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Isocyanate chemicals known to cause adverse health effects when inhaled are essential to making important products and are used in multiple industries. Glutathione (GSH), a major antioxidant of the lower airways with a well described role in xenobiotic metabolism, is a primary reaction target for di-isocyantes. However, GSHs reactivity with poly-isocyanates which have largely replaced diisocyanates (particularly aliphatic) in most end-user settings remains uncertain. We hypothesized aliphatic polyisocyanates would readily react with glutathione under physiologic conditions and the products could be identified using liquid chromatography (LC) coupled-mass spectrometry (MS) and tandem MS/MS. The data identified (tris)GSH-isocyanate adducts as the major reaction product of GSH with the most commonly used contemporary polymeric (tri-isocyanate) formulations of hexamethylene diisocyanate (HDI), the isocyanurate and biuret, as [M+H]+ ions of 1426.53 and 1400.55 m/z respectively in reverse phase LC-MS using electrospray in positive ion mode. The uretdione form of HDI, a stabilized dimer, formed two reaction products with GSH, a tris(GSH)-isocyanate reaction product recognized as a 1258.44 m/z [M+H]+ ion, and a bis(GSH)-isocyanate product identified as a 951.36 m/z [M+H]+ ion. Predicted structures for the newly described GSH-polyisocyanate reaction products, modeled based on collision induced dissociation (CID) fragmentation patterns in tandem MS/MS, support S-linkage of the GSH to N = C = O groups. In summary, industrially-used aliphatic polyisocyanates readily react with GSH to form primarily S-linked tris(GSH)-conjugates, a process that may play an important role in response to respiratory tract exposure.

BackgroundTests for SARS-CoV-2 immunity are needed to help assess responses to vaccination, which can be heterogeneous and may wane over time. The plaque reduction neutralization test (PRNT) is considered the gold standard for measuring serum neutralizing antibodies but requires high level biosafety, live viral cultures and days to complete. We hypothesized that competitive enzyme linked immunoassays (ELISAs) based on SARS-CoV-2 spike protein's receptor binding domain (RBD) attachment to its host receptor, the angiotensin converting enzyme 2 receptor (ACE2r), would correlate with PRNT, given the central role of RBD-ACE2r interactions in infection and published studies to date, and enable evaluation of vaccine responses.Methods and resultsConfiguration and development of a competitive ELISA with plate-bound RBD and soluble biotinylated ACE2r was accomplished using pairs of pre/post vaccine serum. When the competitive ELISA was used to evaluate N = 32 samples from COVID-19 patients previously tested by PRNT, excellent correlation in IC50 results were observed (rs = .83, p < 0.0001). When the competitive ELISA was used to evaluate N = 42 vaccinated individuals and an additional N = 13 unvaccinated recovered COVID-19 patients, significant differences in RBD-ACE2r inhibitory activity were associated with prior history of COVID-19 and type of vaccine received. In longitudinal analyses pre and up to 200 days post vaccine, surrogate neutralizing activity increased markedly after primary and booster vaccine doses, but fell substantially, up to ConclusionsA competitive ELISA based on inhibition of RBD-ACE2r attachment correlates well with PRNT, quantifies significantly higher activity among vaccine recipients with prior COVID (vs. those without), and highlights marked declines in surrogate neutralizing activity over a 6 month period post vaccination. The findings raise concern about the duration of vaccine responses and potential need for booster shots.

SARS-CoV-2 spike antigen-specific IgG and IgA elicited by infection mediate viral neutralization and are likely an important component of natural immunity, however, limited information exists on vaccine induced responses. We measured COVID-19 mRNA vaccine induced IgG and IgA in serum serially, up to 145 days post vaccination in 4 subjects. Spike antigen-specific IgG levels rose exponentially and plateaued 21 days after the initial vaccine dose. After the second vaccine dose IgG levels increased further, reaching a maximum approximately 7-10 days later, and remained elevated (average of 58% peak levels) during the additional >100 day follow up period. COVID-19 mRNA vaccination elicited spike antigen-specific IgA with similar kinetics of induction and time to peak levels, but more rapid decline in serum levels following both the 1st and 2nd vaccine doses (

BackgroundCountries across the globe have mobilized their armed forces in response to COVID-19, placing them at increased risk for viral exposure. Humoral responses to SARS-CoV-2 among military personnel serve as biomarkers of infection and provide a basis for disease surveillance and recognition of work-related risk factors.MethodsEnzyme-linked immunosorbent assays (ELISA) were used to measure SARS-CoV-2 spike antigen-specific IgG in serum obtained from N = 988 US National Guard soldiers between April-June 2020. Occupational information, e.g. military operating specialty (MOS) codes, and demographic data were obtained via questionnaire. Plaque assays with live SARS-CoV-2 were used to assess serum neutralizing capacity for limited subjects (N = 12).ResultsThe SARS-CoV-2 IgG seropositivity rate among the study population was 10.3% and significantly associated with occupation and demographics. Odds ratios were highest for those working in MOS 2T-Transportation (3.6; 95% CI 0.7-18) and 92F-Fuel specialist/ground and aircraft (6.8; 95% CI 1.5-30), as well as black race (2.2; 95% CI 1.2-4.1), household size ≥6 (2.5; 95% CI 1.3-4.6) and known COVID-19 exposure (2.0; 95% CI 1.2-3.3). Seropositivity tracked along major interstate highways and clustered near the international airport and the New York City border. SARS-CoV-2 spike IgG+ serum exhibited low to moderate SARS-CoV-2 neutralizing capacity with IC50s ranging from 1:15 to 1:280. In limited follow-up testing SARS-CoV-2 serum IgG levels remained elevated up to 7 months.ConclusionsThe data highlight increased SARS-CoV-2 seroprevalence among National Guard vs. the local civilian population in association with transportation-related occupations and specific demographics.

Reverse vaccinology is an evolving approach for improving vaccine effectiveness and minimizing adverse responses by limiting immunizations to critical epitopes. Towards this goal, we sought to identify immunogenic amino acid motifs and linear epitopes of the SARS-CoV-2 spike protein that elicit IgG in COVID-19 mRNA vaccine recipients. Paired pre/post vaccination samples from N = 20 healthy adults, and post-vaccine samples from an additional N = 13 individuals were used to immunoprecipitate IgG targets expressed by a bacterial display random peptide library, and preferentially recognized peptides were mapped to the spike primary sequence. The data identify several distinct amino acid motifs recognized by vaccine-induced IgG, a subset of those targeted by IgG from natural infection, which may mimic 3-dimensional conformation (mimotopes). Dominant linear epitopes were identified in the C-terminal domains of the S1 and S2 subunits (aa 558-569, 627-638, and 1148-1159) which have been previously associated with SARS-CoV-2 neutralization in vitro and demonstrate identity to bat coronavirus and SARS-CoV, but limited homology to non-pathogenic human coronavirus. The identified COVID-19 mRNA vaccine epitopes should be considered in the context of variants, immune escape and vaccine and therapy design moving forward.

Recent animal studies, as well as quantitative sodium MRI observations on humans demonstrated that remarkable amounts of sodium can be stored in the skin. It is also known that excess sodium in the tissues leads to inflammation in various organs, but its role in dermal pathophysiology has not been elucidated. Therefore, our aim was to study the effect of dietary salt loading on inflammatory process and related extracellular matrix (ECM) remodeling in the skin. To investigate the effect of high salt consumption on inflammation and ECM production in the skin mice were kept on normal (NSD) or high salt (HSD) diet and then dermatitis was induced with imiquimod (IMQ) treatment. The effect of high salt concentration on dermal fibroblasts (DF) and peripheral blood mononuclear cells (PBMC) was also investigated in vitro. The HSD resulted in increased sodium content in the skin of mice. Inflammatory cytokine Il17 expression was elevated in the skin of HSD mice. Expression of anti-inflammatory Il10 and Il13 decreased in the skin of HSD or HSD IMQ mice. The fibroblast marker Acta2 and ECM component Fn and Col1a1 decreased in HSD IMQ mice. Expression of ECM remodeling related Pdgfb and activation phosphorylated (p)-SMAD2/3 was lower in HSD IMQ mice. In PBMCs, production of IL10, IL13 and PDGFB was reduced due to high salt loading. In cultured DFs high salt concentration resulted in decreased cell motility and ECM production, as well. Our results demonstrate that high dietary salt intake is associated with increased dermal pro-inflammatory status. Interestingly, although inflammation induces the synthesis of ECM in most organs, the expression of ECM decreased in the inflamed skin of mice on high salt diet. Our data suggest that salt intake may alter the process of skin remodeling.

Irrigation water contaminated with Salmonella enterica and Listeria monocytogenes may provide a route of contamination of raw or minimally processed fruits and vegetables. While previous work has surveyed specific and singular types of agricultural irrigation water for bacterial pathogens, few studies have simultaneously surveyed different water sources repeatedly over an extended period of time. This study quantified S. enterica and L. monocytogenes levels (MPN/L) at 6 sites, including river waters: tidal freshwater river (MA04, n = 34), non-tidal freshwater river, (MA05, n = 32), one reclaimed water holding pond (MA06, n = 25), two pond water sites (MA10, n = 35; MA11, n = 34), and one produce wash water site (MA12, n = 10) from September 2016-October 2018. Overall, 50% (84/168) and 31% (53/170) of sampling events recovered S. enterica and L. monocytogenes, respectively. Results showed that river waters supported significantly (p < 0.05) greater levels of S. enterica than pond or reclaimed waters. The non-tidal river water sites (MA05) with the lowest water temperature supported significantly greater level of L. monocytogenes compared to all other sites; L. monocytogenes levels were also lower in winter and spring compared to summer seasons. Filtering 10 L of water through a modified Moore swab (MMS) was 43.5 (Odds ratio, p < 0.001) and 25.5 (p < 0.001) times more likely to recover S. enterica than filtering 1 L and 0.1 L, respectively; filtering 10 L was 4.8 (p < 0.05) and 3.9 (p < 0.05) times more likely to recover L. monocytogenes than 1L and 0.1 L, respectively. Work presented here shows that S. enterica and L. monocytogenes levels are higher in river waters compared to pond or reclaimed waters in the Mid-Atlantic region of the U.S., and quantitatively shows that analyzing 10 L water is more likely recover pathogens than smaller samples of environmental waters.

Many studies on Heracleum have shown poor correspondence between observed molecular clusters and established taxonomic classification amongst closely related species. This might reflect both unresolved taxonomy but perhaps also a lack of good genetic markers. This lack of appropriate and cost effective species-specific genetic markers hinders a resolved relationship for the species complex, and this in turn causes profound management challenges for a genus that contains both endemic species, with important ecological roles, and species with an invasive potential. Microsatellites are traditionally considered markers of choice for comprehensive, yet inexpensive, analyses of genetic variation, including examination of population structure, species identity, linkage map construction and cryptic speciation. In this study, we have used double digest restriction site associated DNA sequencing (ddRADseq) to develop microsatellite markers in Heracleum rechingeri. Genomic DNA from three individuals were digested with Sbf1 and Nde1 and size selected for library construction. The size-selected fragments were sequenced on an Ion Torrent sequencer and a total of 54 microsatellite sequences were bioinformatically confirmed. Twenty five loci were then tested for amplification, resulting in 19 of these being successfully amplified across eight species, comprising both the so-called thick-stemmed species (H. persicum, H. rechingeri, H. gorganicum and H. lasiopetalum), and thin-stemmed species (H. anisactis, H. pastinasifolium and H. transcaucasicum). Both Bayesian and distance-based clustering, and principal coordinate analyses clearly separated these into two groups. Surprisingly, three H. pastinacifolium populations were not separated from populations of the morphologically similar endemic species, H. anisactis, suggesting lack of genetic differentiation. Likewise, high genetic similarity was found between H. persicum and H. rechingeri populations, questioning taxonomic separation at the species level between these taxa. Further analyses are needed to re-evaluate the taxonomic significance of observed morphological variability currently applied to distinguish these sister taxa. Nevertheless, our results represent progress in the effort to develop cost-efficient molecular tools for species discrimination in this genus.

Lifelong noninvasive rejection monitoring in heart transplant patients is a critical clinical need historically poorly met in adults and unavailable for children and infants. Cell-free DNA (cfDNA) donor-specific fraction (DF), a direct marker of selective donor organ injury, is a promising analytical target. Methodological differences in sample processing and DF determination profoundly affect quality and sensitivity of cfDNA analyses, requiring specialized optimization for low cfDNA levels typical of transplant patients. Using next-generation sequencing, we previously correlated elevated DF with acute cellular and antibody-mediated rejection (ACR and AMR) in pediatric and adult heart transplant patients. However, next-generation sequencing is limited by cost, TAT, and sensitivity, leading us to clinically validate a rapid, highly sensitive, quantitative genotyping test, myTAIHEART®, addressing these limitations. To assure pre-analytical quality and consider interrelated cfDNA measures, plasma preparation was optimized and total cfDNA (TCF) concentration, DNA fragmentation, and DF quantification were validated in parallel for integration into myTAIHEART reporting. Analytical validations employed individual and reconstructed mixtures of human blood-derived genomic DNA (gDNA), cfDNA, and gDNA sheared to apoptotic length. Precision, linearity, and limits of blank/detection/quantification were established for TCF concentration, DNA fragmentation ratio, and DF determinations. For DF, multiplexed high-fidelity amplification followed by quantitative genotyping of 94 SNP targets was applied to 1168 samples to evaluate donor options in staged simulations, demonstrating DF call equivalency with/without donor genotype. Clinical validation studies using 158 matched endomyocardial biopsy-plasma pairs from 76 pediatric and adult heart transplant recipients selected a DF cutoff (0.32%) producing 100% NPV for ≥2R ACR. This supports the assay's conservative intended use of stratifying low versus increased probability of ≥2R ACR. myTAIHEART is clinically validated for heart transplant recipients ≥2 months old and ≥8 days post-transplant, expanding opportunity for noninvasive transplant rejection assessment to infants and children and to all recipients >1 week post-transplant.

The coronary artery ligation model in rodents mimics human myocardial infarction (MI). Normally mechanical ventilation and prolonged anesthesia period are needed. Recently, a method has been developed to create MI by popping-out the heart (without ventilation) followed by immediate suture ligation. Mortality is high due to the time-consuming suture ligation process while the heart is exposed. We sought to improve this method and reduce mortality by rapid coronary ligation using a surgical clip instead of a suture.Mice were randomized into 3 groups: clip MI (CMI), suture MI (SMI), or sham (SHAM). In all groups, heart was manually exposed without intubation through a small incision on the chest wall. Unlike the conventional SMI method, mice in the CMI group received a metal clip on left anterior descending artery (LAD), quickly dispensed by an AutoSuture Surgiclip™. The CMI method took only 1/3 of ligation time of the standard SMI method and improved post-MI survival rate. TTC staining and Masson's trichrome staining revealed a similar degree of infarct size in the SMI and CMI groups. Echocardiograph confirmed that both SMI and CMI groups had a similar reduction of ejection fraction and fraction shortening over the time. Histological analysis showed that the numbers of CD68+ macrophages and apoptotic cells (TUNEL-positive) are indistinguishable between the two groups.This new method, taking only less than 3 minutes to complete, represents an efficient myocardial infarction model in rodents.

The reasons for black/white disparities in HIV epidemics among men who have sex with men have puzzled researchers for decades. Understanding reasons for these disparities requires looking beyond individual-level behavioral risk to a more comprehensive framework.From July 2010-December 2012, 803 men (454 black, 349 white) were recruited through venue-based and online sampling; consenting men were provided HIV and STI testing, completed a behavioral survey and a sex partner inventory, and provided place of residence for geocoding. HIV prevalence was higher among black (43%) versus white (13% MSM (prevalence ratio (PR) 3.3, 95% confidence interval (CI): 2.5-4.4). Among HIV-positive men, the median CD4 count was significantly lower for black (490 cells/µL) than white (577 cells/µL) MSM; there was no difference in the HIV RNA viral load by race. Black men were younger, more likely to be bisexual and unemployed, had less educational attainment, and reported fewer male sex partners, fewer unprotected anal sex partners, and less non-injection drug use. Black MSM were significantly more likely than white MSM to have rectal chlamydia and gonorrhea, were more likely to have racially concordant partnerships, more likely to have casual (one-time) partners, and less likely to discuss serostatus with partners. The census tracts where black MSM lived had higher rates of poverty and unemployment, and lower median income. They also had lower proportions of male-male households, lower male to female sex ratios, and lower HIV diagnosis rates.Among black and white MSM in Atlanta, disparities in HIV and STI prevalence by race are comparable to those observed nationally. We identified differences between black and white MSM at the individual, dyadic/sexual network, and community levels. The reasons for black/white disparities in HIV prevalence in Atlanta are complex, and will likely require a multilevel framework to understand comprehensively.

Cytauxzoonosis is an emerging infectious disease of domestic cats (Felis catus) caused by the apicomplexan protozoan parasite Cytauxzoon felis. The growing epidemic, with its high morbidity and mortality points to the need for a protective vaccine against cytauxzoonosis. Unfortunately, the causative agent has yet to be cultured continuously in vitro, rendering traditional vaccine development approaches beyond reach. Here we report the use of comparative genomics to computationally and experimentally interpret the C. felis genome to identify a novel candidate vaccine antigen for cytauxzoonosis. As a starting point we sequenced, assembled, and annotated the C. felis genome and the proteins it encodes. Whole genome alignment revealed considerable conserved synteny with other apicomplexans. In particular, alignments with the bovine parasite Theileria parva revealed that a C. felis gene, cf76, is syntenic to p67 (the leading vaccine candidate for bovine theileriosis), despite a lack of significant sequence similarity. Recombinant subdomains of cf76 were challenged with survivor-cat antiserum and found to be highly seroreactive. Comparison of eleven geographically diverse samples from the south-central and southeastern USA demonstrated 91-100% amino acid sequence identity across cf76, including a high level of conservation in an immunogenic 226 amino acid (24 kDa) carboxyl terminal domain. Using in situ hybridization, transcription of cf76 was documented in the schizogenous stage of parasite replication, the life stage that is believed to be the most important for development of a protective immune response. Collectively, these data point to identification of the first potential vaccine candidate antigen for cytauxzoonosis. Further, our bioinformatic approach emphasizes the use of comparative genomics as an accelerated path to developing vaccines against experimentally intractable pathogens.

A key question in the field of RNA regulation is how some exosome substrates, such as spliceosomal snRNAs and telomerase RNA, evade degradation and are processed into stable, functional RNA molecules. Typical feature of these non-coding RNAs is presence of the Sm complex at the 3'end of the mature RNA molecule. Here, we report that in Saccharomyces cerevisiae presence of intact Sm binding site is required for the exosome-mediated processing of telomerase RNA from a polyadenylated precursor into its mature form and is essential for its function in elongating telomeres. Additionally, we demonstrate that the same pathway is involved in the maturation of snRNAs. Furthermore, the insertion of an Sm binding site into an unstable RNA that is normally completely destroyed by the exosome, leads to its partial stabilization. We also show that telomerase RNA accumulates in Schizosaccharomyces pombe exosome mutants, suggesting a conserved role for the exosome in processing and degradation of telomerase RNA. In summary, our data provide important mechanistic insight into the regulation of exosome dependent RNA processing as well as telomerase RNA biogenesis.

The tropical arboreal environment is a mechanically complex and varied habitat. Arboreal inhabitants must adapt to changes in the compliance and stability of supports when moving around trees. Because the orangutan is the largest habitual arboreal inhabitant, it is unusually susceptible to branch compliance and stability and therefore represents a unique animal model to help investigate how animals cope with the mechanical heterogeneity of the tropical canopy. The aim of this study was to investigate how changes in compliance and time of oscillation of branches are related to easily observable traits of arboreal supports. This should help predict how supports react mechanically to the weight and mass of a moving orangutan, and suggest how orangutans themselves predict branch properties. We measured the compliance and time of oscillation of branches from 11 tree species frequented by orangutans in the rainforest of Sumatra. Branches were pulled at several points along their length using a force balance at the end of a stiff rope, and the local diameter of the branch and the distance to its base and tip were measured. Compliance was negatively associated with both local diameter and length to the tip of the branch, and positively, if weakly, associated with length from the trunk. However, branch diameter not only predicted compliance best, but would also be easiest for an orangutan to observe. In contrast, oscillation times of branches were largely unaffected by local diameter, and only significantly increased at diameters below 2 cm. The results of this study validate previous field research, which related locomotory modes to local branch diameter, while suggesting how arboreal animals themselves sense their mechanical environment.

Angiotensin converting enzyme inhibitors (ACEi) and angiotensin II receptor blockers (ARB) are the standard clinical therapy of diabetic nephropathy (DN), while aldosterone antagonists are only used as adjuncts. Previously in experimental DN we showed that Na/K ATPase (NKA) is mislocated and angiotensin II leads to superimposed renal progression. Here we investigated the monotherapeutic effect of aldosterone blockers on the progression of DN and renal NKA alteration in comparison to ACEi and ARBs. Streptozotocin-diabetic rats developing DN were treated with aldosterone antagonists; ACEi and ARB. Renal function, morphology, protein level and tubular localization of NKA were analyzed. To evaluate the effect of high glucose per se; HK-2 proximal tubular cells were cultured in normal or high concentration of glucose and treated with the same agents. Aldosterone antagonists were the most effective in ameliorating functional and structural kidney damage and they normalized diabetes induced bradycardia and weight loss. Aldosterone blockers also prevented hyperglycemia and diabetes induced increase in NKA protein level and enzyme mislocation. A monotherapy with aldosterone antagonists might be as, or more effective than ACEi or ARBs in the prevention of STZ-induced DN. Furthermore the alteration of the NKA could represent a novel pathophysiological feature of DN and might serve as an additional target of aldosterone blockers.

BackgroundIn 2008, over 300,000 women died during pregnancy or childbirth, mostly in poor countries. While there are proven interventions to make childbirth safer, there is uncertainty about the best way to deliver these at large scale. In particular, there is currently a debate about whether maternal deaths are more likely to be prevented by delivering effective interventions through scaled up facilities or via community-based services. To inform this debate, we examined delivery location and attendance and the reasons women report for giving birth at home.Methodology/principal findingsWe conducted a secondary analysis of maternal delivery data from Demographic and Health Surveys in 48 developing countries from 2003 to the present. We stratified reported delivery locations by wealth quintile for each country and created weighted regional summaries. For sub-Saharan Africa (SSA), where death rates are highest, we conducted a subsample analysis of motivations for giving birth at home. In SSA, South Asia, and Southeast Asia, more than 70% of all births in the lowest two wealth quintiles occurred at home. In SSA, 54.1% of the richest women reported using public facilities compared with only 17.7% of the poorest women. Among home births in SSA, 56% in the poorest quintile were unattended while 41% were attended by a traditional birth attendant (TBA); 40% in the wealthiest quintile were unattended, while 33% were attended by a TBA. Seven per cent of the poorest women reported cost as a reason for not delivering in a facility, while 27% reported lack of access as a reason. The most common reason given by both the poorest and richest women for not delivering in a facility was that it was deemed "not necessary" by a household decision maker. Among the poorest women, "not necessary" was given as a reason by 68% of women whose births were unattended and by 66% of women whose births were attended.ConclusionsIn developing countries, most poor women deliver at home. This suggests that, at least in the near term, efforts to reduce maternal deaths should prioritize community-based interventions aimed at making home births safer.

Bartonella henselae is the zoonotic agent of cat scratch disease and causes potentially fatal infections in immunocompromised patients. Understanding the complex interactions between the host's immune system and bacterial pathogens is central to the field of infectious diseases and to the development of effective diagnostics and vaccines.We report the development of a microarray comprised of proteins expressed from 96% (1433/1493) of the predicted ORFs encoded by the genome of the zoonotic pathogen Bartonella henselae. The array was probed with a collection of 62 uninfected, 62 infected, and 8 "specific-pathogen free" naïve cat sera, to profile the antibody repertoire elicited during natural Bartonella henselae infection.We found that 7.3% of the B. henselae proteins on the microarray were seroreactive and that seroreactivity was not evenly distributed between predicted protein function or subcellular localization. Membrane proteins were significantly most likely to be seroreactive, although only 23% of the membrane proteins were reactive. Conversely, we found that proteins involved in amino acid transport and metabolism were significantly underrepresented and did not contain any seroreactive antigens. Of all seroreactive antigens, 52 were differentially reactive with sera from infected cats, and 53 were equally reactive with sera from infected and uninfected cats. Thirteen of the seroreactive antigens were found to be differentially seroreactive between B. henselae type I and type II. Based on these results, we developed a classifier algorithm that was capable of accurately discerning 93% of the infected animals using the microarray platform. The seroreactivity and diagnostic potential of these antigens was then validated on an immunostrip platform, which correctly identified 98% of the infected cats. Our protein microarray platform provides a high-throughput, comprehensive analysis of the feline humoral immune response to natural infection with the alpha-proteobacterium B. henselae at an antigen-specific, sera-specific, and genome-wide level. Furthermore, these results provide novel insight and utility in diagnostics, vaccine development, and understanding of host-pathogen interaction.

[This corrects the article DOI: 10.1371/journal.pone.0163983.].

Background: Treatment of advanced cutaneous melanoma remains challenging, and new data on melanoma biology are required. The most widely accepted criteria for the prognostic evaluation of melanoma are histopathological and clinical parameters, and the identification of additional tumor markers is thus of paramount importance. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI), an important tool in cancer research, is useful for unraveling the molecular profile of melanoma., Methodology/principal Findings: In this report, we used the melanoma-bearing Libechov minipig (MeLiM), a unique animal model that allows observation of the complete spontaneous regression of invasive cutaneous melanoma, to investigate i) the differences between melanoma and healthy skin protein profiles and ii) the proteins potentially involved in spontaneous regression. The MeLiM tissues were cryosected, histologically characterized, analyzed by MALDI MSI, and immunohistologically stained. Multivariate statistical analyses of the MALDI MSI data revealed ten relevant m/z ions, of which the expression levels varied significantly among the studied MeLiM tissues. These ion peaks were used to create mass ion images/maps and visualize the differences between tumor and healthy skin specimens, as well as among histologically characterized tissue regions., Conclusions/significance: Protein profiles comprising ten statistically significant mass ion peaks useful for differentiating cutaneous melanoma and healthy skin tissues were determined. Peaks at m/z 3044, 6011, 6140 and 10180 were overexpressed in melanoma compared with healthy skin tissue. More specifically, m/z 6140 was expressed at significantly (p < 0.05) higher levels in normally growing melanoma regions than in regions with early and late spontaneous regression. This study demonstrates the clinical utility of MALDI MSI for the analysis of tissue cryosections at a molecular level.

Sparsely tested group of platinum nanoparticles (PtNPs) may have a comparable effect as complex platinum compounds. The aim of this study was to observe the effect of PtNPs in in vitro amplification of DNA fragment of phage λ, on the bacterial cultures (Staphylococcus aureus), human foreskin fibroblasts and erythrocytes. In vitro synthesized PtNPs were characterized by dynamic light scattering (PtNPs size range 4.8-11.7 nm), zeta potential measurements (-15 mV at pH 7.4), X-ray fluorescence, UV/vis spectrophotometry and atomic absorption spectrometry. The PtNPs inhibited the DNA replication and affected the secondary structure of DNA at higher concentrations, which was confirmed by polymerase chain reaction, DNA sequencing and DNA denaturation experiments. Further, cisplatin (CisPt), as traditional chemotherapy agent, was used in all parallel experiments. Moreover, the encapsulation of PtNPs in liposomes (LipoPtNPs) caused an approximately 2.4x higher of DNA damage in comparison with CisPt, LipoCisPt and PtNPs. The encapsulation of PtNPs in liposomes also increased their antibacterial, cytostatic and cytotoxic effect, which was determined by the method of growth curves on S. aureus and HFF cells. In addition, both the bare and encapsulated PtNPs caused lower oxidative stress (determined by GSH/GSSG ratio) in the human erythrocytes compared to the bare and encapsulated CisPt. CisPt was used in all parallel experiments as traditional chemotherapy agent.

The effects of sarcosine on the processes driving prostate cancer (PCa) development remain still unclear. Herein, we show that a supplementation of metastatic PCa cells (androgen independent PC-3 and androgen dependent LNCaP) with sarcosine stimulates cells proliferation in vitro. Similar stimulatory effects were observed also in PCa murine xenografts, in which sarcosine treatment induced a tumor growth and significantly reduced weight of treated mice (p < 0.05). Determination of sarcosine metabolism-related amino acids and enzymes within tumor mass revealed significantly increased glycine, serine and sarcosine concentrations after treatment accompanied with the increased amount of sarcosine dehydrogenase. In both tumor types, dimethylglycine and glycine-N-methyltransferase were affected slightly, only. To identify the effects of sarcosine treatment on the expression of genes involved in any aspect of cancer development, we further investigated expression profiles of excised tumors using cDNA electrochemical microarray followed by validation using the semi-quantitative PCR. We found 25 differentially expressed genes in PC-3, 32 in LNCaP tumors and 18 overlapping genes. Bioinformatical processing revealed strong sarcosine-related induction of genes involved particularly in a cell cycle progression. Our exploratory study demonstrates that sarcosine stimulates PCa metastatic cells irrespectively of androgen dependence. Overall, the obtained data provides valuable information towards understanding the role of sarcosine in PCa progression and adds another piece of puzzle into a picture of sarcosine oncometabolic potential., Competing Interests: The authors have declared that no competing interests exist.

Prostate cancer cells control energy metabolism by chelating intracellular zinc. Thus, zinc delivery has been a popular therapeutic approach for prostate cancer. Here, we propose the use of the membrane-penetrating peptide Novicidin connected to zinc-Schiff base as a carrier vehicle for the delivery of zinc to prostate cells. Mass spectrometry, electrochemistry and spectrophotometry confirmed the formation/stability of this complex and provided insight regarding the availability of zinc for complex interactions. This delivery system showed minor toxicity in normal PNT1A cells and high potency towards PC3 tumor cells. The complex preferentially penetrated PC3 tumor cells in contrast to confinement to the membranes of PNT1A. Furthermore, zinc uptake was confirmed in both cell lines. Molecular analysis was used to confirm the activation of zinc stress (e.g., ZnT-1) and apoptosis (e.g., CASP-1). Our results strongly suggest that the zinc-Schiff base-Novicidin complex has great potential as a novel anticancer drug., Competing Interests: The authors have declared that no competing interests exist.

Background: The environmental impacts of various substances on all levels of organisms are under investigation. Among these substances, endocrine-disrupting compounds (EDCs) present a threat, although the environmental significance of these compounds remains largely unknown. To shed some light on this field, we assessed the effects of 17β-oestradiol on the growth, reproduction and formation of free radicals in Eisenia fetida., Methodology/principal Findings: Although the observed effects on growth and survival were relatively weak, a strong impact on reproduction was observed (50.70% inhibition in 100 μg/kg of E2). We further demonstrated that the exposure of the earthworm Eisenia fetida to a contaminant of emerging concern, 17β-oestradiol (E2), significantly affected the molecules involved in antioxidant defence. Exposure to E2 results in the production of reactive oxygen species (ROS) and the stimulation of antioxidant systems (metallothionein and reduced oxidized glutathione ratio) but not phytochelatins at both the mRNA and translated protein levels. Matrix-assisted laser desorption/ionization (MALDI)-imaging revealed the subcuticular bioaccumulation of oestradiol-3,4-quinone, altering the levels of local antioxidants in a time-dependent manner., Conclusions/significance: The present study illustrates that although most invertebrates do not possess oestrogen receptors, these organisms can be affected by oestrogen hormones, likely reflecting free diffusion into the cellular microenvironment with subsequent degradation to molecules that undergo redox cycling, producing ROS, thereby increasing environmental contamination that also perilously affects keystone animals, forming lower trophic levels.

Resistant cancer phenotype is a key obstacle in the successful therapy of prostate cancer. The primary aim of our study was to explore resistance mechanisms in the advanced type of prostate cancer cells (PC-3) and to clarify the role of autophagy in these processes. We performed time-lapse experiment (48 hours) with ROS generating plumbagin by using multimodal holographic microscope. Furthermore, we also performed the flow-cytometric analysis and the qRT-PCR gene expression analysis at 12 selected time points. TEM and confocal microscopy were used to verify the results. We found out that autophagy (namely mitophagy) is an important resistance mechanism. The major ROS producing mitochondria were coated by an autophagic membrane derived from endoplasmic reticulum and degraded. According to our results, increasing ROS resistance may be also accompanied by increased average cell size and polyploidization, which seems to be key resistance mechanism when connected with an escape from senescence. Many different types of cell-cell interactions were recorded including entosis, vesicular transfer, eating of dead or dying cells, and engulfment and cannibalism of living cells. Entosis was disclosed as a possible mechanism of polyploidization and enabled the long-term survival of cancer cells. Significantly reduced cell motility was found after the plumbagin treatment. We also found an extensive induction of pluripotency genes expression (NANOG, SOX2, and POU5F1) at the time-point of 20 hours. We suppose, that overexpression of pluripotency genes in the portion of prostate tumour cell population exposed to ROS leads to higher developmental plasticity and capability to faster respond to changes in the extracellular environment that could ultimately lead to an alteration of cell fate.

Background: Somatic embryogenesis in conifer species has great potential for the forestry industry. Hence, a number of methods have been developed for their efficient and rapid propagation through somatic embryogenesis. Although information is available regarding the previous process-mediated generation of embryogenic cells to form somatic embryos, there is a dearth of information in the literature on the detailed structure of these clusters., Methodology/principal Findings: The main aim of this study was to provide a more detailed structure of the embryogenic tissue clusters obtained through the in vitro propagation of the Norway spruce (Picea abies (L.) Karst.). We primarily focused on the growth of early somatic embryos (ESEs). The data on ESE growth suggested that there may be clear distinctions between their inner and outer regions. Therefore, we selected ESEs collected on the 56th day after sub-cultivation to dissect the homogeneity of the ESE clusters. Two colourimetric assays (acetocarmine and fluorescein diacetate/propidium iodide staining) and one metabolic assay based on the use of 2,3,5-triphenyltetrazolium chloride uncovered large differences in the metabolic activity inside the cluster. Next, we performed nuclear magnetic resonance measurements. The ESE cluster seemed to be compactly aggregated during the first four weeks of cultivation; thereafter, the difference between the 1H nuclei concentration in the inner and outer clusters was more evident. There were clear differences in the visual appearance of embryos from the outer and inner regions. Finally, a cluster was divided into six parts (three each from the inner and the outer regions of the embryo) to determine their growth and viability. The innermost embryos (centripetally towards the cluster centre) could grow after sub-cultivation but exhibited the slowest rate and required the longest time to reach the common growth rate. To confirm our hypothesis on the organisation of the ESE cluster, we investigated the effect of cluster orientation on the cultivation medium and the influence of the change of the cluster's three-dimensional orientation on its development. Maintaining the same position when transferring ESEs into new cultivation medium seemed to be necessary because changes in the orientation significantly affected ESE growth., Conclusions and Significance: This work illustrated the possible inner organisation of ESEs. The outer layer of ESEs is formed by individual somatic embryos with high metabolic activity (and with high demands for nutrients, oxygen and water), while an embryonal group is directed outside of the ESE cluster. Somatic embryos with depressed metabolic activity were localised in the inner regions, where these embryonic tissues probably have a very important transport function.

Background: The role of genetic variations within the ghrelin gene on cardiometabolic profile and nutritional status is still not clear in humans, particularly in elderly people., Objectives: We investigated six SNPs of the ghrelin gene and their relationship with metabolic syndrome (MS) components., Subjects and Methods: 824 subjects (413 men/411 women, age 77.31±5.04) participating in the Mataró aging study (n = 310) and the Hortega study (n = 514) were analyzed. Anthropometric variables, ghrelin, lipids, glucose and blood pressure levels were measured, and distribution of SNPs -994CT (rs26312), -604GA (rs27647), -501AC (rs26802), R51Q (rs34911341), M72L (rs696217) and L90G (rs4684677) of the ghrelin gene evaluated. Genotypes were determined by multiplex PCR and SNaPshot minisequencing. MS (IDF criteria) was found in 54.9%., Results: No association between any of the SNPs and levels of total fasting circulating ghrelin levels was found. C/A-A/A genotype of M72L was associated with increased risk of central obesity according to IDF criteria, while G/A-G/G genotypes of -604GA with reduced risk. A/A genotype of -501AC polymorphism was associated to decreased BMI. In relation to lipid profile, the same genotypes of -604GA were associated with increased total cholesterol and LDL-cholesterol and -501AC with reduced triglycerides. There were no associations with systolic or diastolic blood pressure levels or with hypertension, glucose levels or diabetes and ghrelin polymorphisms. However, G/G genotype of -604GA was associated with glucose >100 mg/dL. Haplotype analysis showed that only one haplotype is associated with increased risk of waist circumference and central obesity. The analysis of subjects by gender showed an important and different association of these polymorphisms regarding MS parameters., Conclusion: Ghrelin gene variants -604GA, -501AC and M72L are associated with certain components of MS, in particular to BMI and lipid profile in elderly Spanish subjects.

Background and Objectives: Current studies give us inconsistent results regarding the association of neoplasms and zinc(II) serum and tissues concentrations. The results of to-date studies using meta-analysis are summarized in this paper., Methods: Web of Science (Science citation index expanded), PubMed (Medline), Embase and CENTRAL were searched. Articles were reviewed by two evaluators; quality was assessed by Newcastle-Ottawa scale; meta-analysis was performed including meta-regression and publication bias analysis., Results: Analysis was performed on 114 case control, cohort and cross-sectional studies of 22737 participants. Decreased serum zinc level was found in patients with lung (effect size = -1.04), head and neck (effect size = -1.43), breast (effect size = -0.93), liver (effect size = -2.29), stomach (effect size = -1.59), and prostate (effect size = -1.36) cancers; elevation was not proven in any tumor. More specific zinc patterns are evident at tissue level, showing increase in breast cancer tissue (effect size = 1.80) and decrease in prostatic (effect size = -3.90), liver (effect size = -8.26), lung (effect size = -3.12), and thyroid cancer (effect size = -2.84). The rest of the included tumors brought ambiguous results, both in serum and tissue zinc levels across the studies. The association between zinc level and stage or grade of tumor has not been revealed by meta-regression., Conclusion: This study provides evidence on cancer-specific tissue zinc level alteration. Although serum zinc decrease was associated with most tumors mentioned herein, further--prospective--studies are needed.

Single-molecule localization microscopy of biological samples requires a precise knowledge of the employed fluorescent labels. Photoactivation, photoblinking and photobleaching of phototransformable fluorescent proteins influence the data acquisition and data processing strategies to be used in (Fluorescence) Photoactivation Localization Microscopy ((F)-PALM), notably for reliable molecular counting. As these parameters might depend on the local environment, they should be measured in cellulo in biologically relevant experimental conditions. Here, we measured phototransformation quantum yields for Dendra2 fused to actin in fixed mammalian cells in typical (F)-PALM experiments. To this aim, we developed a data processing strategy based on the clustering optimization procedure proposed by Lee et al (PNAS 109, 17436-17441, 2012). Using simulations, we estimated the range of experimental parameters (molecular density, molecular orientation, background level, laser power, frametime) adequate for an accurate determination of the phototransformation yields. Under illumination at 561 nm in PBS buffer at pH 7.4, the photobleaching yield of Dendra2 fused to actin was measured to be (2.5 ± 0.4) × 10(-5), whereas the blinking-off yield and thermally-activated blinking-on rate were measured to be (2.3 ± 0.2) × 10(-5) and 11.7 ± 0.5 s-1, respectively. These phototransformation yields differed from those measured in poly-vinyl alcohol (PVA) and were strongly affected by addition of the antifading agent 1,4-diazabicyclo[2.2.2]octane (DABCO). In the presence of DABCO, the photobleaching yield was reduced 2-fold, the blinking-off yield was decreased more than 3-fold, and the blinking-on rate was increased 2-fold. Therefore, DABCO largely improved Dendra2 photostability in fixed mammalian cells. These findings are consistent with redox-based bleaching and blinking mechanisms under (F)-PALM experimental conditions. Finally, the green-to-red photoconversion quantum yield of Dendra2 was estimated to be (1.4 ± 0.6) × 10(-5) in cellulo under 405 nm illumination.

Metallothionein (MT) has been extensively investigated as a molecular marker of various types of cancer. In spite of the fact that numerous reviews have been published in this field, no meta-analytical approach has been performed. Therefore, results of to-date immunohistochemistry-based studies were summarized using meta-analysis in this review. Web of science, PubMed, Embase and CENTRAL databases were searched (up to April 30, 2013) and the eligibility of individual studies and heterogeneity among the studies was assessed. Random and fixed effects model meta-analysis was employed depending on the heterogeneity, and publication bias was evaluated using funnel plots and Egger's tests. A total of 77 studies were included with 8,015 tissue samples (4,631 cases and 3,384 controls). A significantly positive association between MT staining and tumors (vs. healthy tissues) was observed in head and neck (odds ratio, OR 9.95; 95% CI 5.82-17.03) and ovarian tumors (OR 7.83; 1.09-56.29), and a negative association was ascertained in liver tumors (OR 0.10; 0.03-0.30). No significant associations were identified in breast, colorectal, prostate, thyroid, stomach, bladder, kidney, gallbladder, and uterine cancers and in melanoma. While no associations were identified between MT and tumor staging, a positive association was identified with the tumor grade (OR 1.58; 1.08-2.30). In particular, strong associations were observed in breast, ovarian, uterine and prostate cancers. Borderline significant association of metastatic status and MT staining were determined (OR 1.59; 1.03-2.46), particularly in esophageal cancer. Additionally, a significant association between the patient prognosis and MT staining was also demonstrated (hazard ratio 2.04; 1.47-2.81). However, a high degree of inconsistence was observed in several tumor types, including colorectal, kidney and prostate cancer. Despite the ambiguity in some tumor types, conclusive results are provided in the tumors of head and neck, ovary and liver and in relation to the tumor grade and patient survival.

Background: Proteomics and metalloproteomics are rapidly developing interdisciplinary fields providing enormous amounts of data to be classified, evaluated and interpreted. Approaches offered by bioinformatics and also by biostatistical data analysis and treatment are therefore of extreme interest. Numerous methods are now available as commercial or open source tools for data processing and modelling ready to support the analysis of various datasets. The analysis of scientific data remains a big challenge, because each new task sets its specific requirements and constraints that call for the design of a targeted data pre-processing approach., Methodology/principal Findings: This study proposes a mathematical approach for evaluating and classifying datasets obtained by electrochemical analysis of metallothionein in rat 9 tissues (brain, heart, kidney, eye, spleen, gonad, blood, liver and femoral muscle). Tissue extracts were heated and then analysed using the differential pulse voltammetry Brdicka reaction. The voltammograms were subsequently processed. Classification models were designed making separate use of two groups of attributes, namely attributes describing local extremes, and derived attributes resulting from the level=5 wavelet transform., Conclusions/significance: On the basis of our results, we were able to construct a decision tree that makes it possible to distinguish among electrochemical analysis data resulting from measurements of all the considered tissues. In other words, we found a way to classify an unknown rat tissue based on electrochemical analysis of the metallothionein in this tissue.

Background: Thiol-rich peptides and proteins possess a large number of biological activities and may serve as markers for numerous health problems including cancer. Metallothionein (MT), a small molecular mass protein rich in cysteine, may be considered as one of the promising tumour markers. The aim of this paper was to employ chronopotentiometric stripping analysis (CPSA) for highly sensitive detection of MT., Methodology/principal Findings: In this study, we used adsorptive transfer stripping technique coupled with CPSA for detection of cysteine, glutathione oxidized and reduced, phytochelatin, bovine serum albumin, and metallothionein. Under the optimal conditions, we were able to estimate detection limits down to tens of fg per ml. Further, this method was applied to detect metallothioneins in blood serum obtained from patients with breast cancer and in neuroblastoma cells resistant and sensitive to cisplatin in order to show the possible role of metallothioneins in carcinogenesis. It was found that MT level in blood serum was almost twice higher as compared to the level determined in healthy individuals., Conclusions/significance: This paper brings unique results on the application of ultra-sensitive electroanalytical method for metallothionein detection. The detection limit and other analytical parameters are the best among the parameters of other techniques. In spite of the fact that the paper is mainly focused on metallothionein, it is worth mentioning that successful detection of other biologically important molecules is possible by this method. Coupling of this method with simple isolation methods such as antibody-modified paramagnetic particles may be implemented to lab-on-chip instrument.