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.

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.

An age- and sex-specific biokinetic model for radon.

Publication 137 of the International Commission on Radiological Protection (ICRP) describes a biokinetic model for radon used to derive dose coefficients for occupational intake of radon isotopes. The model depicts transfer of inhaled or ingested radon to blood, exchange of radon between blood and tissues, and gradual loss of radon from the body based on physical laws governing transfer of a non-reactive and soluble gas between materials. This paper describes an age- and sex-specific variation of that model developed for use in an upcoming ICRP series of reports on environmental intake of radionuclides by members of the public titled 'Dose Coefficients for Intakes of Radionuclides by Members of the Public'. The proposed model modifies the model structure and transfer coefficients presented in Publication 137 to allow more realistic dosimetric treatment of bone marrow and breast and expands the model to address pre-adult ages, based on the physical principles used in the development of the model of Publication 137 together with anatomical and physiological changes occurring during human development.

Burden of trichomoniasis among older adults in the United States: a systematic review.

Despite being one of the most common sexually transmitted infections (STIs) in the United States, the epidemiology of trichomoniasis remains understudied. One population that has been historically overlooked regarding STIs is that of older adults, despite many individuals remaining sexually active well into their older years. We investigated the reported prevalence and incidence of trichomoniasis in adults aged ≥45years in the United States using a systematic literature review. Twelve articles were included in the review, all assessing prevalence of trichomoniasis in this age group. Notably, no included articles assessed trichomoniasis incidence. Data collected encompassed several decades, from 1993 to 2016. Estimates of infection prevalence varied widely and ranged from 0.2% to 21.4% in included populations, with the highest prevalence typically seen among individuals seeking diagnostic testing for STIs. Several studies found increased risk for trichomoniasis in older patients compared to younger age groups. This is the first review to examine the risk of trichomoniasis in older adults, and the surprisingly high prevalence suggests that older adults may merit increased screening for trichomoniasis and sexual health education.

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.

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.

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.

Dodecafluoropentane Emulsion in Acute Ischemic Stroke: A Phase Ib/II Randomized and Controlled Dose-Escalation Trial.

PURPOSE: This randomized, placebo-controlled, double-blind, dose-escalation acute ischemic stroke trial was designed to demonstrate maximum tolerated dose, characterize adverse events (AEs), and explore clinical outcomes when intravenous dodecafluoropentane emulsion (DDFPe) was used as neuroprotection. METHODS: Acute ischemic stroke patients (n = 24) with National Institutes of Health Stroke Scale (NIHSS) score of 2-20 were randomized to either 3 doses of intravenous DDFPe or placebo, 1 every 90 minutes, starting within 12 hours of symptom onset. Doses were given without affecting standard stroke care. Each of the 3 dose cohorts included 8 patients, with 2 receiving placebo and 6 receiving DDFPe. Primary outcomes were serious adverse events (SAEs), AEs, NIHSS score, and modified Rankin Score (mRS). RESULTS: No dose-limiting toxicities were encountered, and no maximum tolerated dose was defined. One unrelated delayed death occurred in a DDFPe patient, and another occurred in the placebo group. Group SAEs and AEs were similar in incidence and severity. Early initiation of DDFPe treatment resulted in better NIHSS score response than late initiation (P = .03). Thirty- and 90-day mRS after high-dose therapy suggested clinical improvement (P = .01 and P = .03, respectively). However, the significance of differences in clinical outcomes was limited by small patient numbers and differences in stroke severity between cohorts. CONCLUSIONS: Intravenous DDFPe appears to be safe at all doses tested. Clinical improvements in NIHSS score and mRS were significant but compromised by small sample size.

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.

Preoperative next-generation sequencing of pancreatic cyst fluid is highly accurate in cyst classification and detection of advanced neoplasia.

OBJECTIVE: DNA-based testing of pancreatic cyst fluid (PCF) is a useful adjunct to the evaluation of pancreatic cysts (PCs). Mutations in KRAS/GNAS are highly specific for intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs), while TP53/PIK3CA/PTEN alterations are associated with advanced neoplasia. A prospective study was performed to evaluate preoperative PCF DNA testing. DESIGN: Over 43-months, 626 PCF specimens from 595 patients were obtained by endoscopic ultrasound (EUS)-fine needle aspiration and assessed by targeted next-generation sequencing (NGS). Molecular results were correlated with EUS findings, ancillary studies and follow-up. A separate cohort of 159 PCF specimens was also evaluated for KRAS/GNAS mutations by Sanger sequencing. RESULTS: KRAS/GNAS mutations were identified in 308 (49%) PCs, while alterations in TP53/PIK3CA/PTEN were present in 35 (6%) cases. Based on 102 (17%) patients with surgical follow-up, KRAS/GNAS mutations were detected in 56 (100%) IPMNs and 3 (30%) MCNs, and associated with 89% sensitivity and 100% specificity for a mucinous PC. In comparison, KRAS/GNAS mutations by Sanger sequencing had a 65% sensitivity and 100% specificity. By NGS, the combination of KRAS/GNAS mutations and alterations in TP53/PIK3CA/PTEN had an 89% sensitivity and 100% specificity for advanced neoplasia. Ductal dilatation, a mural nodule and malignant cytopathology had lower sensitivities (42%, 32% and 32%, respectively) and specificities (74%, 94% and 98%, respectively). CONCLUSIONS: In contrast to Sanger sequencing, preoperative NGS of PCF for KRAS/GNAS mutations is highly sensitive for IPMNs and specific for mucinous PCs. In addition, the combination of TP53/PIK3CA/PTEN alterations is a useful preoperative marker for advanced neoplasia.

Long- and short-term outcomes in renal allografts with deceased donors: A large recipient and donor genome-wide association study.

Improvements in immunosuppression have modified short-term survival of deceased-donor allografts, but not their rate of long-term failure. Mismatches between donor and recipient HLA play an important role in the acute and chronic allogeneic immune response against the graft. Perfect matching at clinically relevant HLA loci does not obviate the need for immunosuppression, suggesting that additional genetic variation plays a critical role in both short- and long-term graft outcomes. By combining patient data and samples from supranational cohorts across the United Kingdom and European Union, we performed the first large-scale genome-wide association study analyzing both donor and recipient DNA in 2094 complete renal transplant-pairs with replication in 5866 complete pairs. We studied deceased-donor grafts allocated on the basis of preferential HLA matching, which provided some control for HLA genetic effects. No strong donor or recipient genetic effects contributing to long- or short-term allograft survival were found outside the HLA region. We discuss the implications for future research and clinical application.

Fibrinogen and the prediction of residual obstruction manifested after pulmonary embolism treatment.

Residual pulmonary vascular obstruction (RPVO) and chronic thromboembolic pulmonary hypertension (CTEPH) are both long-term complications of acute pulmonary embolism, but it is unknown whether RPVO can be predicted by variants of fibrinogen associated with CTEPH.We used the Akaike information criterion to select the best predictive models for RPVO in two prospectively followed cohorts of acute pulmonary embolism patients, using as candidate variables the extent of the initial obstruction, clinical characteristics and fibrinogen-related data. We measured the selected models' goodness of fit by analysis of deviance and compared models using the Chi-squared test.RPVO occurred in 29 (28.4%) out of 102 subjects in the first cohort and 46 (25.3%) out of 182 subjects in the second. The best-fit predictive model derived in the first cohort (p=0.0002) and validated in the second cohort (p=0.0005) implicated fibrinogen Bß-chain monosialylation in the development of RPVO. When the derivation procedure excluded clinical characteristics, fibrinogen Bß-chain monosialylation remained a predictor of RPVO in the best-fit predictive model (p=0.00003). Excluding fibrinogen characteristics worsened the predictive model (p=0.03).Fibrinogen Bß-chain monosialylation, a common structural attribute of fibrin, helped predict RPVO after acute pulmonary embolism. Fibrin structure may contribute to the risk of developing RPVO.

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.

Impact of major vascular resection on outcomes and survival in patients with intrahepatic cholangiocarcinoma: A multi-institutional analysis.

BACKGROUND: Major vascular involvement (IVC or portal vein) for intrahepatic cholangiocarcinoma (ICC) has traditionally been considered a contraindication to resection. We sought to define perioperative outcomes and survival of ICC patients undergoing hepatectomy with major vascular resection in a large international multi-institutional database. METHODS: A total of 1087 ICC patients who underwent curative-intent hepatectomy between 1990 and 2016 were identified from 13 institutions. Multivariable logistic and cox regressions were used to determine the impact of major vascular resection on perioperative and survival outcomes. RESULTS: Of 1087 patients who underwent resection, 128 (11.8%) also underwent major vascular resection (21 [16.4%] IVC resections, 98 [76.6%] PV resections, 9 [7.0%] combined resections). Despite more advanced disease, major vascular resection was not associated with the risk of any complication (OR = 0.68, 95%CI 0.32-1.45) or major complications (OR = 0.95, 95%CI 0.49-2.00). Post-operative mortality was also comparable between groups (OR = 1.05, 95%CI 0.32-3.47). In addition, median recurrence-free (14.0 vs 14.7 months, HR = 0.737, 95%CI 0.49-1.10) and overall (33.4 vs 40.2 months, HR = 0.71, 95%CI 0.359-1.40) survival were similar among patients who did and did not undergo major vascular resection (both P > 0.05). CONCLUSION: Among patients with ICC, major vascular resection was not associated with worse perioperative or oncologic outcomes. Concurrent major vascular resection should be considered in appropriately selected patients with ICC undergoing hepatectomy.

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.

Concerted Uranium Research in Europe (CURE): toward a collaborative project integrating dosimetry, epidemiology and radiobiology to study the effects of occupational uranium exposure.

The potential health impacts of chronic exposures to uranium, as they occur in occupational settings, are not well characterized. Most epidemiological studies have been limited by small sample sizes, and a lack of harmonization of methods used to quantify radiation doses resulting from uranium exposure. Experimental studies have shown that uranium has biological effects, but their implications for human health are not clear. New studies that would combine the strengths of large, well-designed epidemiological datasets with those of state-of-the-art biological methods would help improve the characterization of the biological and health effects of occupational uranium exposure. The aim of the European Commission concerted action CURE (Concerted Uranium Research in Europe) was to develop protocols for such a future collaborative research project, in which dosimetry, epidemiology and biology would be integrated to better characterize the effects of occupational uranium exposure. These protocols were developed from existing European cohorts of workers exposed to uranium together with expertise in epidemiology, biology and dosimetry of CURE partner institutions. The preparatory work of CURE should allow a large scale collaborative project to be launched, in order to better characterize the effects of uranium exposure and more generally of alpha particles and low doses of ionizing radiation.

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.

Neutrophil-lymphocyte ratio is a simple and novel biomarker for prediction of survival after radioembolization for metastatic colorectal cancer.

PURPOSE: Previous studies have reported that an elevated preoperative Neutrophil-Lymphocyte Ratio (NLR) is associated with poor prognosis in patients with various solid tumors including colorectal cancer (CRC). Here, we examine whether NLR predicts survival in patients with unresectable CRC metastases undergoing hepatic radioembolization. METHODS: A retrospective review of 104 consecutive patients with unresectable metastatic CRC who were treated with radioembolization after failing first and second-line chemotherapy. RESULTS: Between 2002 and 2012, the median NLR for all patients was 4.6. Using receiver operating curve analysis, there was no difference between using an NLR cut-off of 4.6 or 5. Forty-eight patients had a high NLR of ≥5 and 56 patients had an NLR of <5. Patients in both groups had similar previous extensive chemotherapy and liver-directed interventions. The median survival of patients with high NLR was 5.6 months (range 4.9-7.9 months) compared with 10.6 months (range 8.3-17.0 months) for patients with low NLR; a significant difference was found in overall survival (log-rank test; p = 0.001). Other factors associated with risk of death were extrahepatic spread of disease, presence of pulmonary nodules, previous liver-targeted intervention, and radiographic response. On multivariate analysis, high NLR, progressive radiographic response, and presence of extrahepatic disease remained independently associated with increased risk of death. CONCLUSIONS: NLR is a simply attainable, inexpensive, and useful biomarker to predict outcome in patients with metastatic colorectal cancer receiving radioembolization.