inhibitors that are used in the ex vivo manufacturing of CAR T cells promotes the in vivo expansion of persistent populations of CD4+ memory T cells. By facilitating the probing of cell-state dynamics in vivo, SSN-seq may aid the development of adoptive cell therapies.

Study of a Fluorescent System Based on the Naphthalene Derivative Fluorescent Probe Bound to Al3.

The naphthalene derivative fluorescent probe F6 was synthesized and a 1 × 10-3 mol/L solution of Al3+ and other metals to be tested was prepared for the subsequent experiments. The Al3+ fluorescence system of the naphthalene derivative fluorescent probe F6 was successfully constructed as demonstrated by fluorescence emission spectroscopy. The optimal time, temperature and pH of the reaction were investigated. The selectivity and anti-interference ability of the probe F6 for Al3+ were investigated by fluorescence spectroscopy in a methanol solution. The experiments showed that the probe has high selectivity and anti-interference ability for Al3+. The binding ratio of F6 to Al3+ was 2:1, and the binding constant was calculated to be 1.598 × 105 M-1. The possible mechanism of the binding of the two was speculated. Different concentrations of Al3+ were added to Panax Quinquefolium and Paeoniae Radix Alba. The results showed that the recoveries of Al3+ were 99.75-100.56% and 98.67-99.67%, respectively. The detection limit was 8.73 × 10-8 mol/L. The experiments demonstrated that the formed fluorescence system can be successfully adapted for the determination of Al3+ content in two Chinese herbal medicines, which has good practical application.

Immune checkpoint B7-H3 is a therapeutic vulnerability in prostate cancer harboring PTEN and TP53 deficiencies.

Checkpoint immunotherapy has yielded meaningful responses across many cancers but has shown modest efficacy in advanced prostate cancer. B7 homolog 3 protein (B7-H3/CD276) is an immune checkpoint molecule and has emerged as a promising therapeutic target. However, much remains to be understood regarding B7-H3's role in cancer progression, predictive biomarkers for B7-H3-targeted therapy, and combinatorial strategies. Our multi-omics analyses identified B7-H3 as one of the most abundant immune checkpoints in prostate tumors containing PTEN and TP53 genetic inactivation. Here, we sought in vivo genetic evidence for, and mechanistic understanding of, the role of B7-H3 in PTEN/TP53-deficient prostate cancer. We found that loss of PTEN and TP53 induced B7-H3 expression by activating transcriptional factor Sp1. Prostate-specific deletion of Cd276 resulted in delayed tumor progression and reversed the suppression of tumor-infiltrating T cells and NK cells in Pten/Trp53 genetically engineered mouse models. Furthermore, we tested the efficacy of the B7-H3 inhibitor in preclinical models of castration-resistant prostate cancer (CRPC). We demonstrated that enriched regulatory T cells and elevated programmed cell death ligand 1 (PD-L1) in myeloid cells hinder the therapeutic efficacy of B7-H3 inhibition in prostate tumors. Last, we showed that B7-H3 inhibition combined with blockade of PD-L1 or cytotoxic T lymphocyte-associated protein 4 (CTLA-4) achieved durable antitumor effects and had curative potential in a PTEN/TP53-deficient CRPC model. Given that B7-H3-targeted therapies have been evaluated in early clinical trials, our studies provide insights into the potential of biomarker-driven combinatorial immunotherapy targeting B7-H3 in prostate cancer, among other malignancies.

Decoding the evolutionary response to prostate cancer therapy by plasma genome sequencing.

BACKGROUND: Investigating genome evolution in response to therapy is difficult in human tissue samples. To address this challenge, we develop an unbiased whole-genome plasma DNA sequencing approach that concurrently measures genomic copy number and exome mutations from archival cryostored plasma samples. This approach is applied to study longitudinal blood plasma samples from prostate cancer patients, where longitudinal tissue biopsies from the bone and other metastatic sites have been challenging to collect. RESULTS: A molecular characterization of archival plasma DNA from 233 patients and genomic profiling of 101 patients identifies clinical correlations of aneuploid plasma DNA profiles with poor survival, increased plasma DNA concentrations, and lower plasma DNA size distributions. Deep-exome sequencing and genomic copy number profiling are performed on 23 patients, including 9 patients with matched metastatic tissues and 12 patients with serial plasma samples. These data show a high concordance in genomic alterations between the plasma DNA and metastatic tissue samples, suggesting the plasma DNA is highly representative of the tissue alterations. Longitudinal sequencing of 12 patients with 2-5 serial plasma samples reveals clonal dynamics and genome evolution in response to hormonal and chemotherapy. By performing an integrated evolutionary analysis, minor subclones are identified in 9 patients that expanded in response to therapy and harbored mutations associated with resistance. CONCLUSIONS: This study provides an unbiased evolutionary approach to non-invasively delineate clonal dynamics and identify clones with mutations associated with resistance in prostate cancer.

Evolution of Yin and Yang isoforms of a chromatin remodeling subunit precedes the creation of two genes.

Genes can encode multiple isoforms, broadening their functions and providing a molecular substrate to evolve phenotypic diversity. Evolution of isoform function is a potential route to adapt to new environments. Here we show that de novo, beneficial alleles in the nurf-1 gene became fixed in two laboratory lineages of C. elegans after isolation from the wild in 1951, before methods of cryopreservation were developed. nurf-1 encodes an ortholog of BPTF, a large (>300 kD) multidomain subunit of the NURF chromatin remodeling complex. Using CRISPR-Cas9 genome editing and transgenic rescue, we demonstrate that in C. elegans, nurf-1 has split into two, largely non-overlapping isoforms (NURF-1.D and NURF-1.B, which we call Yin and Yang, respectively) that share only two of 26 exons. Both isoforms are essential for normal gametogenesis but have opposite effects on male/female gamete differentiation. Reproduction in hermaphrodites, which involves production of both sperm and oocytes, requires a balance of these opposing Yin and Yang isoforms. Transgenic rescue and genetic position of the fixed mutations suggest that different isoforms are modified in each laboratory strain. In a related clade of Caenorhabditis nematodes, the shared exons have duplicated, resulting in the split of the Yin and Yang isoforms into separate genes, each containing approximately 200 amino acids of duplicated sequence that has undergone accelerated protein evolution following the duplication. Associated with this duplication event is the loss of two additional nurf-1 transcripts, including the long-form transcript and a newly identified, highly expressed transcript encoded by the duplicated exons. We propose these lost transcripts are non-functional side products necessary to transcribe the Yin and Yang transcripts in the same cells. Our work demonstrates how gene sharing, through the production of multiple isoforms, can precede the creation of new, independent genes.

Monitoring NAD(P)H by an ultrasensitive fluorescent probe to reveal reductive stress induced by natural antioxidants in HepG2 cells under hypoxia.

Reductive stress, the opposite of oxidative stress, represents a disorder in the redox balance state which is harmful to biological systems. For decades, the role of oxidative stress in tumor therapy has been the focus of attention, while the effects of reductive stress have been rarely studied. Here, we report the anti-cancer effects of reductive stress induced by three natural antioxidants (resveratrol, curcumin and celastrol). Considering the fact that the solid tumor microenvironment suffers from hypoxia, we performed cell experiments under hypoxic conditions. In order to observe the reductive stress, we first developed an ultrasensitive fluorescent probe (TCF-MQ) for specifically imaging NAD(P)H which is a marker of reductive stress. TCF-MQ responded to NAD(P)H rapidly and exhibited high sensitivity with a detection limit of 6 nM. With the help of TCF-MQ, we found that upon the treatment of HepG2 cells with pharmacological doses of three natural antioxidants under hypoxic conditions, high levels of NAD(P)H were produced before cell death. The excess NAD(P)H resulted in reductive stress instead of oxidative stress. In contrast, under normoxic conditions, there was no reductive stress involved in the process of cell death induced by three natural antioxidants. Therefore, we hypothesize that the mechanism of cancer cell death induced by natural antioxidants under hypoxia should be attributed to the reductive stress.

Alteration of GyrA amino acid required for ciprofloxacin resistance in Klebsiella pneumoniae isolates in China.

Resistance to ciprofloxacin was detected in 111 (48.1%) isolates of Klebsiella pneumoniae from China. GyrA alterations were identified in the ciprofloxacin-resistant and ciprofloxacin-susceptible isolates. The results, including previously published data, indicate that the single substitution Ser83-->Ile and three types of double mutations at Ser83 and Asp87 were required for ciprofloxacin resistance (P < 0.05).

[Application of nano-sized TiO2 photocatalysis to air purification and sterilization].

OBJECTIVE: To develop and evaluate the efficiency of air purification and sterilization instrument based on nano-sized TiO(2) photocatalytic technique. METHODS: The nano-sized TiO(2) photocatalytic air purification and sterilization instrument was designed and a sample had been prepared. The sterilization efficiencies for E.coli and Klebsiella by the nano-sized TiO(2) photocatalytic instrument and ultraviolet (UV) were measured in closed labs. The on-site efficiency of the instrument was evaluated, too. RESULTS: The nano-sized TiO(2) photocatalytic air purification and sterilization instrument was composed of five units: rough filter, nano-sized TiO(2) photocatalytic unit, activated carbon fiber filter, negative ion generator, and programmed control unit. The E.coli killing rates by the nano-sized TiO(2) photocatalytic instrument were 76.0%, 81.8%, 77.5%, and 80.7% at 30, 60, 90, and 120 minutes, respectively. There was no significant difference between the E.coli killing rates of the instrument and UV (P > 0.05), except the 120 minutes timepoint. The Klebsiella killing rates by the instrument were 78.4%, 79.5%, 67.3%, and 58.5% at 30, 60, 90, and 120 minutes, respectively. The Klebsiella killing efficiencies of the instrument at 30 and 60 minutes were better than that of UV (P < 0.01). There was no significant difference between the Klebsiella killing efficiencies of the instrument and UV (P > 0.05). CONCLUSION: The air sterilization efficiency of the nano-sized TiO(2) photocatalytic instrument should be equivalent or better as compared with the UV. This instrument might be used for the air purification and sterilization of the public locations.

Changes to social feeding behaviors are not sufficient for fitness gains of the Caenorhabditis elegans N2 reference strain.

The standard reference Caenorhabditis elegans strain, N2, has evolved marked behavioral changes in social feeding behavior since its isolation from the wild. We show that the causal, laboratory-derived mutations in two genes, npr-1 and glb-5, confer large fitness advantages in standard laboratory conditions. Using environmental manipulations that suppress social/solitary behavior differences, we show the fitness advantages of the derived alleles remained unchanged, suggesting selection on these alleles acted through pleiotropic traits. Transcriptomics, developmental timing, and food consumption assays showed that N2 animals mature faster, produce more sperm, and consume more food than a strain containing ancestral alleles of these genes regardless of behavioral strategies. Our data suggest that the pleiotropic effects of glb-5 and npr-1 are a consequence of changes to O2 -sensing neurons that regulate both aerotaxis and energy homeostasis. Our results demonstrate how pleiotropy can lead to profound behavioral changes in a popular laboratory model.

Differential expression of bla(SHV) related to susceptibility to ampicillin in Klebsiella pneumoniae.

Klebsiella pneumoniae usually shows intrinsic resistance to ampicillin and other beta-lactams. bla(SHV) is thought to be a key beta-lactamase gene responsible for this intrinsic resistance to ampicillin. Nevertheless, surveys of clinical strains reveal that some isolates of K. pneumoniae that carry bla(SHV) remain susceptible to ampicillin. To explore susceptibility to ampicillin in relation to bla(SHV) in K. pneumoniae, we analysed the existence and transcription of bla(SHV) and determined beta-lactamase activity as well as the susceptibilities to clinically relevant beta-lactams, including ampicillin in 160K. pneumoniae isolates from China. In total, 141 isolates (88.1%) were detected as bla(SHV)-positive, 20 (14.2%) of which were found to be broadly susceptible to all beta-lactams tested, including ampicillin. Among the 20 broadly susceptible isolates, sequencing of bla(SHV) revealed synonymous point mutations in 19 isolates and a premature stop codon in 1 isolate. Reverse transcription-polymerase chain reaction failed to detect bla(SHV) mRNA in five isolates (25%). The results demonstrate that differential expression of bla(SHV) in clinical isolates of K. pneumoniae can affect susceptibility to ampicillin.

Correlations of Genotype with Climate Parameters Suggest Caenorhabditis elegans Niche Adaptations.

Species inhabit a variety of environmental niches, and the adaptation to a particular niche is often controlled by genetic factors, including gene-by-environment interactions. The genes that vary in order to regulate the ability to colonize a niche are often difficult to identify, especially in the context of complex ecological systems and in experimentally uncontrolled natural environments. Quantitative genetic approaches provide an opportunity to investigate correlations between genetic factors and environmental parameters that might define a niche. Previously, we have shown how a collection of 208 whole-genome sequenced wild Caenorhabditis elegans can facilitate association mapping approaches. To correlate climate parameters with the variation found in this collection of wild strains, we used geographic data to exhaustively curate daily weather measurements in short-term (3 month), middle-term (one year), and long-term (three year) durations surrounding the date of strain isolation. These climate parameters were used as quantitative traits in association mapping approaches, where we identified 11 quantitative trait loci (QTL) for three climatic variables: elevation, relative humidity, and average temperature. We then narrowed the genomic interval of interest to identify gene candidates with variants potentially underlying phenotypic differences. Additionally, we performed two-strain competition assays at high and low temperatures to validate a QTL that could underlie adaptation to temperature and found suggestive evidence supporting that hypothesis.

Deep phenotyping unveils hidden traits and genetic relations in subtle mutants.

Discovering mechanistic insights from phenotypic information is critical for the understanding of biological processes. For model organisms, unlike in cell culture, this is currently bottlenecked by the non-quantitative nature and perceptive biases of human observations, and the limited number of reporters that can be simultaneously incorporated in live animals. An additional challenge is that isogenic populations exhibit significant phenotypic heterogeneity. These difficulties limit genetic approaches to many biological questions. To overcome these bottlenecks, we developed tools to extract complex phenotypic traits from images of fluorescently labelled subcellular landmarks, using C. elegans synapses as a test case. By population-wide comparisons, we identified subtle but relevant differences inaccessible to subjective conceptualization. Furthermore, the models generated testable hypotheses of how individual alleles relate to known mechanisms or belong to new pathways. We show that our model not only recapitulates current knowledge in synaptic patterning but also identifies novel alleles overlooked by traditional methods.