Croatian Medical Journal, the official journal of the International Society for Applied Biological Sciences (ISABS), is the tenth special issue over the past eighteen years containing articles prepared for the biennial ISABS conference.This issue of the epitheton ornans of modern life sciences, most scientific conferences are in fact dedicated to specialty or subspecialty areas. ISABS breaks this mold by bringing together forensic, anthropological, and medical geneticists for a five-day celebration of the transformation of their respective fields. By providing a venue for scientists and students from different fields united in their effort to apply molecular genetic principles and techniques to societally relevant issues, the Ninth ISABS Conference aims not only to facilitate discussion of the latest scientific achievements within these three areas of applied genetics, but also to cross-fertilize these fields. This year’s conference will further stimulate the interaction among scientists from the three disciplines by avoiding parallel sessions. We hope that the participants will appreciate and use this opportunity to attend all sessions regardless of their specific expertise.Over the years, ISABS meetings have developed into a unique conference series highlighting advances in three major areas of applied genetics. Although “interdisciplinary” has become an Recognizing the breadth of topics included in the Ninth ISABS Conference and the need to pass the baton to the new generation of leaders, the founding organizers Dragan Primorac , Moses Schanfield , and Stanimir Vuk-Pavlović  tasked two Program Directors with designing the scientific programs in forensic genetics, anthropological genetics, and medical genetics.The introductory session highlights the three major conference topics by lectures on the thirty-year history of forensic DNA analysis, genomics, and individualized medicine, the recent revolution of ancient DNA analysis, and advances in the application of glycobiology to individualized medicine. The keynote address by Anthony Atala , a pioneer in the field, is devoted to regenerative medicine.Manfred Kayser put together six thematic half-day sessions of forensic and anthropological genetics focusing on the most recent developments. The three forensic genetic sessions are devoted to DNA investigative intelligence, next generation DNA sequencing in forensics, and advances in routine forensic DNA analysis, and the three anthropological genetic sessions to the analysis of ancient human genomes, modern human genomes, and human genetic history of the continents. Both programs are driven by recent achievements in massively parallel DNA sequencing (MPS), also referred to as next generation sequencing. MPS has revolutionized anthropological genetics in ancient DNA analysis and holds great promise for population-based anthropological genetics using contemporary samples as well as for forensic genetics, especially if costs can be further reduced. Another recent addition to forensic genetics is the use of DNA and RNA for investigative purposes, a topic to which a conference session is dedicated. Such information allows linking (or excluding a link between) genetically identified sample donors and criminal acts, or leads police investigation to find unknown perpetrators unidentifiable by conventional DNA profiling; hence, it broadens the forensic use of genetics beyond the courtroom.Tamas Ordog organized the medical genetics program, the Mayo Clinic Lectures on Individualized Medicine. The lectures start with an overview of the rapidly expanding use of whole-exome and transcriptome analysis via MPS in individualized patient care. The two half-day sessions include talks on host, microbial, and cancer genetics as well as epigenomics, a rapidly emerging discipline making its way into medical diagnostics and therapy. Recognizing the promise of epigenomics in diagnosis and therapy of cancer and functional and degenerative diseases, emphasized is the role of epigenetic changes in carcinogenesis, epigenetic biomarkers for early, noninvasive detection of cancer and neurodegenerative diseases, and the application of multi-parameter epigenomic testing to individualized medicine. The latter presentation includes the discussion of marker selection, logistics, bioinformatic analyses, as well as current barriers to incorporating epigenomic testing into clinical diagnostic arsenal. Talks on host genomics and epigenomics are complemented by a presentation on the role of microbiome in human physiology and disease. The program of the Ninth ISABS Conference will end with a lecture on twins ‘omics’ and personalized medicine.Similarly to past ISABS meetings, the conference includes special evening events such as the lecture by the Nobel laureate Robert Huber , talks on “celebrity genetics”, poster sessions, Meet-the-Professor sessions, and social events. Meet-the-Professor sessions have been a unique feature of ISABS conferences as they stimulate the interaction of students and other participants with the invited experts in a more relaxed and informal atmosphere than typically available in more conventional settings.In the partnership with PAIN-OMICS, a multidisciplinary consortium of leading clinical, academic and SME researchers in pain and different “omics” technologies, ISABS organizes a session that precedes the Ninth Conference.Before the Ninth ISABS Conference , the American Academy of Forensic Sciences (AAFS) holds its 2015 International Educational Outreach Program in Croatia. The Memorandum of Understanding between AAFS and ISABS will be signed during the official ceremony held at the Ministry of Science, Education and Sports of the Republic of Croatia. For the first time in the history of ISABS, a joint ISABS-AAFS forensic science panel will be organized during the Ninth ISABS Conference.Finally, the program directors and conference organizers express their gratitude to sponsors, academic and commercial, for the continuing support of this truly interdisciplinary, international conference. Bringing together a diverse group of internationally renowned scientists to facilitate research, collaborative endeavors, and education of the next generation of scientists in spite of adverse economic conditions in Croatia (and elsewhere) is a remarkable accomplishment that speaks volumes about the dedication of everyone involved, particularly the local organizers. We hope this tradition continues uninterrupted in the future, for the benefit of all.


Seeds are the major organs responsible for the evolutionary upkeep of angiosperm plants. Seeds accumulate significant amounts of storage compounds used as nutrients and energy reserves during the initial stages of seed germination. The accumulation of storage compounds requires significant amounts of energy, the generation of which can be limited due to reduced penetration of oxygen and light particularly into the inner parts of seeds. In this review, we discuss the adjustment of seed metabolism to limited energy production resulting from the suboptimal penetration of oxygen into the seed tissues. We also discuss the role of photosynthesis during seed development and its contribution to the energy status of developing seeds. Finally, we describe the contribution of amino acid metabolism to the seed energy status, focusing on the Asp-family pathway that leads to the synthesis and catabolism of Lys, Thr, Met, and Ile. Arabidopsis thaliana, amino acid metabolism is modulated so that many amino acids are synthesized and accumulated during seed desiccation . The Asp-family pathway synthesizes the amino acids Lys, Thr, Met, and Ile, which, under conditions of energy shortage, are further catabolized by the TCA cycle to generate energy , where they analyzed developing soybean seeds, which possess active seed photosynthesis  embryos possess active photosynthesis, and RuBisCO acts in a special metabolic context without the Calvin–Benson cycle in order to improve the efficiency of carbon utilization during the synthesis of oil storage  the light reactions that lead to reduction of NADP to NADPH and create a proton gradient across the chloroplast membrane that is used for ATP synthesis; and (ii) the light-independent reactions in which RuBisCO fixes COs sugars . Interes storage . This spAmino acids are constituents of proteins and hence essential components for the life of all organisms. Yet, in response to specific developmental and stress-associated conditions, amino acids also serve as energy donors through their catabolism in the TCA cycle . In vegeFigure 1) is subject to post-translational regulation, where increasing Lys feedback inhibits the activity of dihydrodipicolinate synthase (DHDPS), which catalyzies the first committed step in the Lys biosynthetic branch of the Asp-family pathway. Plants possess two DHDPS isozymes (DHDPS1 and DHDPS2), with DHDPS2 accounting for the majority of the total DHDPS activity , which contains the first two enzymes of Lys catabolism linked as a single polypeptide. In this combined genotype, seed Lys content was nearly 1000-fold higher than wild type (WT), indicating that developing seeds have a strong flux of Lys synthesis as well as catabolism into the TCA cycle  is well connected to the TCA cycle , 2011. Fneration .Figure 1; In the developing seed, the upstream substrate that feeds the Asp-family pathway on route to the TCA cycle is Asn. Asn is transported from vegetative tissues to developing seeds where it is converted by asparaginase into Asp, the starting point metabolite of the Asp-family pathway . This last finding is interesting, as it demonstrates the ability of Lys degradation products to contribute electrons directly to the mitochondrial electron transport chain, as well as supply intermediates to the TCA cycle , accepts electrons from electron transfer flavoprotein (ETF) to reduce ubiquinone. ETF serves as an obligatory electron acceptor for nine mitochondrial flavoprotein dehydrogenases. The ETF/ETFQO system facilitates electron transfer from these flavoprotein dehydrogenases to the main respiratory chain , 2006. HCA cycle .Arabidopsis accessions (Pisum sativum) embryos. When an increase of amino acid delivery was induced, transcripts of the ETF complex were most highly induced  and have been shown to accumulate BCAA under these conditions. These data suggest a possible role for ETHE1 in the ETF/ETFQO system as well as hinting at the importance of the enzyme, per se, during seed development (Although a direct role for the ETF/ETFQO system in seed development has not yet been demonstrated, considerable experimental evidence has accumulated to support this hypothesis. First, the existence of hypoxic conditions and lack of photosynthesis in some seeds point to the need for alternative electron donors in seeds for the mitochondrial electron transport chain. Secondly, analysis of a mutant with high levels of 12 out of the 20 amino acids in seeds was shown to be IVDH defective, further strengthening the role of this enzyme in the regulation of free amino acid homeostasis during seed development . Thirdlycessions . BCAT2 wcessions . Additio induced . It is inditions , 2006. Tnditions . Anotherelopment .In summary, in this review we discussed a number of studies that have enhanced our understanding of the parallel roles of photosynthesis and amino acid-derived respiratory catabolic processes in meeting the demands for normal energy status during seed development. In respect to amino acids, we focused on Lys degradation and highlighted the involvement of the ETF pathway. Technical advances, including improved understanding of sub-cellular compartmentization of metabolism (tabolism , alongsitabolism  are now tabolism , nucleartabolism , and flutabolism , alongsitabolism , will adThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Petroleum hydrocarbons are important energy resources. However, petroleum is also a major pollutant of the environment. Contamination by oil and oil products has caused serious harm, and increasing attention has been paid to the development and implementation of innovative technologies for the removal of these contaminants. Biosurfactants have been extensively used in the remediation of water and soil, as well as in the main stages of the oil production chain, such as extraction, transportation, and storage. This diversity of applications is mainly due to advantages such as biodegradability, low toxicity and better functionality under extreme conditions in comparison to synthetic counterparts. Moreover, biosurfactants can be obtained with the use of agro-industrial waste as substrate, which helps reduce overall production costs. The present review describes the potential applications of biosurfactants in the oil industry and the remediation of environmental pollution caused by oil spills. Petroleum is one of the most important energy resources and a raw material of the chemical industry. The world depends on oil and the use of oil as fuel has contributed to intensive economic development. Although petrochemical plants and oil refineries are beneficial to society, they produce a large amount of hazardous waste. Moreover, oil spills during exploration, transportation, and refining, have caused serious environmental problems ,3,4,5,6.The remediation of contaminated sites can be achieved by physicochemical or biological methods. Conventional physicochemical methods can rapidly remove the majority of spilled oil, but, in most cases, removal simply transfers contaminants from one environmental medium to another and can even produce toxic byproducts. Moreover, crude oil cannot be completely cleaned up with physicochemical methods. Thus, more attention is being given to biological alternatives ,8. BiosuThe diverse structures of biosurfactants have useful properties with industrial potential. The production of surfactants by microorganisms is performed to enhance both the bioavailability of nearly inaccessible substrates and survival under conditions of low moisture. Biosurfactant production generally requires a hydrophobic and hydrophilic carbon source in the culture medium. The process is economically and environmentally attractive when waste products are used ,11. This review addresses the potential roles and applications of biosurfactants, focusing on the petroleum industry and bioremediation processes. The key features of the microbial biosynthesis of biosurfactants, their physicochemical and bioactive properties and the use of industrial wastes as cost-effective alternatives for biosurfactant production are also summarized.The advance of sustainable technologies has driven the search for natural, biodegradable compounds to remediate sites contaminated by hydrocarbons. This has led to the discovery of surfactants of a natural origin. Most of these surfactants are synthesized by living organisms, such as, saponins produced by plants, glycolipids produced by microorganisms, and bile salts produced by animals. Compounds with surfactant properties produced by microorganisms are denominated biosurfactants ,12.Biosurfactants are mainly produced by aerobic microorganisms in aqueous media with a carbon source feedstock, such as carbohydrates, hydrocarbons, fats, and oils. It is believed that biosurfactants are secreted into the culture medium to assist in the growth of the microorganism by facilitating the translocation of insoluble substrates across cell membranes . These cBiosurfactants offer a number of advantages over chemical surfactants, such as biodegradability due to their simple chemical structure, environmental compatibility, low toxicity, which allows use in the cosmetic, pharmaceutical and food industry, high selectivity due to presence of specific functional groups, allowing specificity in the detoxification of specific pollutants, and activity under conditions of extreme temperatures, pH and salinity ,15. ThesPseudomonas spp., Acinetobacter spp., Bacillus spp., and Candida spp., types of biosurfactant, the production process and industrial applications [The fact that biosurfactants are characterized by a vast structural diversity and display a broad range of properties may explain why this group of molecules continues to pique scientific interest ,16,18. Dications . Table 1ications , lists sThe economics of biosurfactant production merit particular attention. The total production of surfactants in 2012 was ~12 million tons, only 3.5 million tons of which were biosurfactants. Moreover, revenues from the bio-based portion of the market were US$ 6.588 million . HoweverEssentially agricultural countries, such as Brazil, have easy access to agro-industrial byproducts. Pseudomonas is known for its capacity to produce extensive quantities of glycolipids. These biosurfactants are classified as rhamnolipids. Bacillus subtilis is another microorganism widely studied for biosurfactant production and is known for its efficiency in producing a lipopeptide with surface activity denominated surfactin or subtilisin [Candidabombicola and Candidalipolytica are among the most commonly studied yeasts for the production of biosurfactants [A number of microorganisms, such as fungi, yeasts, and bacteria, feed on substances that are immiscible in water, producing and using a surface-active substance (biosurfactant) ,17. Amonbtilisin ,31,32,33factants . Table 3factants .The release of contaminants, such as petroleum and petroleum byproducts, into the environment is one of the main causes of global pollution and has become a focus of great concern both in industrialized and developing countries due to the broad environmental distribution in soil, groundwater, and air ,35. The 2 [The media has consistently reported the leakage of thousands of tons of oil that contaminate seawater . One of 2 . In July2 . In Janu2 .Prestige oil spill may be considered as one of the worst. The oil tanker Prestige, loaded with a cargo of 77,000 tons of heavy bunker oil ran into problems off the Galician coast (NW Spain) on 13 November 2002. After several days following an erratic path and spilling 19,000 tons, the tanker finally sank 130 miles west off the southern coast [Over ten events of oil tankers with important wastes have occurred in Europe since 1967. The rn coast .Large amounts of crude oil entering the marine environment, groundwater and soil can cause significant harm to resident organisms . Petroleet al. [Candida sphaerica for the removal of motor oil from soil and seawater and found removal rates of 75% and 92% from clay and silty soil, respectively; in tests carried out with seawater, the biosurfactant exhibited an oil spreading efficiency of 75%, demonstrating its potential for application as an adjuvant in biotechnological processes of environmental decontamination. Batista et al. [Candida tropicalis for removing motor oil from sand and found removal rates of 78% to 97%, demonstrating considerable potential with regard to soil bioremediation. Gusmão et al. [et al. [Candida sphaerica UCP 0995. This biosurfactant removed 95% of motor oil adsorbed to sand, demonstrating considerable potential for use in bioremediation processes. The remediation of hydrocarbons contaminated soils using a cell-bound biosurfactant from Lactobacillus pentosus has been also described [The most common role of biosurfactants is to enhance the dispersal of contaminants in the aqueous phase and increase the bioavailability of the hydrophobic substrate to microorganisms, with subsequent removal of such pollutants through biodegradation ,46. Numeet al.  tested aa et al.  investigo et al.  investig [et al.  evaluateescribed ,48. Tablescribed ,49.Petroleum is one of the major energy sources. The energy demand in the world indicates a 1.7% increase in the number of barrels of oil produced per year, between 2000 and 2030, while consumption is expected to reach 15.3 billion tons of oil per year. Oil reserves allow meeting the world’s demand for approximately 40 years if current levels of consumption are maintained ,50,51. IIn the petroleum industry, biosurfactants have been applied effectively for the exploration of heavy oil, offering advantages over their synthetic counterparts throughout the entire petroleum processing chain . Biosurfactants are used in microbial-enhanced oil recovery, the cleaning of contaminated vessels and to facilitate the transportation of heavy crude oil by pipeline ,54. Tabletc. [Several enhanced oil recovery processes are currently employed worldwide: thermal, chemical, physical, etc. . Howeveretc. . Biosurfetc. . MEOR consists of the tertiary recovery of oil in which microorganisms or their metabolic products are used to recover residual oil. Microorganisms produce polymers and biosurfactants, which reduce oil-rock surface tension by diminishing the capillary forces that impede the movement of oil through the pores of rock. Biosurfactants also aid in the emulsification and breakdown of oil film in rock. MEOR involves different strategies, such as the injection of microorganisms that produce biosurfactants into the reservoir and subsequent spread in situ, the injection of nutrients into the reservoir to stimulate the growth of wild microorganisms that produce biosurfactants or the further production of biosurfactants in reactors and subsequent injection into the reservoir . These pCrude oil often needs to be transported over long distances from the extraction fields to refineries. The transportation of heavy and extra-heavy crude oil entails operational difficulties that limit its economic viability. The major problems are low flowability due to the high degree of viscosity and asphaltene content in heavy crude oil, which leads to inconveniences, such as the deposition of asphaltenes and/or paraffins as well as a drop in pressure that can cause plugging problems in the pipeline ,57. AsphA promising technology involving the production of a stable oil-in-water emulsion that facilitates oil mobility has been recently developed. Biosurfactant-based emulsifiers (bioemulsifiers) are particularly suitable for this application. Bioemulsifiers are high-molecular weight surfactants with different properties in comparison to glycolipids and lipopeptides. These products are not effective at reducing interfacial tensions, but have an excellent capacity to stabilize oil-in-water emulsions. Due to the high number of reactive groups in the molecule, bioemulsifiers bind tightly to oil droplets and form an effective barrier that prevents drop coalescence . EmulsanLarge amounts of crude oil are moved daily, distributed to refineries and placed in storage tanks. The maintenance of these tanks requires periodic washing. However, waste and heavy oil fractions that build up at the bottom and on the walls of storage tanks are highly viscous and become solid deposits that cannot be removed with conventional pumping. The removal of this material requires washing with solvents and manual cleaning, which is a hazardous, time-consuming, labor intensive, expensive procedure, as cleaning operations may include hot water spraying, solvent liquefaction and subsequent land waste disposal ,59. et al. [Gordonia sp. Dispersion activity was greater than that achieved with a chemical or plant-derived surfactant and the biosurfactant remained stable for at least three weeks. Diab and El Din [P.aeruginosa SH 29 applied to the cleaning of oil-contaminated vessels. The oil was recovered from the bottom and walls of the vessels in just fifteen minutes and floated on the supernatant as a distinct phase. According to the authors, this indicates that the biosurfactant in the sterilized supernatant of P.aeruginosa SH 29 can be used directly for cleaning oil storage tanks and other vessels used for the transportation and storage of crude oil. Rocha and Silva et al. [Pseudomonas cepacia CCT6659 for cleaning beaker walls contaminated with a layer of oil and found a removal rate of 80%, which suggests the applicability of this biosurfactant in the cleaning of storage tanks.The use of microbial biosurfactants is an alternative cleaning procedure to decrease the viscosity of sludge and oil deposits through the formation of an oil-in-water emulsion that facilitates the pumping of waste. Moreover, this process allows the recovery of crude oil when the emulsion is broken. Matsui et al.  investigd El Din  evaluatea et al.  tested tet al. [The toxicity of biosurfactants in the environment is not well known. Edwards et al. , in a coet al. .The lower toxicity and higher biodegradability of biological surfactants compared to their chemical counterparts is the main reason for their high acceptability. However, these features are often assumed as only direct consequence of their natural origin. For these reasons, the environmental features of novel biosurfactants should be carefully considered and investigated before their release into the environment . While the microbial toxicity of (bio)surfactants is a possible cause of bioremediation inhibition, many (bio)surfactants are not toxic to microorganisms at concentrations near their CMC values . AnotherRegarding the use of dispersants, several classes of these chemical agents are employed for environmental mitigation and cleanup; however, these industrial chemicals may present risks to aquatic organisms individually and when mixed with oil ,67. The U.S. Clean Water Act and Oil Pollution Act of 1990 requires the maintenance of a National Oil and Hazardous Substances Pollution Contingency Plan (NCP) for response to oil spills that identifies specific commercial products used for control of oil discharges and the quantities and water bodies in which the products may be used . These pMenidia beryllina and Mysidopsis bahia. These CTDs suggest that dispersants alone are generally less toxic than oil. In contrast, most dispersant: oil mixtures are more toxic than oil alone [Probabilistic hazard assessment approaches including Chemical Toxicity Distributions (CTDs) may be useful as an initial step toward prioritizing environmental hazards from the use of dispersants. The CTD approach to two acute toxicity datasets  contain median lethal concentration (LC50) values for dispersants alone and dispersant: oil mixtures, in two standard marine test species, il alone .Rigorous toxicological comparison of untreated and dispersant-treated oil is complicated by the fact that when oil, seawater, and dispersants are mixed, a complex multiphase system results. In this complex system, aquatic organisms can be exposed to many toxicants, in many forms, which can have several modes of action. Moreover, chemical dispersion of oil can yield: (1) dissolved petroleum hydrocarbons; (2) dissolved dispersant surfactants; (3) mixed droplets of bulk oil and surfactants (often in micellar form); and (4) nonmicellar, particulate bulk oil .A second important issue for determining the effects of dispersants, is the separate and combined toxicity of the dispersant and the crude oil droplets. Toxicity became an important issue in the late 1960s and early 1970s when application of toxic products resulted in substantial loss of sea life . Since tBecause the purpose of a dispersant is to facilitate the acceleration of natural attenuation and dilution of spilled oil, the aquatic toxicity of the dispersant: oil mixture is an important consideration. This further complicates a comparative toxicity evaluation, because the course of toxicity in a mixture may be unknown and potentially different for each dispersant. Therefore, although the presence of polycyclic aromatic hydrocarbons does not represent the only factor in determining oil toxicity, evidence links the increased presence of polycyclic aromatic hydrocarbons in chemically dispersed oils to increased toxicity to aquatic organisms ,70.2 production [The general proposal of toxicity tests is to establish the potential impact of chemicals on the biota of a given environment. The information acquired can be used to regulate use of chemical substances and evaluate the necessity for treatment after their release to the environment ,72. An uoduction , enzyme oduction , glucoseoduction , and biooduction .50) is a method that evaluates the rate of population mortality of a species and indicates that the higher the concentration, the lower the toxicity of the surfactant [Several tests have been used to evaluate the toxicity of chemical and biological surfactants on various organisms. The lethal concentration (LCrfactant ,81. The rfactant ,18,20,22This review provided information on the application of biosurfactants as a promising alternative in the petroleum industry and the bioremediation of oil spills. Since biosurfactants are not yet competitive with chemical surfactants from the economic standpoint, a more thorough investigation of biosurfactant production from agro-industrial waste is needed to reduce the production cost and allow the large-scale production of these natural compounds. The versatility and efficiency demonstrated in the application of biosurfactants in the oil production chain and the removal of hydrophobic contaminants make these compounds promising biomolecules.


Autoimmune diseases (AIDs) consist of a group of physiological disorders with highly diversified pathogenesis and clinical manifestations In this special issue, we aimed to give an overview on the most recent progress of biomarker studies on some of the important autoimmune-related diseases, such as systemic lupus erythematosus (SLE), psoriasis, systemic sclerosis (SSc), and primary Sjogren’s syndrome (pSS). Dr. Tianfu Wu’s group The prevalence of pSS is hard to be estimated due to the inconsistent classification criteria of the disease. It also varies largely among surveys in different age groups, ethnicities, and geographical regions with an obvious bias in female population SSc is a chronic multisystem AID with an estimated prevalence in the US to be around 240 cases/million adults SLE is the prototypic AID predominately affecting woman with the production of various autoantibodies as one of the major clinical manifestations. Proteomic microarray, as a powerful high throughput autoantibody screening approach, has been extensively utilized for identification of autoantibodies associated with pathogenesis of SLE. A review from Dr. Quan-Zhen Li’s group The review from Dr. Haitao Niu’s group The multiple omics technologies have greatly facilitated the discovery of biomarkers and improved our knowledge of the etiopathology of AIDs. In the meantime, the advances in high-throughput screening technologies offered excellent opportunities to identify new biomarkers potentially useful for early diagnosis, prognosis, and even prediction of therapeutic responses. Given the high complexities of the immune system and diversification of AIDs, the exploration of novel biomarkers and their underlying molecular mechanism will continue to be hot topics in the study of AIDs. Putting these articles together in this special issue might be helpful to reveal the intercommunication of the AIDs in the new bioinformatics and big data picture and may shed light on other less-studied AIDs or the discovery of universal biomarkers. In addition, novel biomarkers, especially the non-invasive biomarkers, are greatly demanded. Methodologically, application of the state-of-art novel technologies, such as NGS, as well as high-throughput proteomics and metabolomics approaches, in combination with various data analysis platforms, would greatly facilitate the discovery of multiple disease biomarkers and assessment of genes, proteins, metabolites, and network analysis of complex pathways implicated in the specific AID conditions. Comprehensive understanding of the relationship between the biomarkers and AIDs will not only provide valuable tools for disease prediction, early diagnosis, and prognosis, but also shed light on the identification of novel therapeutic target for treatment of the diseases.The authors have declared no competing interests.


Colorectal cancer is a heterogeneous disease that manifests through diverse clinical scenarios. During many years, our knowledge about the variability of colorectal tumors was limited to the histopathological analysis from which generic classifications associated with different clinical expectations are derived. However, currently we are beginning to understand that under the intense pathological and clinical variability of these tumors there underlies strong genetic and biological heterogeneity. Thus, with the increasing available information of inter-tumor and intra-tumor heterogeneity, the classical pathological approach is being displaced in favor of novel molecular classifications. In the present article, we summarize the most relevant proposals of molecular classifications obtained from the analysis of colorectal tumors using powerful high throughput techniques and devices. We also discuss the role that cancer systems biology may play in the integration and interpretation of the high amount of data generated and the challenges to be addressed in the future development of precision oncology. In addition, we review the current state of implementation of these novel tools in the pathological laboratory and in clinical practice. Colorectal cancer is a complex disease with a variable clinical course and with important divergences in the response to treatment, even in tumors with similar histopathological characteristics. Today we know that the most plausible explanation for this erratic behavior may reside in the strong heterogeneity both between and inside of tumors. Within tumors, not only do many different families or clones of cancer cells coexist  but alsoDuring the development of adenocarcinomas, a number of molecular, cellular, and histological alterations give rise to the transition from normal epithelium to adenoma and ultimately to cancer. These alterations can be caused both by genetic and non-genetic events that, in turn, can be classified into deterministic and stochastic. While the deterministic events  contribute to the generation of cell subtypes with phenotype and physiology similar to those found in normal tissues, the stochastic events  account for the cell-to-cell heterogeneity . Even whKRAS, NRAS, and BRAF genes with already known mutations, and the PI3K pathway. The hypermutated tumors were enriched in genomic alterations in genes from the TGF-beta pathway, while mutations in non-hypermutated tumors primarily affected genes from the p53 pathway. Regarding the transcriptional profile, tumors were classified into three distinct subgroups, two of which were respectively associated with MSI/CIMP and CIN features, while the third subgroup showed a mesenchymal invasive phenotype. Moreover, nearly all tumors analyzed show alterations in the expression of MYC transcriptional targets, probably as a result of increased expression and activity of MYC factor.Beyond this first approach to the understanding of colorectal cancer biology, the detailed knowledge on the specific abnormalities within tumors remained elusive until the recent developments of high throughput technologies. However, an increasing number of studies using these novel technologies are now being conducted to discover the molecular alterations behind the colorectal cancer and to find novel biomarkers and therapeutic targets ,12,13,14Special attention should be paid to the fact that two of the previously commented basic ways of alteration in colorectal tumors, the MSI and CIMP pathways, are related to epigenetic modifications, indicating the important role of the malfunction of epigenetic mechanisms in the development and clinical behavior of colorectal tumors. Epigenetic marks occur widely across the genome and extensively regulate the expression of the multitude of genes. The disposition and dosage of these epigenetic marks are strictly controlled in normal non-neoplastic cells but, in cancer cells, their deregulation opens the door to the expression or silencing of oncogenes and tumor suppressor genes, respectively. Therefore, the altered epigenome is a hallmark for many tumors, including colorectal cancer, and constitutes an important cause of heterogeneity. In fact, although the most studied and the major epigenetic events which are believed to play critical roles in colorectal cancer are CpG island methylation and histone modifications , there aAn additional source of heterogeneity resides in the tumor microenvironment  and host-tumor interactions, happening not only within tumors but also in the whole organism as a result of the spreading of cancer cells ,21,22. TTaken together, all these factors make each tumor unique with singular characteristics from the point of view of clinical course, molecular profile, microenvironment, and host-tumor interactions: this is the so-called “unique tumor principle”. Thus, the current classification of colorectal cancer (and other tumors) in a limited number of subtypes should be abandoned and a move made towards the molecular characterization of each tumor as a single entity in order to advance in the implementation of precision oncology .During recent years, several attempts of comprehensive molecular classifications based on transcriptomic profiles have been proposed, and a number of molecular signatures with potential clinical usefulness have been defined for the stratification of patients with different prognosis and treatment response ,37,38,39In one of these studies, De Sousa E Melo and co-workers  investigIn another study, Sadanandam and co-workers  made a cet al. [et al. [et al. [et al. [et al. [et al. [While many of these molecular classifications could be in the future translated to clinical use after further validation, the multiple proposals of classifiers derived from different sources, data and approaches represent an additional issue. In order to circumvent these possible concerns, a colorectal cancer subtyping consortium (CRCSC) has been recently created in an attempt to construct a unified molecular classification . After tet al.  and to t [et al. . Epithel [et al.  and with [et al. . Likewis [et al.  and with [et al. . HoweverThe existence of molecular similarities between normal cell types in colonic crypts and their malignant counterparts in colon cancer can be easily inferred from the analysis performed in one of the above-mentioned studies . This liIn addition, cancer cells may transit by many different phenotypic and molecular states in the course of tumor growth . Proof oIn addition to the variability in morphological, phenotypic, functional, or genetic characteristics, the heterogeneity in colorectal cancer can be also found in aspects related to the expression of molecules involved in antitumor immune-surveillance, specially the major histocompatibility complex (MHC). In colorectal cancer, from the earliest stages of carcinogenesis, clones of tumor cells exhibit altered expression of MHC antigens . These cRegarding MHC, the clones of tumor cells can present two types of alterations  that expThe importance of microenvironment and host-tumor interactions, and specifically the interactions between immune cells and tumor cells, has been highlighted in previous observations in colorectal cancer with MSI phenotype in which tumor-infiltrating lymphocytes were associated with good prognosis . In lineIn colorectal tumors, the heterogeneity is not only limited to primary lesions but also metastases and circulating and disseminated cancer cells may harbor different levels of variability. During the tumor growth, cancer cells detached from primary tumors reach the bloodstream and become circulating tumors cells (CTCs). Before their intravasation into blood vessels, cancer cells must undergo the de-differentiation process known as epithelial-to-mesenchymal transition (EMT) in order to attain the mesenchymal phenotype necessary for their migration and infiltration in surrounding mesenchymal tissues . Once inKRAS mutations in circulating free DNA (cfDNA) in serum of mCRC patients who developed panitumumab resistance [KRAS mutations were detected in 59% of tumor samples from mCRC patients refractory to chemotherapy and targeted agents (bevacizumab and anti-EGFR), whereas the same mutations were detected in 69% of cfDNA samples. Higher frequency of BRAF and PIK3CA mutations in cfDNA were also found regarding matched tumor samples. The increased frequency of mutations in cfDNA might reflect the emergence of mutant subclones during treatment, under-represented when the primary tumor was extracted and evaluated. How this intra-tumor heterogeneity in low-copy mutant subclones affects the clinical response to panitumumab and cetuximab remains controversial [In line with this, current research is moving on the right track exploring the usage of novel sample sources, including among others, plasma and serum . As mainsistance ,64,65. Moversial ,67,68.In addition to DNA, plasma and serum also contain relevant information in other molecular specimens, such as microRNAs  and protKRAS and NRAS mutations) have been demonstrated as being highly concordant between metastases and primary tumors [Since the cell composition and molecular characteristics of metastatic lesions might significantly differ from those found in primary tumors, the analysis of heterogeneity in metastasis could provide relevant information regarding outcome, response to treatment and management of patients with advances tumors. This fact has special relevance for the discovery of prognostic and predictive biomarkers, which are usually developed and tested in primary tumors. Given that metastases are generated by cells or clones that, although under-represented in the primary tumor, display selective advantage to grow at secondary sites ,75. The y tumors ,77,78. Fy tumors ,80,81,82y tumors , in agrey tumors .RAS wild type tumors [KRAS mutations at exon 2 (codons 12 and 13) are found in 45%–50% of mCRC. In addition, a subgroup of 14.7%–17.4% of patients with KRAS exon 2 wild-type mCRC may harbor significant mutations at other KRAS exons  and/or in the RAS isoform NRAS at the same codons assessed in KRAS. Mutations on BRAF have been found in 8%–15% of colorectal cancer and they are mutually exclusive with KRAS mutations. BRAF mutation is clearly associated with a worse prognosis and, at least in pre-treated patients, with a poor response to anti-EGFR therapy [KRAS status has been also defined as a prognostic factor in mCRC patients treated in the first line with oxaliplatin-based chemotherapy and bevacizumab [KRAS exon 2 was independently associated both with poor progression-free and overall survival.Both inter-tumor heterogeneity  and intra-tumor heterogeneity  may have implications on the prognosis, choice of treatment, and emergence of resistances. Oncogenic mutations of components of the RAS-RAF-mitogen-activated protein kinase (MAPK) pathway are common in colorectal cancer. Different clinical trials have indicated that a positive effect of anti-EGFR blockage is restricted to patients with e tumors . KRAS mu therapy ,87. KRASacizumab . In thesRAS mutations, the combined treatment with panitumumab and FOLFOX significantly improved both the progression-free survival and overall survival. In patients receiving FOLFOX4 plus panitumumab, any RAS mutations were negatively associated with outcomes [RAS wild-type subset [The addition of panitumumab to oxaliplatin-based chemotherapy in first line treatment of mCRC was explored in the PRIME trial. Among patients without outcomes . Also, te subset .EGFR ectodomain mutation [KRAS mutations [Interestingly, some patients harbor tumors with low-prevalence mutations (between 0.1% and 5% of mutant DNA to wild type). It has been suggested that selective growth of these mutated subclones could be related to clinical tumor progression. Mathematical modeling and clinical data from plasma cfDNA of mCRC patients demonstrated that most radiographically apparent lesions may contain at least 10 resistant subclones . Expansimutation  and for utations ,64.A number of novel targeted drugs are now in clinical trials in mCRC patients . ClearlyThe biological interpretation of the large amount of data obtained from the previous analyses represents an important challenge to overcome in the coming years. In order to achieve full understanding of the pathological processes underlying the malignant transformation of cancer cells as well as to develop tailored therapies and prognostic and predictive biomarkers for the precision medicine era, we need to go further in the integration of molecular data generated by high throughput technologies. This integration could allow the finding of cause-effect links between the different molecular levels analyzed by “omics”. The discipline that pursues the previous objective is systems biology and its application to the study of tumors is cancer systems biology ,117.However, despite some slight advances ,119, can


Increasing studies in viral ecology are sources of novel knowledge related to the biodiversity of living things, the functioning of ecosystems, and the evolution of the cellular world. This is because viruses are perhaps the most diverse, abundant, and ubiquitous biological entities in the biosphere, although local environmental conditions enrich for certain viral types through selective pressure. They exhibit various lifestyles that intimately depend on the deep-cellular mechanisms, and are ultimately replicated by members of all three domains of cellular life , as well as by giant viruses of some eukaryotic cells. This establishes viral parasites as microbial killers but also as cell partners or metabolic manipulators in microbial ecology. The present chapter sought to review the literature on the diversity and functional roles of viruses of microbes in environmental microbiology, focusing primarily on prokaryotic viruses  in aquatic ecosystems, which form the bulk of our knowledge in modern environmental viral ecology.Since the discovery 2–3 decades ago that viruses of microbes are abundant in marine ecosystems, viral ecology has grown increasingly to reach the status of a full scientific discipline in environmental sciences. A dedicated ISVM society, the International Society for Viruses of Microorganisms, ( As infectious agents of potentially all types of living cells, viruses are the most abundant biological entities in the biosphere. They are ubiquitous components of the microbial food web dynamics in a great variety of environments, including the most extreme ecosystems. Moreover, in spite of the difficulties to routinely observe and describe biological nanoparticles, combined with the absence of conserved evolution tracers such as RNA ribosomal genes, we now consider that viruses represent the greatest reservoir of non-characterized genetic diversity and resources on the earth  but also as cell partners and manipulators  in the world of aquatic ecosystem . The preViruses are biological entities consisting of single- or double-stranded DNA or RNA surrounded by a protein and, for some of them, a lipid coat. In aquatic systems, most viruses are tailed or untailed phages, with a capsid diameter often smaller than 250 nm, based on direct transmission electron microscopy observation. Viruses have no intrinsic metabolism and need the intracellular machinery of a living and sensitive host cell for all processes requiring energy. They have various life cycles, all starting with diffusive passive fixation on specific receptors (often transporter proteins) present at the surface of a host cell, followed by injection of the viral genome into the host cell. In the lytic cycle, the viral genome induces the synthesis of viral constituents, including the replication of the viral genetic material. A number of progeny viruses are then produced and released into the environment by the fatal rupture of the host cell.Emiliania huxleyi  allows cell recovery and the stable coexistence of viruses and their hosts . A variant to the lysogenic cycle is the so-called carrier state or pseudolysogenic cycle, where the viral genome is not integrated with the host genome but rather remains in an “inactive state” within the host cell. There is no replication of the viral genome which is segregated unequally into progeny cells, most likely for a few generations. Pseudolysogenic viruses probably occur in very poor nutrient conditions where host cells are undergoing starvation and cannot offer the energy necessary for viral gene expression.In the lysogenic cycle, the viral genome integrates the genome of the host cell and reproduces as a provirus (or prophage) until an environmental stress to the immune host cell sets off a switch to a lytic cycle. Both the provirus and the host cell benefit from lysogeny. Lysogeny provides a means of persistence for viruses when the abundance of the host cells is very low. Prophages may affect the metabolic properties of host cells which can acquire immunity to superinfections and new phenotypic characteristics such as antibiotic resistance, antigenic changes, and virulence factors, resulting in niche expansion for viral hosts  has revealed highly syntenic hierarchical cores of genes, with DNA replication genes observed in all genomes, followed by previously described, virion-structural and various hypothetical genes. Beyond previously described cyanophage-encoded photosynthetic and phosphate stress genes, genes involved in various putative functions  were indeed described, as well as non-core genes that may drive complex niche diversification  compared to that of most known siphoviruses, with a novel shell symmetry that confers a remarkable stability to a variety of physical, chemical, and environmental factors  of 29 phyla with cultured isolates have sequenced phage representatives, contributing only 0.001% of genome sequence databases . From thfication . The unvfication . Similar factors . Whole gWhole genome comparisons indeed have also shown that there are conserved genes shared among all members within certain viral taxonomic groups. These conserved genes can be targeted using PCR amplification and sequencing for diversity studies of groups of cultured and environmental viruses. Examples of such genes are structural proteins such as gp20, which codes for the capsid formation in T4 phage-like viruses, DNA polymerases for T7-like podophages, or the RNA-dependent RNA polymerase fragment, which has been used to identify novel groups of marine picornaviruses . All of For the whole environmental communities, molecular fingerprinting approaches that separate PCR-generated DNA products, such as denaturing gradient gel electrophoresis DGGE,  and pulsAn introduced fingerprinting approach adapted to viruses, randomly amplified polymorphic DNA-PCR (RAPD-PCR), allows sampling of viruses at the genetic level without requiring viral isolation or previous sequence knowledge . RAPD-PCMetagenomics has revolutionized microbiology by paving the way for a culture-independent assessment and exploitation of microbial and viral communities present in complex environments . MetagenProchlorococcus hosts, suggesting that this virus may influence the abundance, distribution, and diversity of one of the most dominant components of small phytoplankton in oligotrophic oceans  also revealed a high abundance of viral sequences, representing approximately 3% of the total predicted proteins in the 0.1–0.8 μm size fraction of the plankton . Viral sc oceans .Overall, metagenomic analysis of viruses increasingly suggests novel patterns of evolution, changes the existing ideas on the composition of the virus world, and reveals novel groups of viruses and virus-like agents . The genUnexpectedly, metagenomics of marine RNA viruses showed that representatives of one superfamily of eukaryotic viruses, the picorna-like viruses, dominate the RNA virome . Marine Prochlorococcus spp., release extracellular vesicles which have roles in various processes , and were recently demonstrated to have a significant effect on carbon cycling in marine ecosystems  in spite of more than three orders of magnitude variation in the planktonic biomass, as one ranges from either coastal to offshore or from surface to deep-water environments , are provided in an excellent review by According to time, viral abundances fluctuate on diverse scales, from minutes to years, often in association with prokaryotes, which offer the major reservoir for hosts . Surprisronments . This maronments . The higronments . Virus aronments . Evidencic Ocean . Howevereverything is everywhere, but the environment selects” , will offer predictive tools in the context of global change. Microorganisms and their viruses have long been considered as ubiquitous, without a biogeography of any sort. This is because their dispersal is thought to be unlimited due to small size, large absolute abundances and the formation of resistant or dormant stages. The ubiquity tenet for microorganisms is the so-called Baas Becking statement “selects” . The assselects” , primariselects”  or deep-selects” . It was selects” . FurtherFigure 1). Based on the direct observation of infected cells, viral-mediated mortality averages 10–50% of the daily production of heterotrophic prokaryotes and approximately equals the bacterivory from grazers in both fresh and marine waters  upon which the food web dynamic is based . It was cal pool . By explMicromonas pusilla significantly change the DOM composition by increasing the amounts of transparent exopolymer particles (TEP), aromatic amino acids, and humic DOM, with an elevated protein : humus ratio that suggested a higher contribution of labile components in viral-produced DOM than in algal exudates. At the natural community level, these results suggested that viral lysis could decrease the organic matter sedimentation and promotes respiration and nutrient retention whereas, in contrast, the enhanced TEP production could stimulate particle aggregation and export out of the water column. Based on metabolomics approach, Sulfitobacter sp. redirects 75% of nutrients into virions, and 71% of 82 intracellular metabolites were significantly elevated in infected hosts, which also exhibited an elevated metabolic activity compared to non-infected populations. In contrast, more than 70% of 56 compounds in viral lysate decreased in concentration relative to uninfected controls, suggesting that small, labile nutrients from viral lysis are utilized quickly by non-infected cells. The effects of lytic viruses thus directly affect DOM concentration but also its composition. For example, 2H6S), a gas known to influence cloud nucleation , which is massively produced by major bloom-forming species such as M. pusilla, E. huxleyi, and Phaeocystis pouchetii . In addition, viral lysis products contain polymers that can increase gel formation and affect the biological, physicochemical and optical properties of the sea water . The first major way includes the direct effects of the intrinsic activities of viruses: (i) keep in check competitive dominants , (ii) affect the metabolic properties of host cells which can acquire immunity to superinfections and new phenotypic and genotypic traits such as production of toxins , and (iii) transfer both viral and host genes between species , thereby influencing speciation. The second major way comprises the indirect effects of viral activities such as (iv) the structuring effects of lysis products on species composition and richness, (v) the sustenance of the amount of information encoded in genomes that may favor horizontal gene transfer mechanisms, and (vi) the effects of physiological mechanisms involved in the resistance of host against viruses, through the host-pathogen arms race . Together with (i) the high abundance and broad geographical distribution of viruses and viral sequences within microbial fractions, and (ii) the prevalence of genes among typical viral sequences that encode microbial physiological functions, the above-mentioned effects establish environmental viruses as strong vectors that generate genetic variability of aquatic microorganisms and drive both ecological functions and evolutionary changes . This was recently tested by Some viral groups such as the Caudovirales, the tailed double-stranded DNA phages, are probably older than the separation of life into the three now recognized domains of life . This su2 , suggest2 , was proFigure 1). A spectacular case for such a phage conversion is the finding that cholera infection is due to a lysogenic strain of the Vibrio cholerae bacterium. Since the toxin is encoded in the genome of the prophage and is not part of the host genome, non-lysogenic cells do not cause cholera  to fatal lytic infection, with intermediate mutualistic lifestyles  where viral genomes stay within the host and confer new metabolic traits which can increase the fitness of the immune host but also the survival of the phage . The transduction frequency in natural waters ranges from 10-8 to 10-5 per virus, and they might be up to 100 transductants l-1 day-1 . General mechanisms include transformation , conjugation (direct gene transfer from cell-to-cell contact), the activity of GTAs, and transduction, where viruses are the main vectors that move nucleic materials from one cell to another . More interestingly, virally encoded host genes also include crucial photosynthetic genetic elements present in cyanophage genomes, which can be used to maintain the targeted function in dead hosts and accomplish the lytic cycle, and can be transferred between hosts as well  than in their corresponding microbiomes . It has been shown that the presence of grazers in phosphorus-limited microcosms appeared to be a stimulating factor for prokaryotic growth and lytic viral proliferation, with a significant increase in the minor bacterial phylotypes as a consequence of the reduction of resource competition in prokaryotic assemblages (Cytophaga-Flavobacterium (now known as Bacteroidetes) rather changed their community size structure towards grazing-resistant forms .Lysis products can contain phage-borne enzymes which can kill cells and also influence microbial composition . Lysis pHosts and pathogens persist in the environment mainly through a molecular arms race between competing hosts and viruses where, as already discussed, viruses can affect their host in various ways, ranging from the enhancement of the host fitness and metabolic performances to mortality. Because parasites and pathogens tend to have shorter generation time than their hosts, they should evolve more rapidly and maintain advantage in the evolutionary race between defense and counter-defense. The paradox here is how do victim species survive and even thrive in the face of a continuous onslaught of more rapidly evolving enemies? One of the explanations is that the physiological, mechanical and behavioral costs of defense are lower compared to the cost of attack . The virE. huxleyi does not calcify and is “invisible” to viruses, in contrast to the susceptible diploid phase  in the marine archaea Halobacterium salinarum able to protect cells from viral infection. Heterocapsa circularisquama is repressed. Bacteria, cyanobacteria, and archaea are known to prevent viral replication by the acquisition of immune systems consisting of short fragments of foreign nucleic acids into clustered interspaced short palindromic repeats (CRISPRs) in their genomes. CRISPR spacers have homologies with mobile genetic elements such as bacteriophages and plasmids, and those identical to phage sequences provide resistance against viral infection  and is probably the vector of the horizontal gene transfer between the slug and the chloroplasts that provides 80–90% of the genes necessary for photosynthesis  alter the life story of the sea photosynthetic slugs ynthesis . Howeverult slug . PolydnaDaphnia spp. in Oneida and Cayuga lakes (upstate New York). Because Daphnia plays a critical role in many lake ecosystems, such viruses may have important effects on herbivory and thus carbon flow through the lake ecosystem.Aquatic viral ecology is a relatively recent discipline, in increasing development. Viruses are omnipresent in aquatic environments, including the most extreme and worst biotopes, where they often represent the most abundant biological entity. Because all types of cell in the three domains of life have their specific viruses and offer ecological niches to different viral lifestyles, viruses are considered the greatest reservoir of the uncharacterized biological diversity on the earth, which is being probed and described at an increasingly rapid rate, almost exclusively with molecular sequence data. For example, Our conceptual understanding of the function and regulation of aquatic ecosystems, from microbial to global biogeochemical processes, has changed with the study of viruses. Viral-mediated prokaryotic mortality roughly equals bacterivory from protists, which is a significant departure from the traditional view that predation and resource availability are the main factors controlling prokaryotic abundance and production in pelagic systems. Viruses influence both the retention and the export of organic matter in the pelagic realms. Given the prevalence of phage-encoded biological functions within host cells and the occurrence of recombination between phage and host genes, phage populations serve as gene reservoirs that contribute to niche partitioning of microbial species in aquatic ecosystems. Viral-mediated gene transfers include diverse mechanisms  that are known to affect gene evolution in the marine environment. It is thus clear that most of the viruses are not pathogens but mutualistic cell partners that provide helper functions. The discovery of giant viruses of eukaryotes (absent in this review) which encode trademark cellular functions has weakened the gap between inert and living things. Overall, studies in aquatic viral ecology are sources of novel knowledge related to the biodiversity of living things, the functioning of ecosystems, and the evolution of the cellular world. The future challenge is to extend viral ecology studies (i) to all biotopes in the biosphere such as lakes, rivers, underground waters, soils, clouds, air, etc., (ii) to all living organisms which are susceptible to viral attacks , and (iii) the related functions, some of which are crucial to global change , and (iv) to multi-partner symbioses and their effects on the food web dynamics and host maintenance and adaptive evolution.More generally, next-generation sequencing technologies are increasingly revealing that microbial taxa likely to be parasites or symbionts are probably much more prevalent and diverse than previously thought. Every well-studied free-living species has parasites; parasites themselves can be parasitized. As a rule of thumb, there is an estimated four parasitic species for any given host, and the better a host is studied the more parasites are known to infect it. Therefore, parasites and other symbionts should represent a very large number of species and may far outnumber those with “free-living” lifestyles. Paradoxically, free-living hosts, which form the bulk of our knowledge of biology, may be a minority. Microbial parasites offer good experimental models because they are typically characterized by their small size, short generation time, and high rates of reproduction, with simple life cycle occurring generally within a single host. They are diverse and ubiquitous in aquatic ecosystems, comprising viruses, prokaryotes, and eukaryotes. Extensive studies on all aspects of parasites and other symbionts in aquatic microbial ecology are warranted, including method development, life cycle, interactions with hosts and competing microbes, coevolution, effects on food webs, and biogeochemical cycles. I believe that including viruses in the more complex world of parasites and symbionts is promising for biology and ecology in the future.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A large and convincing body of evidence demonstrates that medical conditions associated with chronic inflammatory and immunological abnormalities may represent risk factors for development of neuropsychiatric disorders. Indeed obesity, dysmetabolic conditions, including diabetes, metabolic syndrome, and autoimmune diseases are frequently associated to the development of neurodegenerative and psychiatric illnesses. Recent data have demonstrated that inflammatory cytokines and adipokines can interact with multiple transmitter pathways, neuroendocrine function, synaptic plasticity, and neurocircuits relevant to behavior regulation. Therefore, further understanding of mechanisms linking obesity, dysmetabolism, and (neuro)inflammation to neurological and mental alterations may be especially relevant to the treatment and prevention for such debilitating diseases that represent a major public health concern.Along this line, the current Research Topic provides appreciable evidence showing that activated inflammatory responses can undermine disease-relevant affective, emotional, social, and cognitive functions, so that inflammatory processes may be particularly relevant for the onset and progression of neuropsychiatric disorders.The present volume, at which contributed eminent scholars includes 11 review articles, 1 original research article, and 1 commentary and was organized in the attempt to cover the whole field of the interrelationship between inflammation, dysmetabolism, and neuropsychiatric disorders.Castanon et al. provided converging evidence showing that obesity might be associated with exacerbated neuroinflammation leading to dysfunction in vulnerable brain regions associated with mood regulation, learning, and memory . Their findings shed new light on the pathophysiological mechanisms responsible for brain disorders associated to obesity and suggest innovative strategies for the treatment of neuropsychiatric complications occurring in obese patients.To this regard, a very updated and comprehensive review article by Theoharides et al. demonstrated that pro-inflammatory molecules and related signaling pathways impact on multiple neurotransmitter circuits leading to synaptic damage, neuronal loss, and the activation of other inflammatory participants . In particular, the authors emphasized the role of inflammation in the onset of brain “fog,” which represents a constellation of symptoms that include reduced mental acuity and cognition, inability to concentrate, as well as loss of short- and long-term memory. Brain “fog” may occur in patients suffering from many neuroimmune diseases, systemic mastocytosis, or disorders of mast cells activation, with increase in interleukin-6 (IL-6), tumor necrosis factor (TNF) levels. Moreover, the authors reported that the flavone luteolin might improve attention in children with autism and in mastocytosis patients with brain “fog” by reducing serum levels of TNF and IL-6. Similarly, Muller et al. provided an update about the role of inflammation in the pathogenesis of schizophrenia and the influence of immune activation on brain neurotransmissions. The authors also suggested an immune-based therapeutic approach in a subgroup of schizophrenics in whom an immune dysbalance is associated with chronic inflammation . To this regard, Bonnot et al. emphasized the association between hidden neurometabolic diseases and psychiatric disorders. According to the authors, schizophrenia-like symptoms are by far the most frequent psychiatric warning signs of neurometabolic disorders. These morbidities have a significant impact on the life expectancy of patients, since subjects with chronic psychoses show a 2–3-fold increased risk of death, mostly from cardiovascular and metabolic diseases . Therefore, Ventriglio et al. suggested that clinicians should screen and monitor these comorbidities to reduce mortality rates among patients with psychosis .Recent data, reviewed by Deleidi et al. discussed the link between immune aging and neurological disorders describing how metabolic changes linked to the aging, immunosenescence, and inflammation can enhance the development of neurodegenerative and neuropsychiatric diseases. The authors argued that targeting age-associated (neuro)inflammation could delay the onset or slow disease progression .Aging predisposes to diseases via several mechanisms that in part converge on inflammatory pathways. As age advances, the immune system undergoes a process of senescence accompanied by the increased production of inflammatory cytokines able to affect behavioral and cognitive performances. Steardo et al. explored the dual role of astrocytes in the onset and progression of AD. The authors questioning the validity of the prevalent neuron-centric vision of neuropsychiatric diseases highlighted the role of non-neuronal cells in the pathogenesis and progression of many diseases, also suggesting that astrocytes could be considered as promising target for future AD therapies .In this context, glial cells have been proved to exert a crucial role. In fact, a growing body of evidence shows the remarkable complexity of inflammatory mechanisms in Alzheimer's disease (AD) and how they are often driven by alterations of glial cells functioning. In particular, the review paper by Bedse et al. focused on the role of insulin signaling in the deposition of neuritic plaques and intracellular neurofibrillary tangles . Moreover, the authors proposed intranasal insulin administration as potential therapeutics, since preliminary clinical trials have shown cognition improvement without significant side effects . Similarly, Lutz and Meyer discussed the potential role of the pancreatic peptide amylin as a link between metabolic and neurodegenerative disorders in general, and type 2 diabetes mellitus and AD in particular, emphasizing the possible influence of amylin in the amyloidogenetic process, and providing the rationale for an amylin-based strategy in the treatment of AD (Lutz and Meyer).Recent human and preclinical studies provided convincing proof that AD is a degenerative disease associated with dysmetabolic aspects, including impairments in brain insulin responsiveness, glucose utilization, and energy metabolism .However, although the link between dysmetabolism, obesity, inflammation, and neuropsychiatric/neurodegenerative disorders was extensively investigated in adulthood, there was no convincing literature demonstrating a positive correlation between maternal dysmetabolism and child's mental health. In this perspective, Cifani et al. studied the regulation of gene transcription of several hypothalamic neuropeptides involved in the control of energy balance in rats developing obesity or not, when fed with a high fat diet . The authors provided evidence of selective and time-dependent transcriptional regulation of target genes in obese rats compared to non-obese counterparts .Besides environmental factors , the epigenetic regulation of gene expression has emerged as a potential contributor to the susceptibility and development of obesity. To investigate the individual sensitivity to weight gain/resistance, the research paper by Romano et al. examined the role of oxytocin in a variety of physiological and behavioral processes suggesting that future research will clarify whether oxytocin may represent a promising target for innovative neuropsychiatric treatments .Among the hypothalamic neuropeptides, oxytocin seems to play a crucial role in the regulation of feeding, metabolism, and in several behaviors associated with neuropsychiatric disorders (Romano et al., Taken together, this special issue might provide scientists and clinicians with new insights into this emerging area of research and it might offer more cues for the development of promising therapeutic strategies against such devastating illnesses.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In all living organisms, the phosphorylation of proteins modulates various aspects of their functionalities. In eukaryotes, protein phosphorylation plays a key role in cell signaling, gene expression, and differentiation. Protein phosphorylation is also involved in the global control of DNA replication during the cell cycle, as well as in the mechanisms that cope with stress-induced replication blocks. Similar to eukaryotes, bacteria use Hanks-type kinases and phosphatases for signal transduction, and protein phosphorylation is involved in numerous cellular processes. However, it remains unclear whether protein phosphorylation in bacteria can also regulate the activity of proteins involved in DNA-mediated processes such as DNA replication or repair. Accumulating evidence supported by functional and biochemical studies suggests that phospho-regulatory mechanisms also take place during the bacterial cell cycle. Recent phosphoproteomics and interactomics studies identified numerous phosphoproteins involved in various aspect of DNA metabolism strongly supporting the existence of such level of regulation in bacteria. Similar to eukaryotes, bacterial scaffolding-like proteins emerged as platforms for kinase activation and signaling. This review reports the current knowledge on the phosphorylation of proteins involved in the maintenance of genome integrity and the regulation of cell cycle in bacteria that reveals surprising similarities to eukaryotes. In all living cells, many cellular processes are controlled through the reversible phosphorylation of proteins on serine, threonine and tyrosine (Ser/Thr/Tyr) which results from the opposing action of kinases and phosphatases. While the role of Ser/Thr and Tyr -protein kinases (STYKs) and phosphatases in cellular regulations is extensively documented in eukaryotes, the Ser/Thr/Tyr signaling network in bacteria has a more recent history Hunter, . AnalysiBacillus subtilis by three Ser/Thr kinases, PrkC, PrkD, and YabT, leads to the deregulation of numerous target genes during the transition of vegetative to stationary growth  are histone-like proteins involved in modifying the structural organization of chromosomes by changing its topology at a global or local level  having an active role in signaling while being itself devoid of catalytic activity relevant for signaling, and (ii) being able to promote the interaction of multiple components of signaling cascades at a particular cellular location to regulate their activity , suggesting a potential ability to bring various components of tyrosine, but also serine/threonine phosphorylation pathways in close proximity -mediated control of FtsZ assembly during vegetative growth, to a RacA mediated chromosome anchorage to the pole of the fore spore compartment at the onset of sporulation  to MN and IM and the Vetenskapsrådet (2015-05319) and the Novo Nordisk Foundation to IM. TG is supported by the European Union, Marie Curie ITN AMBER, 317338.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Kluyveromyces marxianus that is widely used in the field of industrial biotechnology.Non-coding RNAs (ncRNAs), which perform diverse regulatory roles, have been found in organisms from all superkingdoms of life. However, there have been limited numbers of studies on the functions of ncRNAs, especially in nonmodel organisms such as K. marxianus by using strand-specific RNA-seq. We found that approximately 60 % of the transcriptome consists of ncRNAs transcribed from antisense and intergenic regions of the genome that were transcribed at lower levels than mRNA. In the transcriptome, a substantial number of long antisense ncRNAs (lancRNAs) are differentially expressed and enriched in carbohydrate and energy metabolism pathways. Furthermore, this enrichment is evolutionarily conserved, at least in yeast. Particularly, the mode of regulation of mRNA/lancRNA pairs is associated with mRNA transcription levels; the correlation between the pairs is positive at high mRNA transcriptional levels and negative at low levels. In addition, significant induction of mRNA and coverage of more than half of the mRNA sequence by a lancRNA strengthens the positive correlation between mRNA/lancRNA pairs.In this study, we measured changes in transcriptome at three time points during the exponential growth phase of K. marxianus in the exponential growth phase reveals pervasive transcription of ncRNAs with evolutionarily conserved functions. Studies of the mode of regulation of mRNA/lancRNA pairs suggest that induction of lancRNA may be associated with switch-like behavior of mRNA/lancRNA pairs and efficient regulation of the carbohydrate and energy metabolism pathways in the exponential growth phase of K. marxianus being used in industrial applications.Transcriptome sequencing of The online version of this article (doi:10.1186/s12864-016-2474-z) contains supplementary material, which is available to authorized users. Kluyveromyces marxianus is a non-conventional yeast species with several advantageous metabolic properties over Saccharomyces cerevisiae, such as fermentation ability at high temperatures, ability to grow on various hexose and pentose sugars, production of less ethanol in the presence of excessive sugar, and weak glucose repression, which enables the fermentation of mixed sugars, such as hemicellulose hydrolysate and inulin, at higher temperatures .The RNASeq dataset supporting the results of this article is available in the Gene Expression Omnibus (GEO) repository, [


Suicide tends to occur in families, and parental psychopathology has been linked to offspring suicidal behaviors. This study explores the influence of parental mental disorders across the lifespan. Data are from the Sao Paulo Megacity Mental Health Survey, a cross-sectional household study with a representative sample of the adult population living in the Sao Paulo Metropolitan Area, Brazil . Survival models examined bivariate and multivariate associations between a range of parental disorders and offspring suicidality. After controlling for comorbidity, number of mental disorders and offspring psychopathology, we found that parental psychopathology influences suicidal behaviors throughout most part of the life cycle, from childhood until young adult years. Generalized anxiety disorder (GAD) and antisocial personality were associated with offspring suicidal ideation , panic and GAD predicted suicidal attempts , and panic was related to the transition from ideation to attempts (OR 2.7). Although noticed in many different stages of the lifespan, this influence is most evident during adolescence. In this period, depression and antisocial personality increased the odds of suicidal ideation , and depression, panic disorder, GAD and substance abuse predicted suicidal attempts (OR varying from 1.7 to 3.8). In short, parental disorders characterized by impulsive-aggression and anxiety-agitation were the main predictors of offspring suicidality across the lifespan. This clinically relevant intergenerational transmission of suicide risk was independent of offspring mental disorders, and this underscores the need for a family approach to psychopathology. Suicide is a major public health concern. Each year, over 800,000 people die by intentional self-harm, making it the fifteenth leading cause of death globally . SuicideSuicide may be best conceived as the most severe outcome of a broader range of behaviors, which also include ideation, plans and attempts. Nonfatal suicidal behavior is 10 to 20 times more frequent than suicide  and consSuicidality tends to run in families –7, but tThis study aims to explore the association between parental psychopathology and offspring suicidality in specific lifecycle periods, examining when this influence is most prominent, its duration, and the primary predictors in each lifecycle stage. We examined these associations in the Sao Paulo Metropolitan Area, Brazil. According to the World Health Organization (WHO), 75% of suicides occur in low- and middle-income countries, and in Brazil, mortality rates have increased 10.4% between 2000 and 2012 . There aData are from the Sao Paulo Megacity Mental Health Survey (SPMHS), a cross-sectional household study of psychiatric morbidity with a representative sample of the adult population living in the Sao Paulo Metropolitan Area, Brazil . This reRespondents were selected by means of a stratified, multistage area probability sample of households. In each household, the interviewer obtained a list of all residents, with information on age, gender and family relationship to the informant. This list was then sorted by gender and inverse order of age, and the eligible respondents were identified, i.e., those who were 18 years or older, Portuguese-speaking and without any disability or handicap that would impair their ability to participate in the study. One resident was then randomly selected by means of a Kish grid, a probabilistic method for selecting household respondents from a table of random numbers . In addiThe interview was administered in two parts. Part I assessed the presence of core mental disorders and suicidal behaviors, as well as sociodemographic variables, while Part II assessed a few non-core diagnoses and potential correlates of psychopathology and suicidality, such as parental mental disorders. Part I was administered to all 5,037 respondents, while Part II was administered to 2,942 subjects, including all of those who met lifetime criteria for Part I disorders and a probability subsample of other respondents. Data were weighted to adjust for the under-sampling of Part II non-cases and to adjust for residual discrepancies between sample and population distributions on a range of socio-demographic variables. Part II respondents are the focus of the current report. Detailed descriptions of sampling and weighting methods are presented elsewhere .The Ethical and Research Committee of the University of Sao Paulo Medical School approved the SPMHS procedures. Respondents were interviewed only after informed written consent was obtained and total confidentiality was assured.Respondents were assessed using the World Mental Health Survey Composite International Diagnostic Interview (WMH-CIDI) , a fullysuicidal ideation in the total sample; suicidal attempt in the total sample; suicidal plan among ideators; and suicidal attempt among ideators.The suicidality module of the WMH-CIDI included assessment of the lifetime occurrence and age-of-onset of suicidal ideation, plan and attempt. Data were provided on the following suicidal behaviors: mood , anxiety , impulse-control  and substance use  disorders. Organic exclusion rules were used in making diagnoses.Respondents were assessed with the WMH-CIDI for the lifetime occurrence of DSM-IV Parental psychopathology was assessed with the expanded version of the Family History Research Diagnostic Criteria Interview ,28. FiveA parental psychiatric disorder was rated present if the respondent gave a positive response to questions on the core symptoms of that particular disorder occurring in the mother or the father. Prior analyses showed that the relationship between parental mental disorders and offspring suicidality was independent of whether the mother, the father or both were affected  and hereThe associations between parental psychopathology and offspring suicidality were estimated in a series of bivariate and multivariate discrete-time survival models, with person-year as the unit of analysis . This me2 tests based on design-corrected coefficient variance-covariance matrices. All significance tests were evaluated using .05 level two-sided tests.To adjust for the weighting and clustering of the stratified multistage sample design, standard errors and significance tests were estimated using the Taylor series method  using SUTo aid interpretation, survival coefficients were converted to odds ratios (ORs) and their standard errors were generated by exponentiating the survival coefficients.The lifetime prevalence of suicidal ideation, plans and attempts was, respectively, 13.84%, 4.95% and 3.39%. Among those with suicidal ideation (n = 585), more than a third also presented suicidal plans; and among those with suicidal plans, 47% attempted suicide. Women exhibited higher rates of all these suicidal outcomes.As shown in Bivariate survival models revealed that each form of parental psychopathology was significantly associated with increased odds of subsequent first onset of both suicidal ideation and attempts (OR varying from 1.5 to 3.4). However, in the multivariate models, only GAD and antisocial personality remained as significant predictors of suicidal ideation ; and only panic disorder and GAD predicted suicidal attempts . No parental disorder was associated with suicidal plans or attempts among ideators .There was a dose-response relationship between the number of parental disorders and suicidal ideation and attempts in the total sample: the greater the number of diagnoses, the greater the prevalence of these suicidal behaviors. There was no significant influence of the number of parental disorders on suicidal plans or attempts among ideators.As seen on Next step, the associations between parental psychopathology and offspring suicidality were explored during childhood (ages 3–12 years) and adolescence (ages 13–19 years), as well as their long-term effects up to young adulthood (ages 20–29 years) and late adulthood (ages 30 years and over) .The most evident influences of parental psychopathology on offspring suicidality could be found during adolescence: Parental depression and antisocial personality increased the odds of suicidal ideation ; and parental depression, panic disorder, GAD and substance abuse predicted suicidal attempts (OR varying from 1.7 to 3.8). During adulthood, parental GAD was associated with suicidal ideation and attempts . No effect of parental psychopathology on offspring suicidality was observed in later adulthood.One must note that during childhood, data indicate that parental GAD increased the odds of suicidal attempts, and that substance abuse and antisocial personality tended to reduce the odds of this behavior. However, the limited number of cases precludes any firm statements.The key results of this study are that parental psychopathology is significantly associated with suicidality in the offspring, and this effect is most evident during adolescence. Comorbidity is a common occurrence, and after controlling for the number and type of disorders and offspring psychopathology, parental anxiety (GAD and panic) and antisocial personality remained related to suicidal ideation and attempts. During adolescence, parental depression also emerged as a significant predictor of suicidal behavior.These findings are similar to those of the pooled analysis of the World Mental Health Survey Initiative. However, in the WMH, all parental disorders examined were associated with offspring suicidality , while iSuicidal behaviors are complex phenomena and result from the interactions of biological, psychological, family, sociocultural and environmental risk factors . As statAs seen, the influence of parental psychopathology on offspring suicidality is most evident during adolescence and this may be due to the convergence of the aforementioned factors during this life stage. Beyond the influence of inherited psychopathological traits and adverse family environments, teenagers are faced with physical modifications of the body and the brain. This is a period of emotional turmoil and heightened impulsivity, at least partly explained by an imbalance in the development of limbic structures  in relation to the prefrontal cortex . FurtherThe findings presented here must be considered taking into account the limitations presented by this study. All data regarding parental psychopathology and suicidal behavior were retrospectively assessed by offspring self-report. Even though childhood events can be recalled with accuracy , the resGlobally, our findings have important implications for research, clinical practice and health policies. They contribute to a greater understanding of the influence of parental psychopathology on offspring suicidality, and indicate that the transmission of suicidal behaviors is independent of the transmission of mental disorders themselves. Hence, future research must go beyond psychiatric categories and include measures of psychopathological traits and dimensions in order to elucidate the mediators and moderators of this association. The most appropriate design would be a prospective, genetically sensitive cohort to allow differentiation of genetic and environmental influences, with inclusion of other parental mental disorders and criteria of severity and persistence of psychopathology. These results are also relevant for clinicians and policy makers, underscoring the need for a family approach to psychopathology. The offspring of individuals with mental disorders is a group at greater odds of suicidality, and must therefore be actively screened for suicidal behaviors and psychopathology. As seen, adolescence is a critical stage, since the influence of parental mental disorders on offspring suicidality is most evident during this period, and suicide is the second most common cause of death in this age group . TherefoS1 Dataset(XLS)Click here for additional data file.


Ureteroarterial fistula (UAF) is a relatively rare condition with about 150 cases reported in the literature. Since it is a potentially life-threatening condition, a prompt diagnosis and treatment are crucial. We present here a rare, challenging case of UAF diagnosed after left nephrectomy, thus involving the ureteral stump. The difficult diagnosis and treatment by contemporary use of endovascular stent placement and ureteral occlusion by mean of metallic coils and Onyx injection are discussed. Ureteroarterial fistula (UAF) , a relatThe chronic use of ureteral stent is accomplished by changes on ureteral wall induced by the foreign body  and periWe present here a challenging case of UAF detected after left nephrectomy and involving the ureteral stump. The proper diagnosis and treatment by mean of endovascular stent placement and endoureteral occlusion by mean of metallic coils and Onyx injection are discussed.A 43-year-old woman underwent open hysterectomy in 2000 for cervical squamous carcinoma, followed in 2002 by salvage lymphadenectomy and chemoradiotherapy for disease relapse. In 2007 she was diagnosed with retroperitoneal fibrosis (RF), conservatively treated with long-term placement of indwelling catheters (8-F double-J catheter) despite her young age, good prognosis, and the possibility of definitive treatment. In January 2014, the patient was admitted in another hospital for the occurrence of massive haematuria and anemia. A CT scan revealed left renal hemorrhage with cloths invading the pelvis and calyces, in absence of active vascular bleeding. She underwent open surgical exploration revealing a massive renal infarction and resulting in left nephrectomy. Despite this, gross hematuria persisted.At admission in our department, she was hemodynamically stable, although intermittent gross haematuria was present. Cystoscopy showed intermittent bleeding from left ureteral orifice; the results of CT scan and elective aortography were unremarkable. The fluky occurrence of hemodynamic stability permitted us to examine in depth the cause of ureteral bleeding, and the patient was scheduled for left ureteroscopy. During the procedure, conducted under fluoroscopic guidance, an unexpected passage of contrast medium in left common iliac artery was identified, thus revealing a UAF. The ureteral catheter was left in place, reaching the upper portion of the ureteral stump, while an intraoperative selective angiography of left common artery was repeated .A stent-graft measuring 10 cm in length and 9 mm in diameter  was used to treat the arterial side of the fistula on the left common iliac artery  lead to surgical exploration and worthless left nephrectomy, which was followed by recurrence of haematuria. Despite the persistence of undiagnosed fistula repeated CT scan and selective angiography were again inadequate to demonstrate the presence of ureteroarterial communication at the ureteral stump level. Only the endoscopy of the ureteral stump with high-pressure contrast injection, acting as a provocative retrograde pyelography, permitted detecting an UAF. During high-pressure ureteroscopy there is the possibility of exacerbating hemorrhage; thus it has been undertaken with the support of a multidisciplinary team ready for emergent treatment.Definitive UAF treatment involves treatment of the artery and the ureter. Arterial treatment included endovascular stenting for poor surgical candidates, arterial occlusion with or without extra-anatomic bypass for marginal candidates, and primary open arterial repair and ureteral procedure  for adequate candidates. Ureteral management includes nephrostomy tube drainage, maintenance of an indwelling ureteral stent, nephroureterectomy, or ureteral ligation with nephrostomy tube drainage in poor surgical candidates.In our case, the vascular side of the fistula was successfully managed by endovascular stent, taking care to prevent lower limb ischemia. The ureteral side of the fistula was more difficult to manage, since no case of bleeding in the ureteral stump was reported. According to the experience of interventional radiologist, and in order to prevent fistulas' recurrence or infection of the endovascular stenting, we decided to manage the ureteral stump by the contemporary use of metallic coils followed by injection of a liquid embolic system. The combination of Onyx and coils may offer some advantages over the use of coils alone , since OIn conclusion, the occurrence of clinical trial of radical pelvic surgery, pelvic radiation, and chronic ureteral stenting for ureteral obstruction increases the risk of UAF, which requires a prompt diagnosis and management. The occurrence of a UAF fistula at ureteral stump, which is the new coming from current experience, is challenging in terms of diagnostic flow chart and management. The combination of endovascular stent placement and ureteral stump occlusion by means of metallic coil and Onyx permitted resolving a singular, life-threatening condition with excellent long-term results.