Qualitative Profiling and Quantification of Neonicotinoid Metabolites in Human Urine by Liquid Chromatography Coupled with Mass Spectrometry

Neonicotinoid pesticides have been widely applied for the production of fruits and vegetables, and occasionally detected in conventionally grown produce. Thus oral exposure to neonicotinoid pesticides may exist in the general population; however, neonicotinoid metabolites in human body fluids have not been investigated comprehensively. The purpose of this study is the qualitative profiling and quantitative analysis of neonicotinoid metabolites in the human spot urine by liquid chromatography coupled with mass spectrometry (LC/MS). Human urine samples were collected from three patients suspected of subacute exposure to neonicotinoid pesticides. A qualitative profiling of urinary metabolites was performed using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) with a database of nominal molecular weights of 57 known metabolites of three neonicotinoid pesticides (acetamiprid, Imidacloprid, and clothianidin), as well as the parent compounds. Then a quantitative analysis of selected urinary metabolites was performed using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a standard pesticide and metabolite, which were detected by the qualitative profiling. The result of qualitative profiling showed that seven metabolites, i.e. an acetamiprid metabolite, N-desmethyl-acetamiprid; three Imidacloprid metabolites, 5-hydroxy-Imidacloprid, 4,5-dihydroxy-imidacloprid, 4,5-dehydro-Imidacloprid; a common metabolite of acetamiprid and Imidacloprid, N-(6-chloronicotinoyl)-glycine; and two clothianidin metabolites, N-desmethyl-clothianidin, N-(2-(methylsulfanyl)thiazole-5-carboxyl)-glycine, as well as acetamiprid, were detected in the urine of three cases. The result of the quantitative analysis showed N-desmethyl-acetamiprid was determined in the urine of one case, which had been collected on the first visit, at a concentration of 3.2 ng/mL. This is the first report on the qualitative and quantitative detection of N-desmethyl-acetamiprid in the human urine. The results suggest that the one case with detection of N-desmethyl-acetamiprid was exposed to acetamiprid through the consumption of contaminated foods. Urinary N-desmethyl-acetamiprid, as well as 5-hydroxy-Imidacloprid and N-desmethyl-clothianidin, may be a good biomarker for neonicotinoid exposure in humans and warrants further investigation.     

Microbial Degradation of Acetamiprid by Ochrobactrum sp. D-12 Isolated from Contaminated Soil

Neonicotinoid insecticides are one of the most important commercial insecticides used worldwide. The potential toxicity of the residues present in environment to humans has received considerable attention. In this study, a novel Ochrobactrum sp. strain D-12 capable of using acetamiprid as the sole carbon source as well as energy, nitrogen source for growth was isolated and identified from polluted agricultural soil. Strain D-12 was able to completely degrade acetamiprid with initial concentrations of 0–3000 mg·L⁻¹ within 48 h. Haldane inhibition model was used to fit the special degradation rate at different initial concentrations, and the parameters q _max, K _s and K _i were determined to be 0.6394 (6 h)⁻¹, 50.96 mg·L⁻¹ and 1879 mg·L⁻¹, respectively. The strain was found highly effective in degrading acetamiprid over a wide range of temperatures (25–35°C) and pH (6–8). The effects of co-substrates on the degradation efficiency of acetamiprid were investigated. The results indicated that exogenously supplied glucose and ammonium chloride could slightly enhance the biodegradation efficiency, but even more addition of glucose or ammonium chloride delayed the biodegradation. In addition, one metabolic intermediate identified as N-methyl-(6-chloro-3-pyridyl)methylamine formed during the degradation of acetamiprid mediated by strain D-12 was captured by LC-MS, allowing a degradation pathway for acetamiprid to be proposed. This study suggests the bacterium could be a promising candidate for remediation of environments affected by acetamiprid.     

Prevalence of Symptomatic Heart Failure with Reduced and with Normal Ejection Fraction in an Elderly General Population–The CARLA Study

Background/Objectives

Chronic heart failure (CHF) is one of the most important public health concerns in the industrialized world having increasing incidence and prevalence. Although there are several studies describing the prevalence of heart failure with reduced ejection fraction (HFREF) and heart failure with normal ejection fraction (HFNEF) in selected populations, there are few data regarding the prevalence and the determinants of symptomatic heart failure in the general population.

Methods

Cross-sectional data of a population-based German sample (1,779 subjects aged 45–83 years) were analyzed to determine the prevalence and determinants of chronic SHF and HFNEF defined according to the European Society of Cardiology using symptoms, echocardiography and serum NT-proBNP. Prevalence was age-standardized to the German population as of December 31st, 2005.

Results

The overall age-standardized prevalence of symptomatic CHF was 7.7% (95%CI 6.0–9.8) for men and 9.0% (95%CI 7.0–11.5) for women. The prevalence of CHF strongly increased with age from 3.0% among 45–54- year-old subjects to 22.0% among 75–83- year-old subjects. Symptomatic HFREF could be shown in 48% (n = 78), symptomatic HFNEF in 52% (n = 85) of subjects with CHF. The age-standardized prevalence of HFREF was 3.8 % (95%CI 2.4–5.8) for women and 4.6 % (95%CI 3.6–6.3) for men. The age-standardized prevalence of HFNEF for women and men was 5.1 % (95%CI 3.8–7.0) and 3.0 % (95%CI 2.1–4.5), respectively. Persons with CHF were more likely to have hypertension (PR = 3.4; 95%CI 1.6–7.3) or to have had a previous myocardial infarction (PR = 2.5, 95%CI 1.8–3.5).

Conclusion

The prevalence of symptomatic CHF appears high in this population compared with other studies. While more women were affected by HFNEF than men, more male subjects suffered from HFREF. The high prevalence of symptomatic CHF seems likely to be mainly due to the high prevalence of cardiovascular risk factors in this population.     

Diagnosis of Bladder Cancer Recurrence Based on Urinary Levels of EOMES,HOXA9, POU4F2, TWIST1, VIM,and ZNF154 Hypermethylation

Background

Non muscle invasive bladder cancer (NMIBC) has the highest recurrence rate of any malignancy and as many as 70% of patients experience relapse. Aberrant DNA methylation is present in all bladder tumors and can be detected in urine specimens. Previous studies have identified DNA methylation markers that showed significant diagnostic value. We evaluated the significance of the biomarkers for early detection of tumor recurrence in urine.

Methodology/Principal Findings

The methylation levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 in urine specimens were measured by real-time PCR (MethyLight). We analyzed 390 urine sediments from 184 patients diagnosed with NMIBC. Urine from 35 age-matched control individuals was used to determine the methylation baseline levels. Recurrence was diagnosed by cystoscopy and verified by histology. Initially, we compared urine from bladder cancer patients and healthy individuals and detected significant hypermethylation of all six markers (P<0.0001) achieving sensitivity in the range 82%–89% and specificity in the range 94%–100%. Following, we validated the urinary hypermethylation for use in recurrence surveillance and found sensitivities of 88–94% and specificities of 43–67%. EOMES, POU4F2, VIM and ZNF154 were more frequently methylated in urine from patients with higher grade tumors (P≤0.08). Univariate Cox regression analysis showed that five markers were significantly associated with disease recurrence; HOXA9 (HR = 7.8, P = 0.006), POU4F2 (HR = 8.5, P = 0.001), TWIST1 (HR = 12.0, P = 0.015), VIM (HR = 8.0, P = 0.001), and ZNF154 (HR = 13.9, P<0.001). Interestingly, for one group of patients (n = 15) we found that hypermethylation was consistently present in the urine samples despite the lack of tumor recurrences, indicating the presence of a field defect.

Conclusion/Significance

Methylation levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 in urine specimens are promising diagnostic biomarkers for bladder cancer recurrence surveillance.     

Toxocariasis Diagnosed in International Travelers at the Institute of Tropical Medicine,Antwerp, Belgium,from 2000 to 2013

Although infection with Toxocara canis or T. catis (commonly referred as toxocariasis) appears to be highly prevalent in (sub)tropical countries, information on its frequency and presentation in returning travelers and migrants is scarce. In this study, we reviewed all cases of asymptomatic and symptomatic toxocariasis diagnosed during post-travel consultations at the reference travel clinic of the Institute of Tropical Medicine, Antwerp, Belgium. Toxocariasis was considered as highly probable if serum Toxocara-antibodies were detected in combination with symptoms of visceral larva migrans if present, elevated eosinophil count in blood or other relevant fluid and reasonable exclusion of alternative diagnosis, or definitive in case of documented seroconversion. From 2000 to 2013, 190 travelers showed Toxocara-antibodies, of a total of 3436 for whom the test was requested (5.5%). Toxocariasis was diagnosed in 28 cases (23 symptomatic and 5 asymptomatic) including 21 highly probable and 7 definitive. All but one patients were adults. Africa and Asia were the place of acquisition for 10 and 9 cases, respectively. Twelve patients (43%) were short-term travelers (< 1 month). Symptoms, when present, developed during travel or within 8 weeks maximum after return, and included abdominal complaints (11/23 symptomatic patients, 48%), respiratory symptoms and skin abnormalities (10 each, 43%) and fever (9, 39%), often in combination. Two patients were diagnosed with transverse myelitis. At presentation, the median blood eosinophil count was 1720/μL [range: 510–14160] in the 21 symptomatic cases without neurological complication and 2080/μL [range: 1100–2970] in the 5 asymptomatic individuals. All patients recovered either spontaneously or with an anti-helminthic treatment (mostly a 5-day course of albendazole), except both neurological cases who kept sequelae despite repeated treatments and prolonged corticotherapy. Toxocariasis has to be considered in travelers returning from a (sub)tropical stay with varying clinical manifestations or eosinophilia. Prognosis appears favorable with adequate treatment except in case of neurological involvement.     

Epidemiology of Antimicrobial Resistance in Escherichia coli Isolates from Raccoons (Procyon lotor) and the Environment on Swine Farms and Conservation Areas in Southern Ontario

Antimicrobial resistance is a global threat to livestock, human and environmental health. Although resistant bacteria have been detected in wildlife, their role in the epidemiology of antimicrobial resistance is not clear. Our objective was to investigate demographic, temporal and climatic factors associated with carriage of antimicrobial resistant Escherichia coli in raccoons and the environment. We collected samples from raccoon paws and feces and from soil, manure pit and dumpsters on five swine farms and five conservation areas in Ontario, Canada once every five weeks from May to November, 2011–2013 and tested them for E. coli and susceptibility to 15 antimicrobials. Of samples testing positive for E. coli, resistance to ≥ 1 antimicrobials was detected in 7.4% (77/1044; 95% CI, 5.9–9.1) of raccoon fecal samples, 6.3% (23/365; 95% CI, 4.0–9.3) of paw samples, 9.6% (121/1260; 8.0–11.4) of soil samples, 57.4% (31/54; 95% CI, 43.2–70.8) of manure pit samples, and 13.8% (4/29; 95% CI, 3.9–31.7) of dumpster samples. Using univariable logistic regression, there was no significant difference in the occurrence of resistant E. coli in raccoon feces on conservation areas versus farms; however, E. coli isolates resistant to ≥ 1 antimicrobials were significantly less likely to be detected from raccoon paw samples on swine farms than conservation areas and significantly more likely to be detected in soil samples from swine farms than conservation areas. Resistant phenotypes and genotypes that were absent from the swine farm environment were detected in raccoons from conservation areas, suggesting that conservation areas and swine farms may have different exposures to resistant bacteria. However, the similar resistance patterns and genes in E. coli from raccoon fecal and environmental samples from the same location types suggest that resistant bacteria may be exchanged between raccoons and their environment.     

Gastrointestinal Infections and Diarrheal Disease in Ghanaian Infants and Children: An Outpatient Case-Control Study

IntroductionDiarrheal diseases are among the most frequent causes of morbidity and mortality in children worldwide, especially in resource-poor areas. This case-control study assessed the associations between gastrointestinal infections and diarrhea in children from rural Ghana.MethodsStool samples were collected from 548 children with diarrhea and from 686 without gastrointestinal symptoms visiting a hospital from 2007–2008. Samples were analyzed by microscopy and molecular methods.ResultsThe organisms most frequently detected in symptomatic cases were Giardia lamblia, Shigella spp./ enteroinvasive Escherichia coli (EIEC), and Campylobacter jejuni. Infections with rotavirus (adjusted odds ratio [aOR] = 8.4; 95% confidence interval [CI]: 4.3–16.6), C. parvum/hominis (aOR = 2.7; 95% CI: 1.4–5.2) and norovirus (aOR = 2.0; 95%CI: 1.3–3.0) showed the strongest association with diarrhea. The highest attributable fractions (AF) for diarrhea were estimated for rotavirus (AF = 14.3%; 95% CI: 10.9–17.5%), Shigella spp./EIEC (AF = 10.5%; 95% CI: 3.5–17.1%), and norovirus (AF = 8.2%; 95% CI 3.2–12.9%). Co-infections occurred frequently and most infections presented themselves independently of other infections. However, infections with E. dispar, C. jejuni, and norovirus were observed more often in the presence of G. lamblia.ConclusionsDiarrheal diseases in children from a rural area in sub-Saharan Africa are mainly due to infections with rotavirus, Shigella spp./EIEC, and norovirus. These associations are strongly age-dependent, which should be considered when diagnosing causes of diarrhea. The presented results are informative for both clinicians treating gastrointestinal infections as well as public health experts designing control programs against diarrheal diseases.     

Association of Vitamin D Receptor Polymorphism with Susceptibility to Symptomatic Pertussis

Pertussis, caused by infection with the gram negative B. pertussis bacterium, is a serious respiratory illness that can last for months. While B. pertussis infection rates are estimated between 1–10% in the general population, notifications of symptomatic pertussis only comprise 0.01–0.1% indicating that most individuals clear B. pertussis infections without developing (severe) clinical symptoms. In this study we investigated whether genetic risk factors are involved in the development of symptomatic pertussis upon B. pertussis infection. Single-nucleotide polymorphisms (SNPs) in candidate genes, MBL2, IL17A, TNFα, VDR, and IL10 were genotyped in a unique Dutch cohort of symptomatic clinically confirmed (ex-)pertussis patients and in a Dutch population cohort. Of the seven investigated SNPs in five genes, a polymorphism in the Vitamin D receptor (VDR) gene (rs10735810) was associated with pertussis. The VDR major allele and its homozygous genotype were more present in the symptomatic pertussis patient cohort compared to the control population cohort. Interestingly, the VDR major allele correlated also with the duration of reported pertussis symptoms. Vitamin D₃ (VD₃) and VDR are important regulators of immune activation. Altogether, these findings suggest that polymorphisms in the VDR gene may affect immune activation and the clinical outcome of B. pertussis infection.     

The metabolism of [2-14C]indole in the rat

1. [2-¹⁴C]Indole has been synthesized from [¹⁴C]formate and o-toluidine via N[¹⁴C]-formyltoluidine. 2. When fed to rats, the ¹⁴C of [¹⁴C]indole (dose 70–80mg./kg. body wt.) is fairly rapidly excreted, and in 2 days an average of 81% appears in the urine, 11% in the faeces and 2·4% as carbon dioxide in the expired air. 3. Radioactivity is excreted in the urine as indoxyl sulphate (50% of the dose), indoxyl glucuronide (11%), oxindole (1·4%), isatin (5·8%), 5-hydroxyoxindole conjugates (3·1%), N-formylanthranilic acid (0·5%) and unchanged indole (0·07%). The faeces contain indoxyl sulphate (0·4% of the dose) and indole (0·2%), but the major metabolites have not been identified. 4. Fed to rats with biliary cannulae an average of 5·6% of a dose of [¹⁴C]indole (20–60mg./kg. body wt.) is excreted in the bile in 2 days. Radioactivity is present as indoxyl sulphate (0·8% dose) and 5-hydroxyoxindole conjugates (0·6%). 5. Rats further metabolize indoxyl into N-formylanthranilic acid and anthranilic acid, and oxindole into 5-hydroxyoxindole. 6. With rat-liver microsomes plus supernatant under aerobic conditions, indole gives indoxyl, oxindole, possibly isatin, N-formylanthranilic acid and anthranilic acid, but under anaerobic conditions gives only oxindole. Similarly, under aerobic conditions, oxindole gives 5-hydroxyoxindole, anthranilic acid and o-aminophenylacetic acid. 7. Indole is metabolized by two pathways, one via indoxyl to isatin, N-formylanthranilic acid and anthranilic acid, and the other via oxindole to 5-hydroxyoxindole and possibly to o-aminophenylacetic and anthranilic acid. 8. The following new compounds are described: 4-hydroxy-2-nitrophenylacetic acid, 3-, 4- and 5-benzyloxy-2-nitrophenylacetic acid, 5- and 7-hydroxyoxindole and 5-aminoacridine indoxyl sulphate.     

Acute and Temporal Expression of Tumor Necrosis Factor (TNF)-α-stimulated Gene 6 Product,TSG6, in Mesenchymal Stem Cells Creates Microenvironments Required for Their Successful Transplantation into Muscle Tissue

Previously, we demonstrated that when mesenchymal stem cells (MSCs) from mouse ES cells were transplanted into skeletal muscle, more than 60% of them differentiated into muscles in the crush-injured tibialis anterior muscle in vivo, although MSCs neither differentiated nor settled in the intact muscle. Microenvironments, including the extracellular matrix between the injured and intact muscle, were quite different. In the injured muscle, hyaluronan (HA), heavy chains of inter-α-inhibitor (IαI), CD44, and TNF-α-stimulated gene 6 product (TSG-6) increased 24–48 h after injury, although basement membrane components of differentiated muscle such as perlecan, laminin, and type IV collagen increased gradually 4 days after the crush. We then investigated the microenvironments crucial for cell transplantation, using the lysate of C2C12 myotubules for mimicking injured circumstances in vivo. MSCs settled in the intact muscle when they were transplanted together with the C2C12 lysate or TSG6. MSCs produced and released TSG6 when they were cultured with C2C12 lysates in vitro. MSCs pretreated with the lysate also settled in the intact muscle. Furthermore, MSCs whose TSG6 was knocked down by shRNA, even if transplanted or pretreated with the lysate, could not settle in the muscle. Immunofluorescent staining showed that HA and IαI always co-localized or were distributed closely, suggesting formation of covalent complexes, i.e. the SHAP-HA complex in the presence of TSG6. Thus, TSG6, HA, and IαI were crucial factors for the settlement and probably the subsequent differentiation of MSCs.     

Workplace Bullying and Mental Health: A Meta-Analysis on Cross-Sectional and Longitudinal Data

Background

A growing body of research has confirmed that workplace bullying is a source of distress and poor mental health. Here we summarize the cross-sectional and longitudinal literature on these associations.

Methods

Systematic review and meta-analyses on the relation between workplace bullying and mental health.

Results

The cross-sectional data (65 effect sizes, N = 115.783) showed positive associations between workplace bullying and symptoms of depression (r = .28, 95% CI = .23–.34), anxiety (r = .34, 95% CI = .29–.40) and stress-related psychological complaints (r = .37, 95% CI = .30–.44). Pooling the literature that investigated longitudinal relationships (26 effect sizes, N = 54.450) showed that workplace bullying was related to mental health complaints over time (r = 0.21, 95% CI = 0.13–0.21). Interestingly, baseline mental health problems were associated with subsequent exposure to workplace bullying (r = 0.18, 95% CI = 0.10–0.27; 11 effect sizes, N = 27.028).

Limitations

All data were self-reported, raising the possibility of reporting- and response set bias.

Conclusions

Workplace bullying is consistently, and in a bi-directional manner, associated with reduced mental health. This may call for intervention strategies against bullying at work.     

Algorithm for diagnosis of early Schistosoma haematobium using prodromal signs and symptoms in pre-school age children in an endemic district in Zimbabwe

IntroductionPrompt diagnosis of acute schistosomiasis benefits the individual and provides opportunities for early public health intervention. In endemic areas schistosomiasis is usually contracted during the first 5 years of life, thus it is critical to look at how the infection manifests in this age group. The aim of this study was to describe the prodromal signs and symptoms of early schistosomiasis infection, correlate these with early disease progression and risk score to develop an easy to use clinical algorithm to identify early Schistosoma haematobium infection cases in resource limited settings.MethodologyTwo hundred and four, preschool age children who were lifelong residence of a schistosomiasis endemic district and at high risk of acquiring schistosomiasis were followed up from July 2019 to December 2019, during high transmission season. The children received interval and standard full clinical evaluations and laboratory investigations for schistosomiasis by clinicians blinded from their schistosomiasis infection status. Diagnosis of S. haematobium was by urine filtration collected over three consecutive days. Signs and symptoms of schistosomiasis at first examination visit were compared to follow-up visits. Signs and symptoms common on the last schistosomiasis negative visit (before a subsequent positive) were assigned as early schistosomiasis infection (ESI), after possible alternative causes were ruled out. Logistic regression identified clinical predictors. A model based score was assigned to each predictor to create a risk for every child. An algorithm was created based on the predictor risk scores and validated on a separate cohort of 537 preschool age children.ResultsTwenty-one percent (42) of the participants were negative for S. haematobium infection at baseline but turned positive at follow-up. The ESI participants at the preceding S. haematobium negative visit had the following prodromal signs and symptoms in comparison to non-ESI participants; pruritic rash adjusted odds ratio (AOR) = 21.52 (95% CI 6.38–72.66), fever AOR = 82 (95% CI 10.98–612), abdominal pain AOR = 2.6 (95% CI 1.25–5.43), pallor AOR = 4 (95% CI 1.44–11.12) and a history of facial/body swelling within the previous month AOR = 7.31 (95% CI 3.49–15.33). Furthermore 16% of the ESI group had mild normocytic anaemia, whilst 2% had moderate normocytic anaemia. A risk score model was created using a rounded integer from the relative risks ratios. The diagnostic algorithm created had a sensitivity of 81% and a specificity of 96.9%, Positive predictive value = 87.2% and NPV was 95.2%. The area under the curve for the algorithm was 0.93 (0.90–0.97) in comparison with the urine dipstick AUC = 0.58 (0.48–0.69). There was a similar appearance in the validation cohort as in the derivative cohort.ConclusionThis study demonstrates for the first time prodromal signs and symptoms associated with early S. haematobium infection in pre-school age children. These prodromal signs and symptoms pave way for early intervention and management, thus decreasing the harm of late diagnosis. Our algorithm has the potential to assist in risk-stratifying pre-school age children for early S. haematobium infection. Independent validation of the algorithm on another cohort is needed to assess the utility further.     

GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate) Is the Preferred Substrate for Chondroitin N-Acetylgalactosaminyltransferase-1

A deficiency in chondroitin N-acetylgalactosaminyltransferase-1 (ChGn-1) was previously shown to reduce the number of chondroitin sulfate (CS) chains, leading to skeletal dysplasias in mice, suggesting that ChGn-1 regulates the number of CS chains for normal cartilage development. Recently, we demonstrated that 2-phosphoxylose phosphatase (XYLP) regulates the number of CS chains by dephosphorylating the Xyl residue in the glycosaminoglycan-protein linkage region of proteoglycans. However, the relationship between ChGn-1 and XYLP in controlling the number of CS chains is not clear. In this study, we for the first time detected a phosphorylated tetrasaccharide linkage structure, GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate), in ChGn-1^−/− growth plate cartilage but not in ChGn-2^−/− or wild-type growth plate cartilage. In contrast, the truncated linkage tetrasaccharide GlcUAβ1–3Galβ1–3Galβ1–4Xyl was detected in wild-type, ChGn-1^−/−, and ChGn-2^−/− growth plate cartilage. Consistent with the findings, ChGn-1 preferentially transferred N-acetylgalactosamine to the phosphorylated tetrasaccharide linkage in vitro. Moreover, ChGn-1 and XYLP interacted with each other, and ChGn-1-mediated addition of N-acetylgalactosamine was accompanied by rapid XYLP-dependent dephosphorylation during formation of the CS linkage region. Taken together, we conclude that the phosphorylated tetrasaccharide linkage is the preferred substrate for ChGn-1 and that ChGn-1 and XYLP cooperatively regulate the number of CS chains in growth plate cartilage.     

How Have We Diagnosed Early-Stage Lung Cancer without Radiographic Screening? A Contemporary Single-Center Experience

Background

The National Lung Screening Trial (NLST), which demonstrated a reduction in lung cancer mortality, may result in widespread computed tomography (CT)-based screening of select populations. How early-stage lung cancer has been diagnosed without screening, and what proportion of these cases would be captured by a screening program modeled on the NLST, is not currently known. We therefore evaluated current patterns of early-stage lung cancer presentation.

Methodology/Principal Findings

We performed a single-institution retrospective analysis of patients diagnosed with stage I–II non-small cell lung cancer (NSCLC) from 2000–2009. Associations between patient and imaging characteristics were assessed using univariate and multivariate analyses. A total of 412 patients met criteria for analysis. Among those with available reason for initial imaging, the reason was symptoms in 51%, follow-up of other conditions in 43%, and screening in 6%. Reason for imaging was associated with race (P<0.001), insurance type (P = 0.005), and disease stage (P<0.001). Type of initial imaging was associated with reason for imaging (P<0.001), year (chest x-ray 67% in 2000–2004 vs. 49% in 2005–2009; P<0.001), and disease stage (P = 0.005). Among patients with available quantified smoking history, 48% were age 55–74 years and smoked 30-plus pack-years, therefore meeting NLST entry criteria.

Conclusions/Significance

Symptoms remain a dominant but declining reason for detection of early-stage NSCLC. The proportion of cases detected initially by CT scan without antecedent chest x-ray has increased considerably. Because as few as half of cases meet NLST eligibility criteria, clinicians should remain aware of the diverse circumstances of early-stage lung cancer presentation to expedite therapy.     

The Metabolic Origins of Mannose in Glycoproteins

Mannose in N-glycans is derived from glucose through phosphomannose isomerase (MPI, Fru-6-P ↔ Man-6-P) whose deficiency causes a congenital disorder of glycosylation (CDG)-Ib (MPI-CDG). Mannose supplements improve patients'' symptoms because exogenous mannose can also directly contribute to N-glycan synthesis through Man-6-P. However, the quantitative contributions of these and other potential pathways to glycosylation are still unknown. We developed a sensitive GC-MS-based method using [1,2-¹³C]glucose and [4-¹³C]mannose to measure their contribution to N-glycans synthesized under physiological conditions (5 mm glucose and 50 μm mannose). Mannose directly provides ∼10–45% of the mannose found in N-glycans, showing up to a 100-fold preference for mannose over exogenous glucose based on their exogenous concentrations. Normal human fibroblasts normally derive 25–30% of their mannose directly from exogenous mannose, whereas MPI-deficient CDG fibroblasts with reduced glucose flux secure 80% of their mannose directly. Thus, both MPI activity and exogenous mannose concentration determine the metabolic flux into the N-glycosylation pathway. Using various stable isotopes, we found that gluconeogenesis, glycogen, and mannose salvaged from glycoprotein degradation do not contribute mannose to N-glycans in fibroblasts under physiological conditions. This quantitative assessment of mannose contribution and its metabolic fate provides information that can help bolster therapeutic strategies for treating glycosylation disorders with exogenous mannose.     

Plant height–crown radius and canopy coverage–density relationships determine above-ground biomass–density relationship in stressful environments

Debate continues in theoretical ecology over whether and why the scaling exponent of biomass–density (M–N) relationship varies along environmental gradients. By developing a novel geometric model with assumptions of allometric growth at the individual level and open canopy at the stand level, we propose that plant height–crown radius and canopy coverage–density relationships determine the above-ground M–N relationship in stressful environments. Results from field investigation along an aridity gradient (from eastern to western China) confirmed our model prediction and showed that the above-ground M–N scaling exponent increased with drought stress. Therefore, the ‘universal’ scaling exponents (−3/2 or −4/3) of the M–N relationship predicted by previous models may not hold for above-ground parts in stressful environments.     

Determinants of Antimicrobial Resistance in Escherichia coli Strains Isolated from Faeces and Urine of Women with Recurrent Urinary Tract Infections

For women with recurrent urinary tract infections (rUTI), the contribution of antibiotic use versus patient-related factors in determining the presence of antimicrobial resistance in faecal and urinary Escherichia coli, obtained from the same patient population, has not been assessed yet. Within the context of the ‘Non-antibiotic prophylaxis for recurrent urinary tract infections’ (NAPRUTI) study, the present study assessed determinants of antimicrobial resistance in E. coli isolated from urinary and faecal samples of women with rUTIs collected at baseline. Potential determinants of resistance were retrieved from self-administered questionnaires. From 434 asymptomatic women, 433 urinary and 424 faecal samples were obtained. E. coli was isolated from 146 (34%) urinary samples and from 336 (79%) faecal samples, and subsequently tested for antimicrobial susceptibility. Multivariable analysis showed trimethoprim/sulfamethoxazole (SXT) use three months prior to inclusion to be associated with urine E. coli resistance to amoxicillin (OR 3.6, 95% confidence interval: 1.3–9.9), amoxicillin-clavulanic acid (OR 4.4, 1.5–13.3), trimethoprim (OR 3.9, 1.4–10.5) and SXT (OR 3.2, 1.2–8.5), and with faecal E. coli resistance to trimethoprim (OR 2.0, 1.0–3.7). The number of UTIs in the preceding year was correlated with urine E. coli resistance to amoxicillin-clavulanic acid (OR 1.11, 1.01–1.22), trimethoprim (OR 1.13, 1.03–1.23) and SXT (OR 1.10, 1.01–1.19). Age was predictive for faecal E. coli resistance to amoxicillin (OR 1.02, 1.00–1.03), norfloxacin and ciprofloxacin (both OR 1.03, 1.01–1.06). In conclusion, in women with rUTI different determinants were found for urinary and faecal E. coli resistance. Previous antibiotic use and UTI history were associated with urine E. coli resistance and age was a predictor of faecal E. coli resistance. These associations could best be explained by cumulative antibiotic use.     

Frozen Cropland Soil in Northeast China as Source of N2O and CO2 Emissions

Agricultural soils are important sources of atmospheric N₂O and CO₂. However, in boreal agro-ecosystems the contribution of the winter season to annual emissions of these gases has rarely been determined. In this study, soil N₂O and CO₂ fluxes were measured for 6 years in a corn-soybean-wheat rotation in northeast China to quantify the contribution of wintertime N₂O and CO₂ fluxes to annual emissions. The treatments were chemical fertilizer (NPK), chemical fertilizer plus composted pig manure (NPKOM), and control (Cont.). Mean soil N₂O fluxes among all three treatments in the winter (November–March), when soil temperatures are below −7°C for extended periods, were 0.89–3.01 µg N m⁻² h⁻¹, and in between the growing season and winter (October and April), when freeze-thaw events occur, 1.73–5.48 µg N m⁻² h⁻¹. The cumulative N₂O emissions were on average 0.27–1.39, 0.03–0.08 and 0.03–0.11 kg N₂O_–N ha⁻¹ during the growing season, October and April, and winter, respectively. The average contributions of winter N₂O efflux to annual emissions were 6.3–12.1%. In all three seasons, the highest N₂O emissions occurred in NPKOM, while NPK and Cont. emissions were similar. Cumulative CO₂ emissions were 2.73–4.94, 0.13–0.20 and 0.07–0.11 Mg CO₂-C ha⁻¹ during growing season, October and April, and winter, respectively. The contribution of winter CO₂ to total annual emissions was 2.0–2.4%. Our results indicate that in boreal agricultural systems in northeast China, CO₂ and N₂O emissions continue throughout the winter.     

Shifts in the Antibiotic Susceptibility,Serogroups, and Clonal Complexes of Neisseria meningitidis in Shanghai,China: A Time Trend Analysis of the Pre-Quinolone and Quinolone Eras

Background

Fluoroquinolones have been used broadly since the end of the 1980s and have been recommended for Neisseria meningitidis prophylaxis since 2005 in China. The aim of this study was to determine whether and how N. meningitidis antimicrobial susceptibility, serogroup prevalence, and clonal complex (CC) prevalence shifted in association with the introduction and expanding use of quinolones in Shanghai, a region with a traditionally high incidence of invasive disease due to N. meningitidis.

Methods and Findings

A total of 374 N. meningitidis isolates collected by the Shanghai Municipal Center for Disease Control and Prevention between 1965 and 2013 were studied. Shifts in the serogroups and CCs were observed, from predominantly serogroup A CC5 (84%) in 1965–1973 to serogroup A CC1 (58%) in 1974–1985, then to serogroup C or B CC4821 (62%) in 2005–2013. The rates of ciprofloxacin nonsusceptibility in N. meningitidis disease isolates increased from 0% in 1965–1985 to 84% (31/37) in 2005–2013 (p < 0.001). Among the ciprofloxacin-nonsusceptible isolates, 87% (27/31) were assigned to either CC4821 (n = 20) or CC5 (n = 7). The two predominant ciprofloxacin-resistant clones were designated China^{CC4821-R1-C/B} and China^CC5-R14-A. The China^{CC4821-R1-C/B} clone acquired ciprofloxacin resistance by a point mutation, and was present in 52% (16/31) of the ciprofloxacin-nonsusceptible disease isolates. The China^CC5-R14-A clone acquired ciprofloxacin resistance by horizontal gene transfer, and was found in 23% (7/31) of the ciprofloxacin-nonsusceptible disease isolates. The ciprofloxacin nonsusceptibility rate was 47% (7/15) among isolates from asymptomatic carriers, and nonsusceptibility was associated with diverse multi-locus sequence typing profiles and pulsed-field gel electrophoresis patterns. As detected after 2005, ciprofloxacin-nonsusceptible strains were shared between some of the patients and their close contacts. A limitation of this study is that isolates from 1986–2004 were not available and that only a small sample of convenience isolates from 1965–1985 were available.

Conclusions

The increasing prevalence of ciprofloxacin resistance since 2005 in Shanghai was associated with the spread of hypervirulent lineages CC4821 and CC5. Two resistant meningococcal clones China^{CC4821-R1-C/B} and China^CC5-R14-A have emerged in Shanghai during the quinolone era. Ciprofloxacin should be utilized with caution for the chemoprophylaxis of N. meningitidis in China.