Microbial individuality: how single-cell heterogeneity enables population level strategies.

Much of our knowledge of microbial life is only a description of average population behaviours, but modern technologies provide a more inclusive view and reveal that microbes also have individuality. It is now acknowledged that isogenic cell-to-cell heterogeneity is common across organisms and across different biological processes. This heterogeneity can be regulated and functional, rather than just reflecting tolerance to noisy biochemistry. Here, we review recent advances in our understanding of microbial heterogeneity, with an emphasis on the pervasiveness of heterogeneity, the mechanisms that sustain it, and how heterogeneity enables collective function.The research has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement 338060. The work in the Locke laboratory is also supported by a fellowship from the Gatsby Foundation (GAT3273/GLC).This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.mib.2015.01.00

Widespread inter-individual gene expression variability in Arabidopsis thaliana.

A fundamental question in biology is how gene expression is regulated to give rise to a phenotype. However, transcriptional variability is rarely considered although it could influence the relationship between genotype and phenotype. It is known in unicellular organisms that gene expression is often noisy rather than uniform, and this has been proposed to be beneficial when environmental conditions are unpredictable. However, little is known about inter-individual transcriptional variability in multicellular organisms. Using transcriptomic approaches, we analysed gene expression variability between individual Arabidopsis thaliana plants growing in identical conditions over a 24-h time course. We identified hundreds of genes that exhibit high inter-individual variability and found that many are involved in environmental responses, with different classes of genes variable between the day and night. We also identified factors that might facilitate gene expression variability, such as gene length, the number of transcription factors regulating the genes and the chromatin environment. These results shed new light on the impact of transcriptional variability in gene expression regulation in plants

Co-expression Networks From Gene Expression Variability Between Genetically Identical Seedlings Can Reveal Novel Regulatory Relationships.

Co-expression networks are a powerful tool to understand gene regulation. They have been used to identify new regulation and function of genes involved in plant development and their response to the environment. Up to now, co-expression networks have been inferred using transcriptomes generated on plants experiencing genetic or environmental perturbation, or from expression time series. We propose a new approach by showing that co-expression networks can be constructed in the absence of genetic and environmental perturbation, for plants at the same developmental stage. For this, we used transcriptomes that were generated from genetically identical individual plants that were grown under the same conditions and for the same amount of time. Twelve time points were used to cover the 24-h light/dark cycle. We used variability in gene expression between individual plants of the same time point to infer a co-expression network. We show that this network is biologically relevant and use it to suggest new gene functions and to identify new targets for the transcriptional regulators GI, PIF4, and PRR5. Moreover, we find different co-regulation in this network based on changes in expression between individual plants, compared to the usual approach requiring environmental perturbation. Our work shows that gene co-expression networks can be identified using variability in gene expression between individual plants, without the need for genetic or environmental perturbations. It will allow further exploration of gene regulation in contexts with subtle differences between plants, which could be closer to what individual plants in a population might face in the wild

Frequency doubling in the cyanobacterial circadian clock

Organisms use circadian clocks to generate 24-h rhythms in gene expression. However, the clock can interact with other pathways to generate shorter period oscillations. It remains unclear how these different frequencies are generated. Here, we examine this problem by studying the coupling of the clock to the alternative sigma factor $\textit{sigC}$ in the cyanobacterium $\textit{Synechococcus elongatus}$. Using single-cell microscopy, we find that $\textit{psbAI}$, a key photosynthesis gene regulated by both $\textit{sigC}$ and the clock, is activated with two peaks of gene expression every circadian cycle under constant low light. This two-peak oscillation is dependent on $\textit{sigC}$, without which $\textit{psbAI}$ rhythms revert to one oscillatory peak per day. We also observe two circadian peaks of elongation rate, which are dependent on $\textit{sigC}$, suggesting a role for the frequency doubling in modulating growth. We propose that the two-peak rhythm in $\textit{psbAI}$ expression is generated by an incoherent feedforward loop between the clock, $\textit{sigC}$ and $\textit{psbAI}$. Modelling and experiments suggest that this could be a general network motif to allow frequency doubling of outputs.This research was made possible by the award of a European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement 338060. The work in the Locke laboratory is further supported by a fellowship from the Gatsby Foundation (GAT3272/GLC) and a Fellowship from the Human Frontier Science Program (CDA00068/2012)

Coordinated circadian timing through the integration of local inputs in Arabidopsis thaliana.

Individual plant cells have a genetic circuit, the circadian clock, that times key processes to the day-night cycle. These clocks are aligned to the day-night cycle by multiple environmental signals that vary across the plant. How does the plant integrate clock rhythms, both within and between organs, to ensure coordinated timing? To address this question, we examined the clock at the sub-tissue level across Arabidopsis thaliana seedlings under multiple environmental conditions and genetic backgrounds. Our results show that the clock runs at different speeds (periods) in each organ, which causes the clock to peak at different times across the plant in both constant environmental conditions and light-dark (LD) cycles. Closer examination reveals that spatial waves of clock gene expression propagate both within and between organs. Using a combination of modeling and experiment, we reveal that these spatial waves are the result of the period differences between organs and local coupling, rather than long-distance signaling. With further experiments we show that the endogenous period differences, and thus the spatial waves, can be generated by the organ specificity of inputs into the clock. We demonstrate this by modulating periods using light and metabolic signals, as well as with genetic perturbations. Our results reveal that plant clocks can be set locally by organ-specific inputs but coordinated globally via spatial waves of clock gene expression

A one-piece 3D printed flexure translation stage for open-source microscopy.

Open source hardware has the potential to revolutionise the way we build scientific instruments; with the advent of readily available 3D printers, mechanical designs can now be shared, improved, and replicated faster and more easily than ever before. However, printed parts are typically plastic and often perform poorly compared to traditionally machined mechanisms. We have overcome many of the limitations of 3D printed mechanisms by exploiting the compliance of the plastic to produce a monolithic 3D printed flexure translation stage, capable of sub-micron-scale motion over a range of 8 × 8 × 4 mm. This requires minimal post-print clean-up and can be automated with readily available stepper motors. The resulting plastic composite structure is very stiff and exhibits remarkably low drift, moving less than 20 μm over the course of a week, without temperature stabilisation. This enables us to construct a miniature microscope with excellent mechanical stability, perfect for time-lapse measurements in situ in an incubator or fume hood. The ease of manufacture lends itself to use in containment facilities where disposability is advantageous and to experiments requiring many microscopes in parallel. High performance mechanisms based on printed flexures need not be limited to microscopy, and we anticipate their use in other devices both within the laboratory and beyond.We would like to thank Paula Rudall (Jodrell Laboratory, Royal Botanic Gardens, Kew, UK) for preparing the Pollia condensata samples. RWB was supported by Research Fellowships from Queens’ College, Cambridge and the Royal Commission for the Exhibition of 1851, and partial support was provided by EPSRC EP/L027151/1, the University Teaching and Learning Innovation Fund and the SynBioFund initiative.This is the final version of the article. It first appeared from AIP Publishing via http://dx.doi.org/10.1063/1.4941068 Data supporting this publication is available at http://www.repository.cam.ac.uk/handle/1810/253294. Design files and assembly instructions are available at http://docubricks.com/projects/ openflexure-microscope

Rapid detection of BRCA1/2 recurrent mutations in Chinese breast and ovarian cancer patients with multiplex SNaPshot genotyping panels.

BRCA1/2 mutations are significant risk factors for hereditary breast and ovarian cancer (HBOC), its mutation frequency in HBOC of Chinese ethnicity is around 9%, in which nearly half are recurrent mutations. In Hong Kong and China, genetic testing and counseling are not as common as in the West. To reduce the barrier of testing, a multiplex SNaPshot genotyping panel that targeted 25 Chinese BRCA1/2 mutation hotspots was developed, and its feasibility was evaluated in a local cohort of 441 breast and 155 ovarian cancer patients. For those who tested negative, they were then subjected to full-gene testing with next-generation sequencing (NGS). BRCA mutation prevalence in this cohort was 8.05% and the yield of the recurrent panel was 3.52%, identifying over 40% of the mutation carriers. Moreover, from 79 Chinese breast cancer cases recruited overseas, 2 recurrent mutations and one novel BRCA2 mutation were detected by the panel and NGS respectively. The developed genotyping panel showed to be an easy-to-perform and more affordable testing tool that can provide important contributions to improve the healthcare of Chinese women with cancer as well as family members that harbor high risk mutations for HBOC

Short-chain fatty acid level and field cancerization show opposing associations with enteroendocrine cell number and neuropilin expression in patients with colorectal adenoma

BACKGROUND: Previous reports have suggested that the VEGF receptor neuropilin-1 (NRP-1) is expressed in a singly dispersed subpopulation of cells in the normal colonic epithelium, but that expression becomes dysregulated during colorectal carcinogenesis, with higher levels in tumour suggestive of a poor prognosis. We noted that the spatial distribution and morphology if NRP-1 expressing cells resembles that of enteroendocrine cells (EEC) which are altered in response to disease state including cancer and irritable bowel syndrome (IBS). We have shown that NRP-1 is down-regulated by butyrate in colon cancer cell lines in vitro and we hypothesized that butyrate produced in the lumen would have an analogous effect on the colon mucosa in vivo. Therefore we sought to investigate whether NRP-1 is expressed in EEC and how NRP-1 and EEC respond to butyrate and other short-chain fatty acids (SCFA - principally acetate and propionate). Additionally we sought to assess whether there is a field effect around adenomas. METHODOLOGY: Biopsies were collected at the mid-sigmoid, at the adenoma and at the contralateral wall (field) of 28 subjects during endoscopy. Samples were fixed for IHC and stained for either NRP-1 or for chromogranin A (CgA), a marker of EEC. Stool sampling was undertaken to assess individuals' butyrate, acetate and propionate levels. RESULT: NRP-1 expression was inversely related to SCFA concentration at the colon landmark (mid-sigmoid), but expression was lower and not related to SCFA concentration at the field. Likewise CgA+ cell number was also inversely related to SCFA at the landmark, but was lower and unresponsive at the field. Crypt cellularity was unaltered by field effect. A colocalisation analysis showed only a small subset of NRP-1 localised with CgA. Adenomas showed extensive, weaker staining for NRP-1 which contrastingly correlated positively with butyrate level. Field effects cause this relationship to be lost. Adenoma tissue shows dissociation of the co-regulation of NRP-1 and EEC. CONCLUSION: NRP-1 is inversely associated with levels of butyrate and other SCFA in vivo and is expressed in a subset of CgA expressing cells. EEC number is related to butyrate level in the same way

Cerebral fat embolism and the "starfield" pattern: a case report

Nearly all long-bone fractures are accompanied by some form of fat embolism. The rare complication of clinically significant fat embolism syndrome, however, occurs in only 0.9-2.2% of cases. The clinical triad of fat embolism syndrome consists of respiratory distress, altered mental status, and petechial rash. Cerebral fat embolism causes the neurologic involvement seen in fat embolism syndrome. A 19-year-old African-American male was admitted with gunshot wounds to his right hand and right knee. He had diffuse hyperactive deep tendon reflexes, bilateral ankle clonus and decerebrate posturing with a Glasgow Coma Scale (GCS) score of 4T. Subsequent MRI of the brain showed innumerable punctate areas of restricted diffusion consistent with "starfield" pattern. On a 10-week follow up he has a normal neurological examination and he is discharged home. Despite the severity of the neurologic insult upon initial presentation, the majority of case reports on cerebral fat embolism illustrate that cerebral dysfunction associated with cerebral fat embolism is reversible. When neurologic deterioration occurs in the non-head trauma patient, then a systemic cause such as fat emboli should be considered. We describe a patient with non-head trauma who demonstrated the classic "starfield" pattern on diffusion-weighted MRI imaging

Bioactivity-guided identification and cell signaling technology to delineate the immunomodulatory effects of Panax ginseng on human promonocytic U937 cells

<p>Abstract</p> <p>Background</p> <p>Ginseng is believed to have beneficial effects against human diseases, and its active components, ginsenosides, may play critical roles in its diverse physiological actions. However, the mechanisms underlying ginseng's effects remain to be investigated. We hypothesize some biological effects of ginseng are due to its anti-inflammatory effects.</p> <p>Methods</p> <p>Human promonocytic U937 cells were used to investigate the immunomodulatory effects of ginseng following TNF-α treatment. A global gene expression profile was obtained by using genechip analysis, and specific cytokine expression was measured by quantitative RT-PCR and ELISA. HPLC was used to define the composition of ginsenosides in 70% ethanol-water extracts of ginseng. Activation of signalling kinases was examined by Western blot analysis.</p> <p>Results</p> <p>Seventy percent ethanol-water extracts of ginseng significantly inhibited the transcription and secretion of CXCL-10 following TNF-α stimulation. Nine ginsenosides including Rb₁, Rb₂, Rc, Rd, Re, Rf, Rg₁, Rg₃and Rh₁were identified in our extract by HPLC. Seven out of nine ginsenosides could significantly inhibit TNF-α-induced CXCL-10 expression in U937 cells and give comparable inhibition of CXCL-10 transcription to those with the extract. However, the CXCL-10 suppressive effect of individual ginsenosides was less than that of the crude extract or the mixture of ginsenosides. The CXCL-10 suppression can be correlated with the inactivation of ERK1/2 pathways by ginseng.</p> <p>Conclusion</p> <p>We showed ginseng suppressed part of the TNF-α-inducible cytokines and signalling proteins in promonocytic cells, suggesting that it exerts its anti-inflammatory property targeting at different levels of TNF-α activity. The anti-inflammatory role of ginseng may be due to the combined effects of ginsenosides, contributing in part to the diverse actions of ginseng in humans.</p