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Background & Aims: HBV coinfection is common among people living with HIV (PLWH) and is the most important cause of hepatocellular carcinoma (HCC). While risk prediction tools for HCC have been validated in patients with HBV monoinfection, they have not been evaluated in PLWH. Thus, we performed an external validation of PAGE-B in people with HIV/HBV coinfection.Methods: We included data on PLWH from four European cohorts who were positive for HBsAg and did not have HCC before starting tenofovir. We estimated the predictive performance of PAGE-B for HCC occurrence over 15 years in patients receiving tenofovir-containing antiretroviral therapy. Model discrimination was assessed after multiple imputation using Cox regression with the prognostic index as a covariate, and by calculating Harrell's c-index. Calibration was assessed by comparing our cumulative incidence with the PAGE-B derivation study using Kaplan-Meier curves.Results: In total, 2,963 individuals with HIV/HBV coinfection on tenofovir-containing antiretroviral therapy were included. PAGE-B was Conclusions: For individuals with HIV/HBV coinfection, PAGE-B is a valid tool to determine the need for HCC screening. Impact and implications: Chronic HBV infection is the most important cause of hepatocellular carcinoma (HCC) among people living with HIV. Valid risk prediction may enable better targeting of HCC screening efforts to high-risk individuals. We aimed to validate PAGE-B, a risk prediction tool that is based on age, sex, and platelets, in 2,963 individuals with HIV/HBV coinfection who received tenofovir-containing antiretroviral therapy. In the present study, PAGE-B showed good discrimination, adequate calibration, and a cut-off of

The CRISPR-Cas9 system is a powerful tool for studying gene functions and has tremendous potential for disease treatment. However, precise genome editing requires thorough assessments to minimize unintended on- and off-target effects. Here, we report an unexpected deletion of a 287 kb region on Chromosome 10 (10q23.31) in chronic myelogenous leukemia HAP1 cells, which are frequently used in CRISPR screens. The deleted region encodes regulatory genes, includingPAPSS2, ATAD1, KLLN, andPTEN. We found that this deletion was not a direct consequence of CRISPR-Cas9 off-targeting but rather occurred frequently by the process of generating CRISPR-Cas9-modifed cells. The deletion was associated with global changes in histone acetylation and gene expression, affecting fundamental cellular processes such as cell cycle and DNA replication. We detected this deletion in cancer patient genomes. As in HAP1 cells, the deletion contributed to similar gene expression patterns among cancer patients despite interindividual differences. Overall, our findings suggest that the unintended deletion of 10q23.31 can confound CRISPR-Cas9 studies, highlights the importance of assessing unintended genomic changes in CRISPR-Cas9-modified cells and may have clinical significance in cancer research.HighlightsCRISPR-Cas9-modified HAP1 cells carry an unexpected large genomic deletion at 10q23.31 encompassing four protein-coding genes frequently expressed across various cell types.The 10q23.31 deletion is accompanied by global changes in histone modification and transcriptomes.The generation of CRISPR-Cas9-modified cells rather than Cas9 activity increases the frequencies of the deletion at 10q23.31.The 10q23.31 deletion identified in HAP1 cells resembles a commonly occurring deletion pattern in cancer patients.

Enhanced biological phosphorus removal (EBPR) can recover significant quantities of wastewater phosphorus. However, this resource recovery process realizes limited use largely due to process stability concerns. The research evaluated the effects of anaerobic HRT (τAN ) and VFA concentration-critical operational parameters that can be externally controlled-on EBPR performance. Evaluated alone, τAN (1-4 h) exhibited no statistical effect on effluent phosphorus. However, PHA increased with VFA loading and biomass accumulated more phosphorus. Regarding resiliency, under increasing VFA loads PAOs hydrolyzed more phosphorus to uptake/catabolize VFAs; moreover, PHA synthesis normalized to VFA loading increased with τAN , suggesting fermentation. Kinetically, PAOs exhibited a Monod-like relationships for qPHAAN and qVFAAN as a function of anaerobic P release; additionally, qPAE exhibited a Monod-like relationship with end-anaerobic PHA concentration. A culminating analysis affirmed the relationship between enhanced aerobic P uptake, and net P removal, with a parameter (phosphorus removal propensity factor) that combines influent VFA concentration with τAN . PRACTITIONER POINTS: Evaluated alone τAN exhibits no statistical effect on effluent phosphorus in an EBPR configuration. Increased PHA synthesis, associated with increased VFAs and/or extended τAN, enhances aerobic phosphorus removal. PHA synthesis normalized to VFA loading increased with τAN , suggesting fermentation in the EBPR anaerobic zone. Aerobic phosphorus uptake increases linearly with anaerobic phosphorus release, with the slope exceeding unity. Increased VFAs can be substituted for shorter anaerobic HRTs, and vice versa, to enhance EBPR performance.

Background & Aims: HCV has emerged as a sexually transmitted infection (STI) among HIV-positive men who have sex with men (MSM). We evaluated HCV incidence and its risk factors among HIV-negative MSM using HIV pre-exposure prophylaxis (PrEP). Methods: Participants of the Amsterdam PrEP project were tested for HCV antibodies or HCV-RNA every 6 months. Participants used daily or event-driven PrEP and could switch regimens during follow-up. We calculated incidence rates (IRs) for overall HCV infection and separately for primary and re-infection. A univariable Bayesian exponential survival model was used to identify risk factors associated with incident HCV infection. The HCV NS5B gene fragment (709 bp) was sequenced and compared to HCV isolates from HIV-positive MSM and other risk groups (n = 419) using phylogenetic analysis. Results: Among 350 participants contributing 653.6 person-years (PYs), we detected 15 HCV infections in 14 participants (IR = 2.30/100PY). There were 8 primary infections (IR = 1.27/100PY) and 7 re-infections (IR = 27.8/100PY). IR was 2.71/100PY in daily and 1.15/100PY in event-driven PrEP users. Factors associated with incident HCV infection were higher number of receptive condomless anal sex acts with casual partners (posterior hazard ratio [HR] 1.57 per ln increase; 95% credibility interval [CrI] 1.09–2.20), anal STI (posterior HR 2.93; 95% CrI 1.24–7.13), injecting drug use (posterior HR 4.69; 95% CrI 1.61–12.09) and sharing straws when snorting drugs (posterior HR 2.62; 95% CrI 1.09–6.02). We identified robust MSM-specific HCV clusters of subtypes 1a, 4d, 2b and 3a, which included MSM with and without HIV. Conclusions: HIV-negative MSM using PrEP are at risk of incident HCV infection, while identified risk factors are similar to those in HIV-positive MSM. Regular HCV testing is needed, especially for those with a previous HCV infection and those reporting risk factors. Lay summary: We report that hepatitis C virus infections are frequently acquired among HIV-negative men who have sex with men (MSM) using pre-exposure prophylaxis to prevent HIV infection. New infections occurred more frequently in those reporting receptive anal sex without using condoms, having an anal sexually transmitted infection, injecting drugs, and sharing straws when snorting drugs. The viruses found in HIV-negative men using pre-exposure prophylaxis are genetically similar to those in HIV-positive men, but not in other hepatitis C risk groups, suggesting that (sexual) transmission is occurring between HIV-positive MSM and HIV-negative MSM using pre-exposure prophylaxis. Clinical Trial number: Dutch trial registration number NTR5411.

DNA double-strand breaks (DSBs) can be repaired through various pathways. Understanding how these pathways are regulated is of great interest for cancer research and optimization of gene editing. The local chromatin environment can affect the balance between repair pathways, but this is still poorly understood. Here we provide a detailed protocol for DSB-TRIP, a technique that utilizes the specific DNA scars left by DSB repair pathways to study pathway usage throughout the genome. DSB-TRIP randomly integrates a repair reporter into many genomic locations, followed by the induction of DSBs in the reporter. Multiplexed sequencing of the resulting scars at all integration sites then reveals the balance between several repair pathways, which can be linked to the local chromatin state of the integration sites. Here we present a step-by-step protocol to perform DSB-TRIP in K562 cells and to analyse the data by a dedicated computational pipeline. We discuss strengths and limitations of the technique, as well as potential additional applications to study DNA repair.

Water resource recovery facilities (WRRFs) increasingly must maximize nitrogen and phosphorus removal, but concurrently face challenges to reduce their energy usage and environmental footprint. In particular, biological nutrient removal (BNR), which targets removal of phosphorus and nitrogen, exhibits a large energy demand. However, a BNR process achieving partial oxidation of NH

Background Doravirine is a NNRTI with demonstrated effect as third agent in treatment naive and experienced HIV patients. Aim To evaluate outcomes of cART experienced patients switching their therapy to DOR/TDF/3TC or doravirine based regimens, with focus on efficacy, tolerability and costs. Methods Baseline characteristics like treatment history, and laboratory values were retrospectively collected with follow up data at 6 months. If patients stopped therapy, reasons were evaluated. Baseline and follow-up data were compared in patients that continued therapy using IBM SPSS. Potential cost savings compared to prior cART were estimated for the cohort that continued DOR/TDF/3TC for follow-up period and extrapolated to the average per year. Results A total of 377 patients (91% men) were included that completed outpatient clinic visit or/and laboratory data at 6 months: 97.1% switched to Doravirine/tenofovir/lamivudine (DOR/TDF/3TC) and 2.9% to other doravirine based regimens. Doravirine was prematurely stopped in 58/377 patients (15.4%). Reason to stop was a patient-reported adverse event in 43/377 patients (11.4%); most frequently gastrointestinal (3.8%) or insomnia/sleep disturbances (3.4%). Other reasons were virologic failure (0,8%) with only 1 case demonstrating resistance (V106VA, V108VI), decreased renal function (0,8%), and ALAT grade 2 (0,5%). Within the group that continued doravirine grade 1 ALAT elevation was more frequently observed at 6 months compared to baseline (14.2% vs. 7.9% (p=0.004), n=318); no grade 2/3/4 increase was observed. A decrease in LDL-c was seen (3.50 (±1.10) mmol/L vs. 2.97 (±0.79) mmol/L; n=42, p Conclusion Doravirine is a suitable therapy for maintenance treatment and shows enormous savings. In addition, our data shows that it is also effective and well tolerated by 85% in our patient group.

Contains fulltext : 232443.pdf (Publisher’s version ) (Closed access) BACKGROUND: In the Netherlands, access to direct-acting antivirals (DAAs) against hepatitis C virus (HCV) has been unrestricted for chronic infection since 2015. We evaluated whether the nationwide incidence of HCV infections in individuals with HIV has changed since 2015. METHODS: In this retrospective cohort study, data from the ATHENA cohort of people with HIV aged 18 years or older attending any of the 24 HIV treatment centres in the Netherlands between 2000 and 2019 were assessed. We used parametric proportional hazards models with a piecewise exponential survival function to model HCV primary infection and reinfection incidence per 1000 person-years. FINDINGS: Of the 23 590 individuals without previous HCV infection, 1269 cases of HCV primary infection were documented (incidence 5·2 per 1000 person-years [95% CI 5·0-5·5]). The highest incidence was observed in men who have sex with men (MSM; 7·7 per 1000 person-years [7·3-8·2]) and was lower in people who inject drugs (PWID; 1·7 per 1000 person-years [0·7-4·1]) and other key populations (1·0 per 1000 person-years [0·8-1·2]). In MSM, incidence increased in 2007 to 14·3 per 1000 person-years and fluctuated between 8·7 and 13·0 per 1000 person-years from 2008 to 2015. In 2016, incidence declined to 6·1 cases per 1000 person-years and remained steady between 4·1 and 4·9 per 1000 person-years from 2017 to 2019. Of the 1866 individuals with a previous HCV infection, 274 reinfections were documented (incidence 26·9 per 1000 person-years [95% CI 23·9-30·3]). The highest incidence rate was observed in MSM (38·5 per 1000 person-years [33·9-43·7]) and was lower in PWID (10·9 per 1000 person-years [3·5-33·8]) and other key populations (8·9 per 1000 person-years [6·3-12·5]). In MSM, reinfection incidence fluctuated between 38·0 and 88·9 per 1000 person-years from 2006 to 2015, reaching 55·6 per 1000 person-years in 2015. In 2016, reinfection incidence declined to 41·4 per 1000 person-years, followed by further decreases to 24·4 per 1000 person-years in 2017 and 11·4 per 1000 person-years in 2019. INTERPRETATION: The sharp decline in HCV incidence in MSM with HIV shortly after restrictions on DAAs were lifted suggests a treatment-as-prevention effect. HCV incidence was already low in PWID and other groups before unrestricted access. Ongoing HCV transmission is occurring in MSM, as illustrated by a declining but high rate of reinfection, stressing the need for additional preventive measures. FUNDING: Dutch Ministry of Health, Welfare, and Sport.

Successful treatment of chronic hepatitis C with direct-acting antiviral agents (DAAs) is expected to lead to improvement in liver fibrosis in most of the patients. However, limited data are available on the improvement of advanced liver fibrosis and cirrhosis, measured by transient elastography after treatment. This study assessed the change in liver stiffness measurements after successful treatment with DAAs in patients with pre-treatment advanced fibrosis or cirrhosis.This observational retrospective cohort study included 514 mono-infected chronic hepatitis C patients, treated with all possible DAA-regimes in the Amsterdam region, the Netherlands. Liver stiffness was measured using FibroScan® at baseline and during follow-up. Cut-off values for staging liver fibrosis were ≥ 9.5 kPa for advanced fibrosis (F3) and ≥ 14.6 kPa for cirrhosis (F4).Liver stiffness decreased significantly from a median of 15.6 kPa (IQR 11.4-25.4) to 9.4 kPa (IQR 6.2-17.0) in 197 patients with pre-treated advanced fibrosis or cirrhosis. In 50.3% of these patients, liver stiffness improved to a value fitting with mild to moderate fibrosis (9.5 kPa, F0-F2) after successful treatment. Multivariate analysis demonstrated that a pre-treatment FibroScan® value of ≥ 20.0 kPa was associated with persisting advanced fibrosis or cirrhosis after treatment (OR 29.07, p0.001).Liver stiffness improves significantly after successful direct-acting antiviral agent treatment in chronic hepatitis C patients with advanced fibrosis or cirrhosis prior to DAA treatment. Long-term outcomes regarding occurrence of hepatocellular carcinoma (HCC) in these patients are required to determine whether they can be safely discharged from HCC surveillance.

Histoplasma capsulatum is an endemic fungus in especially tropical areas. While mostly asymptomatic, histoplasmosis can be life-threatening in immunocompromised patients.A 60-year-old woman of Suriname origin, with a history of renal transplantation and use of mycophenolate mofetil and prednisone, presented with abdominal pain and diarrhea. Colonoscopy revealed ulcerative ileocolitis and biopsy showed active granulomatous inflammation. Morbus Crohn was considered the most plausible diagnosis after ruling out several infectious and pharmacological causes. Despite prednisone treatment, symptoms persisted and infliximab was initiated. The patient developed constitutional symptoms and radiological examination revealed disseminated granulomatous disease. Liver biopsy and re-evaluation of previous intestinal histopathology confirmed suspected histoplasmosis.Histoplasmosis should be considered in immunocompromised patients with ileocolitis who have been in endemic regions (South America). Physicians need to assess the risk of previous exposure to histoplasmosis before starting anti-TNF-α therapy.

Highlights • After the global push for the use of Hydroxychloroquine and Chloroquine there is ongoing discussion about the effectivity of these drugs. • Findings of this observational study provide crucial data on a potential protective effect of Hydroxychloroquine in non-ICU, hospitalized COVID-19 patients. • Early treatment with HCQ on the first day of admission is associated with a reduced risk of 53% in transfer to the ICU for mechanical ventilation. • This protective effect was not observed for Chloroquine, therefore these drugs cannot be regarded as interchangeable., Background The global push for the use of hydroxychloroquine (HCQ) and chloroquine (CQ) against COVID-19 resulted in an ongoing discussion about the effectivity and toxicity of these drugs. Recent studies report no effect of (H)CQ on 28 day-mortality. We investigated the effect of HCQ and CQ in hospitalized patients on the non-ICU COVID-ward. Methods A nationwide, observational cohort study was performed in The Netherlands. Hospitals were given the opportunity to decide independently on the use of three different COVID-19 treatment strategies: HCQ or CQ, or no treatment. We compared the outcome between these groups. The primary outcomes were 1) death on the COVID-19 ward, and 2) transfer to the Intensive Care Unit (ICU). Results The analysis contained 1064 patients from 14 hospitals: 566 patients received treatment with either HCQ (n = 189) or CQ (n = 377), and 498 patients received no treatment. In a multivariate propensity matched weighted competing regression analysis, there was no significant effect of (H)CQ on mortality on the COVID-ward. HCQ however was associated with a significant decreased risk of transfer to the ICU (Hazard ratio (HR) = 0.47, 95%CI = 0.27–0.82, p = 0.008), when compared to controls. This effect was not found in the CQ group (HR = 0.80; 95%CI = 0.55–1.15, p = 0.207), and remained significant after competing risk analysis. Conclusion The results of this observational study demonstrate a lack of effect of (H)CQ on non-ICU mortality. However, we show that the use of HCQ - but not CQ - is associated with 53% decreased risk of transfer of COVID-19 patients from the regular ward to the ICU. Recent prospective studies have reported on 28 days all-cause mortality only, therefore additional prospective data on the early effect of HCQ in preventing transfer to the ICU is still needed.

DNA double-strand break (DSB) repair is mediated by multiple pathways, including classical non-homologous end-joining pathway (NHEJ) and several homology-driven repair pathways. This is particularly important for Cas9-mediated genome editing, where the outcome critically depends on the pathway that repairs the break. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a newly developed multiplexed reporter assay in combination with Cas9 cutting, we systematically measured the relative activities of three DSB repair pathways as function of chromatin context in >1,000 genomic locations. This revealed that NHEJ is broadly biased towards euchromatin, while microhomology-mediated end-joining (MMEJ) is more efficient in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 shifts the balance towards NHEJ. Single-strand templated repair (SSTR), often used for precise CRISPR editing, competes with MMEJ, and this competition is weakly associated with chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance, and guidance for the design of Cas9-mediated genome editing experiments.

Chen et al. present TSA-Seq, a new mapping method that measures cytological distances relative to spatially distinct nuclear subcompartments. From novel nuclear organization maps of human cells, they identify transcription hot zones of high gene density that are near nuclear speckles and enriched in highly expressed genes, housekeeping genes, and genes with low transcriptional pausing., While nuclear compartmentalization is an essential feature of three-dimensional genome organization, no genomic method exists for measuring chromosome distances to defined nuclear structures. In this study, we describe TSA-Seq, a new mapping method capable of providing a “cytological ruler” for estimating mean chromosomal distances from nuclear speckles genome-wide and for predicting several Mbp chromosome trajectories between nuclear compartments without sophisticated computational modeling. Ensemble-averaged results in K562 cells reveal a clear nuclear lamina to speckle axis correlated with a striking spatial gradient in genome activity. This gradient represents a convolution of multiple spatially separated nuclear domains including two types of transcription “hot zones.” Transcription hot zones protruding furthest into the nuclear interior and positioning deterministically very close to nuclear speckles have higher numbers of total genes, the most highly expressed genes, housekeeping genes, genes with low transcriptional pausing, and super-enhancers. Our results demonstrate the capability of TSA-Seq for genome-wide mapping of nuclear structure and suggest a new model for spatial organization of transcription and gene expression., Graphical Abstract

Enhanced biological phosphorus removal (EBPR) is an engineered water resource recovery facility (WRRF) process configuration that can produce effluent P < 0.5 mg/L. To consistently achieve low effluent P concentrations, EBPR requires volatile fatty acids (VFAs) to induce requisite biochemical reactions. Moreover, returned activated sludge (RAS) nitrate concentrations must be minimized. Returned activated sludge fermentation can potentially address process needs. However, research detailed herein highlights concerns with RAS fermentation integrated with EBPR. Under 2 and 4 hours of RAS fermentation periods, no consequential VFA production was observed; similar results were observed in batch tests with RAS from a full-scale EBPR WRRF. More critically, EBPR performance was poor, with average effluent concentrations of 1.0 to 2.4 mg/L. Furthermore, the glycogen accumulating organism (GAO) fraction under RAS fermentation was 4.3 to 8.7 times higher than in a conventional EBPR mixed microbial consortium (MMC). Integrated RAS fermentation-EBPR only performed well under "high" RAS nitrate; thus, should RAS fermentation be implemented, careful control to prevent anaerobic conditions in the fermentation zone is required.

// Ann E. Zeleniak 1, 2 , Wei Huang 2, 3 , Mary K. Brinkman 2, 3 , Melissa L. Fishel 4, 5, 6 , Reginald Hill 2, 3 1 Integrated Biomedical Sciences Program, University of Notre Dame, South Bend, Indiana, USA 2 Harper Cancer Research Institute, University of Notre Dame, South Bend, Indiana, USA 3 Department of Biological Sciences, University of Notre Dame, South Bend, Indiana, USA 4 Indiana University School of Medicine, Department of Pharmacology and Toxicology, Indianapolis, Indiana, USA 5 Indiana University School of Medicine, Department of Pediatrics, Wells Center for Pediatric Research, Indianapolis, Indiana, USA 6 Pancreatic Cancer Signature Center, Indianapolis, Indiana, USA Correspondence to: Reginald Hill, email: rhill3@nd.edu Keywords: pancreatic cancer, metastasis, MTSS1, inflammation, tumor microenvironment Received: September 18, 2016 Accepted: January 19, 2017 Published: January 27, 2017 ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of 7%. This dismal prognosis is largely due to the inability to diagnose the disease before metastasis occurs. Tumor cell dissemination occurs early in PDAC. While it is known that inflammation facilitates this process, the underlying mechanisms responsible for this progression have not been fully characterized. Here, we functionally test the role of metastasis suppressor 1 (MTSS1) in PDAC. Despite evidence showing that MTSS1 could be important for regulating metastasis in many different cancers, its function in PDAC has not been studied. Here, we show that loss of MTSS1 leads to increased invasion and migration in PDAC cell lines. Moreover, PDAC cells treated with cancer-associated fibroblast-conditioned media also have increased metastatic potential, which is augmented by loss of MTSS1. Finally, overexpression of MTSS1 in PDAC cell lines leads to a loss of migratory potential in vitro and an increase in overall survival in vivo . Collectively, our data provide insight into an important role for MTSS1 in suppressing tumor cell invasion and migration driven by the tumor microenvironment and suggest that therapeutic strategies aimed at increasing MTSS1 levels may effectively slow the development of metastatic lesions, increasing survival of patients with PDAC.

The anthropogenic discharge of phosphorus (P) into surface waters can induce the proliferation of cyanobacteria and algae, which can negatively impact water quality. Enhanced biological P removal (EBPR) is an engineered process that can be employed to efficiently remove significant quantities of P from wastewater. Within this engineered system, the mixed microbial consortium (MMC) becomes enriched with polyphosphate accumulating organisms (PAOs). To date much knowledge has been developed on PAOs, and the EBPR process is generally well understood; nonetheless, the engineered process remains underutilized. In this study, investigations were conducted using qPCR and Illumina MiSeq to assess the impacts of wastewater (synthetic vs. real) on EBPR microbial ecology. While a strong relationship was demonstrated between EBPR metrics (P:C; influent VFA:P) and excellent P removal across diverse EBPR systems and MMCs, no such correlations existed with the specific MMCs. Moreover, MMCs exhibited distinct clusters based on substrate, and qPCR results based on the putative PAO Accumulibacter did not correlate with BLASTN eubacterial results for either Accumulibacter or Rhodocyclaceae. More critically, PAO-based sequences aligned poorly with Accumulibacter for both eubacterial and PAO primer sets, which strongly suggests that the conventional PAO primers applied in FISH and qPCR analysis do not sufficiently target the putative PAO Accumulibacter. In particular, negligible alignment was observed for PAO amplicons obtained from a MMC performing excellent EBPR on crude glycerol (an atypical substrate). A synthetic wastewater-based MMC exhibited the best observed BLASTN match of the PAO amplicons, raising concerns about the potential relevance in using synthetic substrates in the study of EBPR.

Polyhydroxyalkanoates (PHAs) are biodegradable polymers that can substitute for petroleum-based plastics in a variety of applications. One avenue to commercial PHA production involves coupling waste-based synthesis with the use of mixed microbial consortia (MMC). In this regard, production requires maximizing the enrichment of a MMC capable of feast-famine PHA synthesis, with the metabolic response induced through imposition of aerobic-dynamic feeding (ADF) conditions. However, the concept of PHA production in complex matrices remains unrefined; process operational improvements are needed, along with an enhanced understanding of the MMC. Research presented herein investigated the effect of aeration on feast-famine PHA synthesis, with four independent aeration state systems studied; MMC were fed volatile fatty acid (VFA)-rich fermented dairy manure. Regardless of the aeration state, all MMC exhibited a feast-famine response based on observed carbon cycling. Moreover, there was no statistical difference in PHA synthesis rates, with qPHA ranging from 0.10 to 0.19 CmmolPHA gVSS−1 min−1; VFA uptake rates exhibited similar statistical indifferences. PHA production assessments on the enriched MMC resulted in maximum intracellular concentrations ranging from 22.5 to 90.7% (mgPHA mgVSS−1); at maximum concentration, the mean hydroxyvalerate mol content was 73 ± 0.6%. While a typical feast-famine dissolved oxygen (DO) pattern was observed at maximum aeration, less resolution was observed at decreasing aeration rates, suggesting that DO may not be an optimal process monitoring parameter. At lower aeration states, nitrogen cycling patterns, supported by molecular investigations targeting AOBs and NOBs, indicate that NO2 and NO3 sustained feast-famine PHA synthesis. Next-generation sequencing analysis of the respective MMC revealed numerous and diverse genera exhibiting the potential to achieve PHA synthesis, suggesting functional redundancy embedded in the diverse MMC. Ultimately, results demonstrate that aeration can be controlled in waste-based ADF systems to sustain PHA production potential, while enriching for a diverse MMC that exhibits potential functional redundancy. Reduced aeration could also enhance cost competitiveness of waste-based PHA production, with potential further benefits associated with nitrogen treatment.

Background Obstetric guidelines are useful to improve the quality of care. Availability of international guidelines has rapidly increased, however the contextualization to enhance feasibility of implementation in health facilities in low and middle-income settings has only been described in literature in a few instances. This study describes the approach and lessons learned from the ‘bottom-up’ development process of context-tailored national obstetric guidelines in middle-income country Suriname. Methods Local obstetric health care providers initiated the guideline development process in Suriname in August 2016 for two common obstetric conditions: hypertensive disorders of pregnancy (HDP) and post partum haemorrhage (PPH). Results The process consisted of six steps: (1) determination of how and why women died, (2) interviews and observations of local clinical practice, (3) review of international guidelines, (4) development of a primary set of guidelines, (5) initiation of a national discussion on the guidelines content and (6) establishment of the final guidelines based on consensus. Maternal enquiry of HDP- and PPH-related maternal deaths revealed substandard care in 90 and 95% of cases, respectively. An assessment of the management through interviews and labour observations identified gaps in quality of the provided care and large discrepancies in the management of HDP and PPH between the hospitals. International recommendations were considered unfeasible and were inconsistent when compared to each other. Local health care providers and stakeholders convened to create national context-tailored guidelines based on adapted international recommendations. The guidelines were developed within four months and locally implemented. Conclusion Development of national context-tailored guidelines is achievable in a middle-income country when using a ‘bottom-up’ approach that involves all obstetric health care providers and stakeholders in the earliest phase. We hope the descriptive process of guideline development is helpful for other countries in need of nationwide guidelines.

Current genome editing tools enable targeted mutagenesis of selected DNA sequences in many species. However, the efficiency and the type of introduced mutations by the genome editing method are largely dependent on the target site. As a consequence, the outcome of the editing operation is difficult to predict. Therefore, a quick assay to quantify the frequency of mutations is vital for a proper assessment of genome editing actions. We developed two methods that are rapid, cost-effective, and readily applicable: (1) TIDE, which can accurately identify and quantify insertions and deletions (indels) that arise after introduction of double strand breaks (DSBs); (2) TIDER, which is suited for template-mediated editing events including point mutations. Both methods only require a set of PCR reactions and standard Sanger sequencing runs. The sequence traces are analyzed by the TIDE or TIDER algorithm (available at https://tide.nki.nl or https://deskgen.com ). The routine is easy, fast, and provides much more detailed information than current enzyme-based assays. TIDE and TIDER accelerate testing and designing of DSB-based genome editing strategies.

Summary DNA double-strand break (DSB) repair is mediated by multiple pathways. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a multiplexed reporter assay in combination with Cas9 cutting, we systematically measure the relative activities of three DSB repair pathways as a function of chromatin context in >1,000 genomic locations. This reveals that non-homologous end-joining (NHEJ) is broadly biased toward euchromatin, while the contribution of microhomology-mediated end-joining (MMEJ) is higher in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 reverts the balance toward NHEJ. Single-stranded template repair (SSTR), often used for precise CRISPR editing, competes with MMEJ and is moderately linked to chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance and guidance for the design of Cas9-mediated genome editing experiments., Graphical abstract, Highlights • Sequencing-based reporter detects activities of multiple DSB repair pathways • Multiplexing approach to integrate reporter in >1,000 genomic locations • Overlay with epigenome data reveals effects of chromatin context on pathway balance • Pathway balance differs between heterochromatin and euchromatin, Schep et al. designed a reporter to probe the activities of three DNA double-strand break repair pathways. Integration of this reporter in >1,000 genomic locations in a human cell line provided a detailed view of the impact of local chromatin context on the balance between the repair pathways.

Polyhydroxyalkanoate (PHA) production from waste feedstocks using mixed microbial consortia (MMCs) is a promising alternative to conventional commercial production from refined feedstocks with axenic cultures. Prior work has reinforced this potential with nutrient-limited waste feedstocks and stringently controlled operation. The process would be more competitive, however, without these restrictions. To that end, a pilot-scale three-stage feast-famine MMC PHA system using dairy manure was operated with relaxed control for 87 days. All three stages absorbed operational fluctuations and exhibited stable performance comparable to tightly controlled analogs. PHA mass fractions (0.29 to 0.40 g/g; volatile solids basis) approached commercial viability and the purified PHA (21 ± 3% 3-hydroxyvalerate; mole basis) exhibited similarly stable material properties. Finally, characterization of the MMC's PHA synthesizing capability suggested an innate potential for medium-chain-length monomers. Collectively, these results demonstrate the potential for PHA production from a feedstock without a strict nutrient limitation and the process's resilience against operational variability.

Dairy manure management is increasingly becoming an environmental challenge. In this regard, manure anaerobic digestion (AD) can be applied to address environmental concerns; however, dairy manure AD remains economically uncompetitive. Ongoing research is focused on enhanced resource recovery from manure, including maximizing AD methane yield through a novel multi-stage AD configuration. Research presented herein centered on the hypothesis that separately digesting fine and coarse solids from fermented dairy manure would improve methane production; the hypothesis was disproven. While maximum methane concentration was realized on fine solids, combined solids AD yielded enhanced VS destruction. The diverse combined-solids substrate enriched for a more heterogeneous bacterial/archaeal consortium that balanced fermentation and methanogenesis to yield maximum product (methane). However, results suggest that targeted AD of the fat-rich fine solids could be a more optimal approach for processing manure; alternate (non-AD) methods could then be applied to extract value from the fibrous fraction.

SummaryWhile nuclear compartmentalization is an essential feature of three-dimensional genome organization, no genomic method exists for measuring chromosome distances to defined nuclear structures. Here we describe TSA-Seq, a new mapping method able to estimate mean chromosomal distances from nuclear speckles genome-wide and predict several Mbp chromosome trajectories between nuclear compartments without sophisticated computational modeling. Ensemble-averaged results reveal a clear nuclear lamina to speckle axis correlated with a striking spatial gradient in genome activity. This gradient represents a convolution of multiple, spatially separated nuclear domains, including two types of transcription “hot-zones”. Transcription hot-zones protruding furthest into the nuclear interior and positioning deterministically very close to nuclear speckles have higher numbers of total genes, the most highly expressed genes, house-keeping genes, genes with low transcriptional pausing, and super-enhancers. Our results demonstrate the capability of TSA-Seq for genome-wide mapping of nuclear structure and suggest a new model for nuclear spatial organization of transcription.

Summary The RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals variable and often slow repair rates, with half-life times up to ∼10 hr. Furthermore, repair of the DSBs tends to be error prone. Both classical and microhomology-mediated end joining pathways contribute to the erroneous repair. Estimation of their individual rate constants indicates that the balance between these two pathways changes over time and can be altered by additional ionizing radiation. Our approach provides quantitative insights into DSB repair kinetics and fidelity in single loci and indicates that Cas9-induced DSBs are repaired in an unusual manner., Graphical Abstract, Highlights • Approach to measure single-locus DSB repair kinetics after Cas9-induced breaks • Multiple repair pathways can act on a single locus with distinct kinetics • Repair tends to be slow and error prone, although this depends on the locus, Brinkman et al. report a strategy to determine the rate and fidelity of double-strand break repair at single loci cut by Cas9. They also infer the contributions from different repair pathways. Cas9-induced breaks are repaired at variable but often slow rates, and the repair tends to be error prone.

Template-directed CRISPR/Cas9 editing is a powerful tool for introducing subtle mutations in genomes. However, the success rate of incorporation of the desired mutations at the target site is difficult to predict and therefore must be empirically determined. Here, we adapted the widely used TIDE method for quantification of templated editing events, including point mutations. The resulting TIDER method is a rapid, cheap and accessible tool for testing and optimization of template-directed genome editing strategies. A free web tool for TIDER data analysis is available at http://tide.nki.nl.

BACKGROUND In 2012, 9.3 million head of dairy cows in the USA produced an estimated 20 million metric tons of manure solids, but little value was gained from this manure. There is a pressing need to enhance manure resource recovery efforts, as dairy manure has potentially significant environmental impacts. This study evaluated components of an integrated suite of biological processes designed to maximize resource recovery from dairy manure, in which algae grown on polyhydroxyalkanoate (PHA) production effluent (PHA-algae) were fermented and anaerobically digested to determine process impacts. RESULTS A 10% PHA-algae supplement produced 11% more volatile fatty acids (VFA) during fermentation and 11% more methane during anaerobic digestion (AD) (vs. dairy manure); the PHA-algae biogas also contained a higher percentage (62.7 vs. 59.1%) of methane than manure biogas. Algal augmentation exhibited no negative effect on fermenter or AD operation. Quantitative polymerase chain reaction (PCR) showed that the ADs contained substantial populations of both acetoclastic and hydrogenotrophic methanogens, which, given the heterogeneous substrate, enhanced process stability. There were significant differences between PHA-algae batches, and large quantities of COD were released during algae freezing. CONCLUSION PHA-algae yielded more VFA during fermentation, and a more methane-rich biogas following AD than dairy manure. A 10% PHA-algae supplement caused no process disturbance in normal manure flora. © 2015 Society of Chemical Industry

SummaryThe RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals a relatively slow repair rate (~6h). Furthermore, the double strand break is predominantly repaired in an error-prone fashion (at least 70%). Both classical and microhomology-mediated end-joining pathways are active and contribute to the repair in a stochastic manner. However, the balance between these two pathways changes over time and can be altered by chemical inhibition of DNAPKcs or additional ionizing radiation. Our strategy is generally applicable to study DSB repair kinetics and fidelity in single loci, and demonstrates that Cas9-induced DSBs are repaired in an unusual manner.

In response to UV light, single-stranded DNA intermediates coated with replication protein A (RPA) are generated, which trigger the ATR-Chk1 checkpoint pathway. Recruitment and/or activation of several checkpoint proteins at the damaged sites is important for the subsequent cell cycle arrest. Surprisingly, upon UV irradiation, Rad9 and RPA only minimally accumulate at DNA lesions in G2 phase, suggesting that only a few single-stranded DNA intermediates are generated. Also, little phosphorylated Chk1 is observed in G2 phase after UV-irradiation, and UV light fails to elicit efficient accumulation of typical DNA damage response proteins at sites of damage in this phase. By contrast, p38 MAPK is phosphorylated in G2 phase cells after UV damage. Interestingly, despite the lack of an obvious activation of the ATR-Chk1 pathway, only the combined inhibition of the ATR- and p38-dependent pathways results in a complete abrogation of the UV-induced G2/M arrest. This suggests that UV light induces less hazardous lesions in G2 phase or that lesions created in this phase are less efficiently processed, resulting in a low activation of the ATR-Chk1 pathway. UV-induced G2 checkpoint activation in this situation therefore relies on signalling via the p38 MAPK and ATR-Chk1 signalling cascades.

Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context.Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio–temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.

The purpose of this research was to develop a better understanding of the dynamic effects of anaerobic hydraulic retention time (HRT) on both enhanced biological phosphorus removal (EBPR) performance and enrichment of phosphorus accumulating organisms (PAOs). The research was conducted using laboratory-scale sequencing batch reactors inoculated with mixed microbial consortia and fed real wastewater. Exposing microorganisms to extended anaerobic HRTs is not recommended for EBPR configured systems. In this research, however, longer anaerobic exposure did not negatively affect performance even if volatile fatty acids were depleted. Further, extended anaerobic HRTs may positively affect phosphorus removal through enhanced aerobic uptake. The EBPR consortia also appear to maintain reserve energetic capacity in the form of polyphosphate that can be used to survive and grow under variable operational and environmental conditions. Finally, the tested EBPR systems yield mixed microbial consortia enriched with PAOs (specifically Candidatus Accumulibacter phosphatis) at approximately 7.1 to 21.6% of the total population.

Recent publications have reported superior efficacy of telaprevir- or boceprevir-based triple therapy over conventional peginterferon-alfa/ribavirin therapy, albeit with varying rates of adverse events and treatment discontinuations in HIV/HCV coinfected patients. Therefore, the aim of this study is to describe the effectiveness of triple therapy in an HIV/HCV coinfection cohort in the Netherlands.HIV-infected patients with chronic HCV genotype 1 starting triple therapy including either boceprevir or telaprevir were enrolled, 26% had F3-F4 fibrosis. Data were assessed at Week 4, 8, 12, 24, 48 and SVR12 (i.e. absence of detectable plasma HCV RNA 12 weeks after completion of treatment). Failure was defined as discontinuation of treatment due to virological failure, adverse events or loss to follow-up.A total of 53 HIV/HCV coinfected patients started peginterferon-alfa/ribavirin therapy with either boceprevir (n = 29) or telaprevir (n = 24). SVR12 was achieved in 19 (66%) of the boceprevir-treated and 15 (63%) of the telaprevir-treated patients. Both prior relapse and achievement of a rapid virological response were associated with a higher SVR12 rate. Non- response, breakthrough and relapse occurred in 4, 1 and 5 patients on boceprevir and 3, 2, 2 on telaprevir, respectively. One patient was lost to follow-up and one patient died due to progression of liver failure. Except for these two patients, no treatment discontinuations were observed due to adverse events.In HIV/HCV coinfected patients, boceprevir or telaprevir triple therapy was well tolerated and resulted in favourable SVR12 rates comparable with previous publications concerning HCV mono-infected patients.

Enhanced biological phosphorus removal (EBPR) is predicated on exposing bacteria to cyclical anaerobic/aerobic environments while providing volatile fatty acids (VFAs). Combined, this environment enriches for phosphorus accumulating organisms (PAOs) and induces metabolisms to ensure excess phosphorus removal. Crude glycerol (CG), a byproduct of biodiesel manufacturing, is an alternate waste stream that could be substituted to achieve excess phosphorus removal; research into the use of CG yielded unexpected findings. While CG was an excellent substrate to accomplish and/or help achieve excess phosphorus removal, CG-fed bacteria did not consistently exhibit theoretical EBPR metabolisms. Specifically, anaerobic phosphorus release was not required for successful EBPR; however, carbon cycling patterns were consistent with theory. Analysis of results suggests that PAOs will first leverage carbon to generate energy anaerobically; only as needed will the bacteria utilize polyphosphate reserves anaerobically. Results also demonstrated that excess phosphorus removal can be achieved with a small fraction of PAOs.

Conventional anaerobic digesters (ADs) treating dairy manure are fed with raw or fermented manure rich in volatile fatty acids (VFAs). In contrast, pre-fermented AD (PF-AD) is fed with the more recalcitrant, fiber-rich fraction of manure that has been pre-fermented and depleted of VFAs. Thus, the substrate of PF-AD may be likened to a lean diet rich in fibers while the pre-fermentation stage fermenter is fed a relatively rich diet containing labile organic substances. Previous results have shown that conventional and pre-fermented ADs fed with raw or pre-fermented manure, respectively, produced comparable methane yields. The primary objective of this study was to characterize, using next-generation DNA sequencing, the bacterial communities in various bioreactors (pre-fermentation stage fermenter; various operational arrangements PF-AD; conventional single-stage AD; and a full scale AD) and compare the Firmicutes to Bacteroidetes (F/B) ratios in these different systems. Firmicutes and Bacteroidetes constituted the two most abundant phyla in all AD samples analyzed, as well as most of the samples analyzed in the fermenters and manure samples. Higher relative abundance of Bacteroidetes, ranging from 26% to 51% of bacteria, tended to be associated with PF-AD samples, while the highest relative abundance of Firmicutes occurred in the fermenter (maximum of 76% of bacteria) and manure (maximum of 66% of bacteria) samples. On average, primary stage fermenters exhibited microbiological traits linked to obesity: higher F/B ratios and a ‘diet’ that is less fibrous and more labile compared to that fed to PF-AD. On the other hand, microbial characteristics associated with leanness (lower F/B ratios combined with fibrous substrate) were associated with PF-AD. We propose that bacterial communities in AD shift depending on the quality of substrate, which ultimately results in maintaining VFA yields in PF-AD, similar to the role of bacterial communities and a high fiber diet in lean mice.

A 67-year-old man was treated with maintenance intravesical BCG for superficial bladder cancer. As a culture-proven complication of this therapy, he developed general malaise, high fever, granulomatous hepatitis and a mycotic aneurysm in his left knee. All complications were treated successfully with antituberculous therapy. No vascular surgery was necessary. This case report again stresses the necessity to recognise complications of BCG treatment and to start adequate therapy as soon as possible.

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments. A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native β-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed. To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources. The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFDα and β, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFDα and β improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed. Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

Herpes Simplex Virus type 1 (HSV-1) establishes a latent infection in the sensory neurons of the peripheral nervous system of humans. Although about 80 genes are expressed during the lytic cycle of the virus infection, essentially only one gene is expressed during the latent cycle. This gene is known as the latency associated transcript (LAT) and it appears to play a role in the latency cycle through an anti-apoptotic function in the 5’ end of the gene and miRNA encoded along the length of the transcript which down regulate some of the viral immediate early (IE) gene products. The LAT gene is about 8.3 kb long and consists of two exons separated by an unusual intron. The intron between the exons consists of two nested introns. This arrangement of introns has been called a twintron. Furthermore, the larger (2 kb) intron has been shown to be very stable. In this study we measure the stability of the shorter 1.5 kb nested intron and find its half-life is similar to the longer intron. This was achieved by deleting the 0.5 kb overlapping intron from a plasmid construct designed to express the LAT transcript from a tet-inducible promoter, and measuring the half-life of the 1.5 kb intron in tissue culture cells. This finding supports the hypothesis that it is the common branch-point region of these nested introns that is responsible for their stability.

Over 9 million dairy cows generate an estimated 226 billion kg of wet manure annually in the US. The purpose of this study was to demonstrate the viability of a novel two-stage anaerobic digestion (AD) process for producing methane-rich biogas on pre-fermented dairy manure. In summary, it was observed that AD of thickened pre-fermented manure can generate comparable biogas quantities to AD using raw manure, with enhanced methane content. Despite receiving a lower quality (i.e., partially biodegraded) substrate, biogas stoichiometry and overall process stability in the two-stage system was also comparable to AD receiving raw manure. Finally, the two-stage AD was more enriched with the acetoclastic methanogen Methanosarcinaceae (Msc; compared to AD of raw manure) and biogas production appeared closely linked with the Msc fraction. In fact, the enhanced enrichment of Msc likely contributed to the successful and stable operations.

The objective of this research was to advance a fundamental understanding of a unique post-anoxic denitrification process for achieving biological nutrient removal (BNR), with an emphasis on elucidating the impacts of surface oxygen transfer (SOT), variable process loadings, and bioreactor operational conditions on nitrogen and phosphorus removal. Two sequencing batch reactors (SBRs) were operated in an anaerobic/aerobic/anoxic mode for over 250 days and fed real municipal wastewater. One SBR was operated with a headspace open to the atmosphere, while the other had a covered liquid surface to prevent surface oxygen transfer. Process performance was assessed for mixed volatile fatty acid (VFA) and acetate-dominated substrate, as well as daily/seasonal variance in influent phosphorus and ammonia loadings. Results demonstrated that post-anoxic BNR can achieve near-complete (>99%) inorganic nitrogen and phosphorus removal, with soluble effluent concentrations less than 1.0 mgN L−1 and 0.14 mgP L−1. Observed specific denitrification rates were in excess of typical endogenous values and exhibited a linear dependence on the glycogen concentration in the biomass. Preventing SOT improved nitrogen removal but had little impact on phosphorus removal under normal loading conditions. However, during periods of low influent ammonia, the covered reactor maintained phosphorus removal performance and showed a greater relative abundance of polyphosphate accumulating organisms (PAOs) as evidenced by quantitative real-time PCR (qPCR). While GAOs were detected in both reactors under all operational conditions, BNR performance was not adversely impacted. Finally, secondary phosphorus release during the post-anoxic period was minimal and only occurred if nitrate/nitrite were depleted post-anoxically.

The long-term toxicity of highly active antiretroviral therapy (HAART), especially the development of both the lipodystrophy syndrome and hyperlipidemia, has been linked to the use of HIV protease inhibitors in most studies. However, the broad spectrum of toxicities associated with one of the other classes of drugs, the nucleoside analogue reverse transcriptase inhibitors (NRTIs), can display complications almost as serious and with more fatal outcomes. The common pathway of NRTI toxicities is induction of mitochondrial dysfunction [1, 2], because NRTIs inhibit DNA polymerase-y, an essential enzyme for the replication of mitochondrial DNA (mtDNA). Depletion of mtDNA will follow, successively leading to depletion of mtDNA-encoded proteins and to mitochondrial dysfunction. All long-term side effects of NRTIs that have been described can be attributed to this mitochondrial toxicity, and recently we hypothesized that the HAART-related lipodystrophy is induced via this mechanism as well [3].

Antibodies to DNA are quite specific for systemic lupus erythematosus (SLE) and occur in the majority of SLE patients. Therefore, their detection is an important diagnostic aid to the clinician. Detection of anti-dsDNA may precede the diagnosis of SLE by more than a year. Fluctuations in the level of anti-dsDNA in an individual patient may give important information on the clinical status of the patient. Four of the most important methods developed for the measurement of anti-dsDNA antibodies will be discussed in this paper: the Farr assay, the PEG assay, the indirect immunofluorescence test on Crithidia luciliae and the ELISA. They will also be compared with one commercially available (Farr) assay, the Amersham anti-dsDNA kit. Each method, detects a part of the spectrum of anti-dsDNA antibodies produced by a patient. The Farr assay is the most specific for SLE; however, milder forms of the disease in which patients have only low avidity anti-dsDNA may easily be missed by this technique. Clinically, high avidity anti-dsDNA is related more frequently to the occurrence of nephritis, whereas low avidity anti-dsDNA antibodies are found more often in patients with central nervous system involvement. Traditionally, SLE is considered an immune-complex disease, in which inflammatory processes are initiated by local deposition of DNA/anti-dsDNA complexes. More recently, a major role was thought to be played by crossreactions of anti-dsDNA with tissue constituents. Our current view, however, is that such a crossreactivity plays only a minor role; we postulate that binding to glomerular constituents is caused by anti-dsDNA antibodies complexed with DNA and histones.