Toward Microbiome Engineering: Expanding the Repertoire of Genetically Tractable Members of the Human Gut Microbiome.

Genetic manipulation is necessary to interrogate the functions of microbes in their environments, such as the human gut microbiome. Yet, the vast majority of human gut microbiome species are not genetically tractable. Here, we review the hurdles to seizing genetic control of more species. We address the barriers preventing the application of genetic techniques to gut microbes and report on genetic systems currently under development. While methods aimed at genetically transforming many species simultaneously in situ show promise, they are unable to overcome many of the same challenges that exist for individual microbes. Unless a major conceptual breakthrough emerges, the genetic tractability of the microbiome will remain an arduous task. Increasing the list of genetically tractable organisms from the human gut remains one of the highest priorities for microbiome research and will provide the foundation for microbiome engineering.

Microbiome engineering: Taming the untractable.

In this issue of Cell, Jin et al. describe several innovative tools for microbiome engineering to enable in situ editing of complex communities. However, challenges remain to overcome the widespread genetic intractability of microbiome constituents.

EFFECTIVE DOSE COEFFICIENTS FOR RADON AND PROGENY: A REVIEW OF ICRP AND UNSCEAR VALUES.

The International Commission on Radiological Protection (ICRP) publishes guidance on protection against radon exposure in homes and workplaces. ICRP Publication 137 recommends a dose coefficient of 3 mSv per mJ h m-3 (~10 mSv WLM-1) to be used in most circumstances of radon exposure, for workers in buildings and in underground mines. Recently, United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) reviewed radon epidemiology and dosimetry and concluded that its established dose coefficient of 1.6 mSv per mJ h m-3 (5.7 mSv WLM-1) should be retained for use in its comparisons of radiation exposures from different sources in a population. This paper explains and compares the reviews of the scientific evidence from UNSCEAR and ICRP. It is shown that the UNSCEAR and ICRP reviews are consistent and support the use of the ICRP reference dose coefficients for radiation protection purposes. It is concluded that the ICRP dose coefficient should be used to calculate doses to workers.

Chromatin accessibility dynamics of Chlamydia-infected epithelial cells.

Chlamydia are Gram-negative, obligate intracellular bacterial pathogens responsible for a broad spectrum of human and animal diseases. In humans, Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and is the causative agent of trachoma (infectious blindness) in disadvantaged populations. Over the course of its developmental cycle, Chlamydia extensively remodels its intracellular niche and parasitises the host cell for nutrients, with substantial resulting changes to the host cell transcriptome and proteome. However, little information is available on the impact of chlamydial infection on the host cell epigenome and global gene regulation. Regions of open eukaryotic chromatin correspond to nucleosome-depleted regions, which in turn are associated with regulatory functions and transcription factor binding. We applied formaldehyde-assisted isolation of regulatory elements enrichment followed by sequencing (FAIRE-Seq) to generate temporal chromatin maps of C. trachomatis-infected human epithelial cells in vitro over the chlamydial developmental cycle. We detected both conserved and distinct temporal changes to genome-wide chromatin accessibility associated with C. trachomatis infection. The observed differentially accessible chromatin regions include temporally-enriched sets of transcription factors, which may help shape the host cell response to infection. These regions and motifs were linked to genomic features and genes associated with immune responses, re-direction of host cell nutrients, intracellular signalling, cell-cell adhesion, extracellular matrix, metabolism and apoptosis. This work provides another perspective to the complex response to chlamydial infection, and will inform further studies of transcriptional regulation and the epigenome in Chlamydia-infected human cells and tissues.

Sleep problems in advanced cancer patients and their caregivers: Who is disturbing whom?

Background The aims of the study were to understand sleep problems and their effects in advanced cancer patients and spousal and intimate partner caregivers and to examine the directionality of the link between patients' and caregivers' sleep problems. Methods Fifty-four advanced cancer patients and their spousal and intimate partners were administered a battery of questionnaires that included the Pittsburgh Sleep Quality Index and the Center for Epidemiological Studies at the patients' cancer diagnosis and at 2, 4, and 6 months after diagnosis. Results Patients' and caregivers' sleep duration was significantly related. Using cross-lagged panel analyses, caregivers' sleep quality significantly predicted patients' sleep quality and patients' sleep quality subsequently predicted caregivers' sleep quality. Patients' sleep latency significantly was found to significantly predict caregivers' sleep latency. Conclusion Patients diagnosed with cancer and their intimate partners have poor sleep quality and sleep patterns are related.

Early Transcriptional Landscapes of Chlamydia trachomatis-Infected Epithelial Cells at Single Cell Resolution.

Chlamydia are Gram-negative obligate intracellular bacterial pathogens responsible for a variety of disease in humans and animals worldwide. Chlamydia trachomatis causes trachoma in disadvantaged populations, and is the most common bacterial sexually transmitted infection in humans, causing reproductive tract disease. Antibiotic therapy successfully treats diagnosed chlamydial infections, however asymptomatic infections are common. High-throughput transcriptomic approaches have explored chlamydial gene expression and infected host cell gene expression. However, these were performed on large cell populations, averaging gene expression profiles across all cells sampled and potentially obscuring biologically relevant subsets of cells. We generated a pilot dataset, applying single cell RNA-Seq (scRNA-Seq) to C. trachomatis infected and mock-infected epithelial cells to assess the utility, pitfalls and challenges of single cell approaches applied to chlamydial biology, and to potentially identify early host cell biomarkers of chlamydial infection. Two hundred sixty-four time-matched C. trachomatis-infected and mock-infected HEp-2 cells were collected and subjected to scRNA-Seq. After quality control, 200 cells were retained for analysis. Two distinct clusters distinguished 3-h cells from 6- and 12-h. Pseudotime analysis identified a possible infection-specific cellular trajectory for Chlamydia-infected cells, while differential expression analyses found temporal expression of metallothioneins and genes involved with cell cycle regulation, innate immune responses, cytoskeletal components, lipid biosynthesis and cellular stress. We find that changes to the host cell transcriptome at early times of C. trachomatis infection are readily discernible by scRNA-Seq, supporting the utility of single cell approaches to identify host cell biomarkers of chlamydial infection, and to further deconvolute the complex host response to infection.

Dual RNA-Seq of Chlamydia and Host Cells.

During the infection of a host cell by a bacterial pathogen, a cascading series of gene expression changes occurs as each organism manipulates or responds to the other via defense or survival strategies. Unraveling this complex interplay is key for our understanding of bacterial virulence and host response pathways for the development of novel therapeutics. Dual RNA sequencing (dual RNA-Seq) has recently been developed to simultaneously capture host and bacterial transcriptomes from an infected cell. Leveraging the sensitivity and resolution allowed by RNA-seq, dual RNA-Seq can be applied to any bacteria-eukaryotic host interaction. We pioneered dual RNA-Seq to simultaneously capture Chlamydia and host expression profiles during an in vitro infection as proof of principle. Here we provide a detailed laboratory protocol and bioinformatics analysis guidelines for dual RNA-seq experiments focusing on Chlamydia as the organism of interest.

Structure-activity analysis of peptidic Chlamydia HtrA inhibitors.

Chlamydia trachomatis high temperature requirement A (CtHtrA) is a serine protease that performs proteolytic and chaperone functions in pathogenic Chlamydiae; and is seen as a prospective drug target. This study details the strategies employed in optimizing the irreversible CtHtrA inhibitor JO146 [Boc-Val-Pro-ValP(OPh)2] for potency and selectivity. A series of adaptations both at the warhead and specificity residues P1 and P3 yielded 23 analogues, which were tested in human neutrophil elastase (HNE) and CtHtrA enzyme assays as well as Chlamydia cell culture assays. Trypsin and chymotrypsin inhibition assays were also conducted to measure off-target selectivity. Replacing the phosphonate moiety with α-ketobenzothiazole produced a reversible analogue with considerable CtHtrA inhibition and cell culture activity. Tertiary leucine at P3 (8a) yielded approximately 33-fold increase in CtHtrA inhibitory activity, with an IC50 = 0.68 ±â€¯0.02 µM against HNE, while valine at P1 retained the best anti-chlamydial activity. This study provides a pathway for obtaining clinically relevant inhibitors.

Socioeconomic disparities of depressive symptoms and cytokines in hepatocellular carcinoma.

OBJECTIVE: To examine the associations among socioeconomic factors, depressive symptoms, and cytokines in patients diagnosed with hepatocellular carcinoma (HCC). METHODS: A total of 266 patients diagnosed with HCC were administered a battery of questionnaires including a sociodemographic questionnaire and the Center for Epidemiologic StudiesDepression (CES-D) scale. Blood samples were collected to assess serum levels of cytokines using Luminex. Descriptive statistics, Mann-Whitney U, Kruskal-Wallis, linear regression, and Bonferroni corrections were performed to test the hypotheses. RESULTS: Of the 266 patients, 24% reported depressive symptoms in the clinical range (CES-D ≥ 22). Females had higher CES-D score than males (Mann-Whitney U = 7135, P = .014, Padj  = .028). Being unemployed/disabled (Kruskal-Wallis = 14.732, P = .001, Padj  = .005) was found to be associated with higher depressive symptoms in males but not in females. Serum level of IL-2 (Kruskal-Wallis = 17.261, P = .001, Padj  = .005) were found to be negatively associated with education level. Gender (ß = .177, P = .035), income (ß = -.252, P = .004), whether the patient's income met their basic needs (ß = .180, P = .035), and IL-1ß (ß = -.165, P = .045) independently predicted depressive symptoms and together explained 19.4% of variance associated with depressive symptoms. CONCLUSIONS: Sociodemographic and socioeconomic factors were predictive of inflammation and depressive symptoms. Recommendations include the development of gender-targeted interventions for patients diagnosed with HCC who have low socioeconomic status (SES) and may suffer from depressive symptoms.

Prospective Analyses of Cytokine Mediation of Sleep and Survival in the Context of Advanced Cancer.

OBJECTIVE: The aims of this study were to examine the potential association between sleep problems, symptom burden, and survival in patients with advanced cancer. METHODS: A prospective study of 294 patients with gastrointestinal cancer administered questionnaires assessing sleep, depression, anxiety, stress, pain, fatigue, and health-related quality of life. Serum levels of cytokines including interleukin (IL)-1α, IL-1ß, tumor necrosis factor α, IL-10, IL-2, and interferon-γ were measured to assess biological mediation between sleep and survival. Survival was measured as time from diagnosis to death. RESULTS: Fifty-nine percent of patients reported poor sleep quality, 53% reported poor sleep efficiency, 39% reported sleep latency greater than 30 minutes, and 45% reported sleeping less than 6 hours or greater than 10 hours. We found a significant association between sleep duration and symptom burden. Shorter sleep duration was significantly associated with higher levels of fatigue (r = -0.169, p = .01), pain (r = -0.302, p = .01), anxiety (r = -0.182, p = .01), depression (r = -0.172, p = .003), and lower levels of quality of life (r = 0.240, p = .01). After adjustment for demographic, psychological, and disease-specific factors, short sleep duration was associated with reduced survival (hazard ratio [HR] linear = 0.485, 95% confidence interval = 0.275-0.857) and there was also evidence for a quadratic pattern (HR quadrati = 1.064, 95% confidence interval = 1.015-1.115) suggesting a curvilinear relationship between sleep duration and survival. Interleukin 2 was the only cytokine significantly related to survival (HR = 1.01, p = .003) and sleep duration (ß = -30.11, p = .027). When of IL-2 was added to the multivariable model, short and long sleep (ß = -0.557, p = .097; ß = 0.046, p = .114) were no longer significantly related to survival, suggesting mediation by IL-2. CONCLUSION: Sleep duration was associated with symptom burden and poorer survival and IL-2 was found to mediate the association between sleep and survival. Screening and treatment of sleep problems in patients diagnosed with cancer are warranted.

Bioinformatic analysis of bacteria and host cell dual RNA-sequencing experiments.

Bacterial pathogens subvert host cells by manipulating cellular pathways for survival and replication; in turn, host cells respond to the invading pathogen through cascading changes in gene expression. Deciphering these complex temporal and spatial dynamics to identify novel bacterial virulence factors or host response pathways is crucial for improved diagnostics and therapeutics. Dual RNA sequencing (dRNA-Seq) has recently been developed to simultaneously capture host and bacterial transcriptomes from an infected cell. This approach builds on the high sensitivity and resolution of RNA sequencing technology and is applicable to any bacteria that interact with eukaryotic cells, encompassing parasitic, commensal or mutualistic lifestyles. Several laboratory protocols have been presented that outline the collection, extraction and sequencing of total RNA for dRNA-Seq experiments, but there is relatively little guidance available for the detailed bioinformatic analyses required. This protocol outlines a typical dRNA-Seq experiment, based on a Chlamydia trachomatis-infected host cell, with a detailed description of the necessary bioinformatic analyses with currently available software tools.

A Laboratory Methodology for Dual RNA-Sequencing of Bacteria and their Host Cells In Vitro.

Dual RNA-Sequencing leverages established next-generation sequencing (NGS)-enabled RNA-Seq approaches to measure genome-wide transcriptional changes of both an infecting bacteria and host cells. By simultaneously investigating both organisms from the same biological sample, dual RNA-Seq can provide unique insight into bacterial infection processes and reciprocal host responses at once. However, the difficulties involved in handling both prokaryotic and eukaryotic material require distinct, optimized procedures. We previously developed and applied dual RNA-Seq to measure prokaryotic and eukaryotic expression profiles of human cells infected with bacteria, using in vitro Chlamydia-infected epithelial cells as proof of principle. Here we provide a detailed laboratory protocol for in vitro dual RNA-Seq that is readily adaptable to any host-bacteria system of interest.

Copper(II)-bis(thiosemicarbazonato) complexes as anti-chlamydial agents.

Lipophilic copper (Cu)-containing complexes have shown promising antibacterial activity against a range of bacterial pathogens. To examine the susceptibility of the intracellular human pathogen Chlamydia trachomatis to copper complexes containing bis(thiosemicarbazone) ligands [Cu(btsc)], we tested the in vitro effect of CuII-diacetyl- and CuII-glyoxal-bis[N(4)-methylthiosemicarbazonato] (Cu(atsm) and Cu(gtsm), respectively) on C. trachomatis. Cu(atsm) and to a greater extent, Cu(gtsm), prevented the formation of infectious chlamydial progeny. Impacts on host cell viability and respiration were also observed in addition to the Chlamydia impacts. This work suggests that copper-based complexes may represent a new lead approach for future development of new therapeutics against chlamydial infections, although host cell impacts need to be fully explored.

CtHtrA: the lynchpin of the chlamydial surface and a promising therapeutic target.

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection worldwide and the leading cause of preventable blindness. Reports have emerged of treatment failure, suggesting a need to develop new antibiotics to battle Chlamydia infection. One possible candidate for a new treatment is the protease inhibitor JO146, which is an effective anti-Chlamydia agent that targets the CtHtrA protein. CtHtrA is a lynchpin on the chlamydial cell surface due to its essential and multifunctional roles in the bacteria's stress response, replicative phase of development, virulence and outer-membrane protein assembly. This review summarizes the current understanding of CtHtrA function and presents a mechanistic model that highlights CtHtrA as an effective target for anti-Chlamydia drug development.

RADON DOSIMETRY FOR WORKERS: ICRP'S APPROACH.

The International Commission on Radiological Protection (ICRP) has recently published two reports on radon exposure; Publication 115 on lung cancer risks from radon and radon progeny and Publication 126 on radiological protection against radon exposure. A specific graded approach for the control of radon in workplaces is recommended where a dose assessment is required in certain situations. In its forthcoming publication on Occupational Intakes of Radionuclides (OIR) document, Part 3, effective dose coefficients for radon and thoron will be provided. These will be calculated using ICRP reference biokinetic and dosimetric models. Sufficient information and dosimetric data will be given so that site-specific dose coefficients can be calculated based on measured aerosol parameter values. However, ICRP will recommend a single dose coefficient of 12 mSv per working level month (WLM) for inhaled radon progeny to be used in most circumstances. This chosen reference value was based on both dosimetry and epidemiological data. In this paper, the application and use of dose coefficients for workplaces are discussed including the reasons for the choice of the reference value. Preliminary results of dose calculations for indoor workplaces and mines are presented. The paper also briefly describes the general approach for the management of radon exposure in workplaces based both on ICRP recommendations and the European directive (2013/59/EURATOM).

Sleep duration is associated with survival in advanced cancer patients.

OBJECTIVE: Sleep problems have been linked to increased risk of mortality in the general population. Limited evidence suggests similar relationships among people diagnosed with cancer. The aims of the present study were to investigate the type and rates of sleep problems in advanced cancer patients and examine whether sleep problems are associated with survival. METHODS: A prospective study of 292 patients with advanced cancers affecting the hepatobiliary and pancreatic systems were administered a battery of questionnaires measuring sociodemographic information, sleep, and depression. Descriptive statistics, ANOVA, Chi-square, Kaplan-Meier survival, and Cox regression analyses were performed to test the aims. RESULTS: The majority of patients were male (64%) and the mean age was 62 years (SD = 11). Fifty-nine percent of patients reported poor sleep quality; 43% reported sleeping ≤6 h and 2% ≥10 h; 40% reported sleep latency of 30 min or greater; average sleep efficiency was 80%. Of the 292 patients, 58% reported clinically levels of depression and depressive symptoms were related to shorter sleep duration (p = 0.02). After adjusting for factors known to contribute to survival, a curvilinear relationship was observed between sleep duration and mortality: short and long sleep duration were associated with increased mortality [linear term: hazard ratio (HR) = 0.485, 95% confidence interval (CI) = 0.275-0.857; quadratic term: HR = 1.064, 95% CI = 1.015-1.115]. CONCLUSIONS: Consistent with findings in the general population, a curvilinear relationship between sleep duration and mortality was observed in advanced cancer patients. The high prevalence of sleep problems and link with mortality warrants routine screening and development of evidence-based treatments for sleep problems in the oncology setting.

Leukaemia mortality and low-dose ionising radiation in the WISMUT uranium miner cohort (1946-2013).

OBJECTIVES: To examine the risk of death from leukaemia in relation to occupational chronic low-level external and internal radiation exposure in a cohort of 58 972 former German uranium miners with mortality follow-up from 1946 to 2013. METHODS: The red bone marrow (RBM) dose from low-linear energy transfer (LET) (mainly external γ-radiation) and high-LET (mainly radon gas) radiation was estimated based on a job-exposure matrix and biokinetic/dosimetric models. Linear excess relative risks (ERR) and 95% CIs were estimated via Poisson regression for chronic lymphatic leukaemia (CLL) and non-CLL. RESULTS: The mean cumulative low-LET and high-LET RBM doses among the 86% radiation-exposed workers were 48 and 9 mGy, respectively. There was a positive non-significant dose-response for mortality from non-CLL (n=120) in relation to low-LET (ERR/Gy=2.18; 95% CI -0.41 to 6.37) and high-LET radiation (ERR/Gy=16.65; 95% -1.13 to 46.75). A statistically significant excess was found for the subgroup chronic myeloid leukaemia (n=31) in relation to low-LET radiation (ERR/Gy=7.20; 95% CI 0.48 to 24.54) and the subgroup myeloid leukaemia (n=99) (ERR/Gy=26.02; 95% CI 2.55 to 68.99) for high-LET radiation. The ERR/Gy tended to be about five to ten times higher for high-LET versus low-LET radiation; however, the CIs largely overlapped. Results indicate no association of death from CLL (n=70) with either type of radiation. CONCLUSIONS: Our findings indicate an increased risk of death for specific subtypes from non-CLL in relation to chronic low-LET and high-LET radiation, but no such relation for CLL.

Longterm survival outcomes of patients undergoing treatment with radiofrequency ablation for hepatocellular carcinoma and metastatic colorectal cancer liver tumors.

BACKGROUND: We aim to investigate long-term survival outcomes in patients undergoing radiofrequency ablation (RFA), based on our longitudinal 5 and 10 year follow-up data. METHODS: All patients who underwent RFA for hepatocellular carcinoma (HCC) and colorectal liver metastasis (CLM) between 1999 and 2010. RESULTS: 320 patients were included with oncologic diagnoses of HCC in 122 (38.1%) and CLM in 198 (61.9%). The majority of patients had a single tumor ablation (71% RFA 1 lesion). Minimum 5 year follow-up information was available in 89% patients, with a median follow-up of 115.3 months. In patients with HCC, disease eventually recurred in 73 (64%) patients. In patients with CLM, disease recurrence was ultimately seen in 143 (84.1%) patients. In the HCC group, the 5- and 10-year overall survivals were 38.5% and 23.4%, while in the CLM group, the 5- and 10-year overall survivals were 27.6% and 15%, respectively. CONCLUSIONS: The use of RFA as a part of treatment strategy for primary and metastatic liver tumors imparts 10-year overall survivals of >23% and 15%, respectively. This study indicates that long-term survival is possible with RFA treatment.

A Chlamydia trachomatis strain with a chemically generated amino acid substitution (P370L) in the cthtrA gene shows reduced elementary body production.

BACKGROUND: Chlamydia (C.) trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness. Genetic approaches to investigate C. trachomatis have been only recently developed due to the organism's intracellular developmental cycle. HtrA is a critical stress response serine protease and chaperone for many bacteria and in C. trachomatis has been previously shown to be important for heat stress and the replicative phase of development using a chemical inhibitor of the CtHtrA activity. In this study, chemically-induced SNVs in the cthtrA gene that resulted in amino acid substitutions (A240V, G475E, and P370L) were identified and characterized. METHODS: SNVs were initially biochemically characterized in vitro using recombinant protein techniques to confirm a functional impact on proteolysis. The C. trachomatis strains containing the SNVs with marked reductions in proteolysis were investigated in cell culture to identify phenotypes that could be linked to CtHtrA function. RESULTS: The strain harboring the SNV with the most marked impact on proteolysis (cthtrA P370L) was detected to have a significant reduction in the production of infectious elementary bodies. CONCLUSIONS: This provides genetic evidence that CtHtrA is critical for the C. trachomatis developmental cycle.