Immunisation Information Systems (IIS) are computerised confidential population based-systems containing individual-level information on vaccines received in a given area. They benefit individuals directly by ensuring vaccination according to the schedule and they provide information to vaccine providers and public health authorities responsible for the delivery and monitoring of an immunisation programme. In 2016, the European Centre for Disease Prevention and Control (ECDC) conducted a survey on the level of implementation and functionalities of IIS in 30 European Union/European Economic Area (EU/EEA) countries. It explored the governance and financial support for the systems, IIS software, system characteristics in terms of population, identification of immunisation recipients, vaccinations received, and integration with other health record systems, the use of the systems for surveillance and programme management as well as the challenges involved with implementation. The survey was answered by 27 of the 30 EU/EEA countries having either a system in production at national or subnational levels (n = 16), or being piloted (n = 5) or with plans for setting up a system in the future (n = 6). The results demonstrate the added-value of IIS in a number of areas of vaccination programme monitoring such as monitoring vaccine coverage at local geographical levels, linking individual immunisation history with health outcome data for safety investigations, monitoring vaccine effectiveness and failures and as an educational tool for both vaccine providers and vaccine recipients. IIS represent a significant way forward for life-long vaccination programme monitoring. Immunisation Information Systems (IIS) are defined as confidential, population-based, computerised databases that record all immunisation doses administered by participating providers to persons residing within a given geopolitical area . At the Following the introduction of a vaccine, its uptake and benefit-risk profile requires continuous assessment in order to monitor the performance of vaccination programmes ,3 and toFor an IIS to fully support vaccination programmes, there are various features that are considered important. These can include: (i) complete and accurate denominator populations from different sources; (ii) secure vaccine recipient and record identification through uniform unique identifiers (UID); (iii) complete, timely and correct vaccination records with real-time electronic access to the IIS; (iv) recording of vaccinations given to the recipient and vaccine details  facilitated by pre-entered information, selection menus and reading of barcodes; (v) production of automated outputs; (vi) the facility to offer services that are useful to all parties including vaccine recipients, parents and vaccine providers. This includes for example: recall functions, trusted medical information, and the possibility for parents and vaccine recipients to request certified records of immunisation history.The European Council conclusions on childhood immunisation in 2011 and on vaccinations as an effective tool in public health in 2014 both recommend the adoption of such systems and the World Health Organization European Vaccine Action Plan 2015–2020 recognises IIS as ‘an integral part of well-functioning health systems’ .This article presents the findings of a survey conducted by the European Centre for Disease Prevention and Control (ECDC) across EU/European Economic Area (EEA) countries that assessed the level of implementation of IIS and their functionalities, as well as the challenges encountered during the design and implementation. The aim of the survey was to share knowledge about IIS in the EU/EEA in order to build consensus on the characteristics of an optimal system and to describe differences in core functionalities and standards across countries.Following a review of the literature and in consultation with subject-matter experts, two cross-sectional surveys were developed to assess the status of IIS implementation and functionalities in EU/EEA countries ,19.The first, more comprehensive survey, which included 100 questions, targeted countries with an IIS in operation or being piloted. The other, briefer survey (including nine questions), targeted countries with no IIS or IIS at a very early stage of implementation. The surveys can be found on the ECDC website . RespondThe full comprehensive survey explored the current status of IIS implementation, governance, regulation and financial sustainability, population covered, nature of the data recorded, technical solutions used, linkage with other health information systems, outputs generated, and challenges and barriers to implementation.The briefer survey examined the current status of IIS implementation, barriers to the planning or implementing of IIS, plans for the future, and if there was a strategy for e-Health in place.The surveys opened on 1 May 2016 and closed on 20 May 2016. Countries that could not complete either of the two surveys by the deadline were asked to complete a basic set of five questions.In May 2016, the 28 EU Member States plus two EEA countries (Norway and Iceland) were invited to participate in the surveys. Respondents were identified through the ECDC National Focal Points (NFPs) for Vaccine Preventable Diseases (VPD).The EU survey tool was used to administer the survey . In counThe United States Centers for Disease Prevention and Control (US CDC) definition of an IIS was used as a reference in this survey  Box). . Box). BoxIIS are confidential, population-based, computerized databases that record all immunisation doses administered by participating providers to persons residing within a given geopolitical area. At the point of clinical care, an IIS can provide consolidated immunisation histories for use by a vaccination provider in determining appropriate client vaccinations. At the population level, an IIS provides aggregate data on vaccinations for use in surveillance and programme operations, and in guiding public health action with the goals of improving vaccination rates and reducing vaccine-preventable disease.Information was received from 27 countries of the 30 contacted, with 16 countries answering the full comprehensive survey, nine countries answering the brief survey and two countries (Luxembourg and Slovakia) answering only to the basic set of five questions.The list of responding institutions and which survey they completed is shown in Among the 27 countries who responded to either the comprehensive or brief survey or the basic set of five questions, 17 provided information on governance and financial support. In the survey, governance was defined as ‘the body at national or regional level that is in charge of the day-to-day management of the IIS and of the data contained in the system’.For eight of the 13 countries with national systems, governance of the IIS is the sole responsibility of the National Institute of Public Health (NIPH). For two countries governance is held by the Ministry of Health (MoH), for Latvia it is held by the National Health Service (NHS), in Romania it is held by both the NIPH and MoH, and in Slovakia it is held by the National Health Information System (NHIS). Among the four countries with subnational systems, i.e. Belgium (described through Flanders), Spain  and the United Kingdom (UK) (described through England), governance is held by subnational or regional health authorities. In Portugal (described through mainland) it is held by both the NIPH and MoH .Financial support for the IIS comes from the national government for thirteen countries. In Latvia and Slovakia the IIS is funded by the national government and EU funds. The regional government finances the IIS in Belgium (Flanders) and Spain .The status of implementation of IIS in the 27 countries is as follows . Eight countries have a currently operating national system that meets the US CDC definition of an IIS, i.e. Denmark, Finland, Iceland, Ireland, Malta, the Netherlands, Norway and Romania. In Finland the IIS includes more features than specified in the US CDC definition.Two countries (Germany and Sweden) have national systems in place that do not fully meet the US CDC definition of an IIS. In particular, their systems have no ability to consolidate immunisation histories for use at point of clinical care and only provide aggregated data on vaccinations at population level. Five countries have more than one subnational IIS, including Austria (number not specified), Belgium , Portugal (mainland and Madeira), Spain  and the UK . For Belgium, Portugal, Spain and the UK, the survey describes the systems in operation in Flanders, mainland Portugal, Andalucía and England respectively. The systems in Belgium, Portugal and Spain fulfil the criteria of the US CDC IIS definition. The UK systems vary, some systems do meet the CDC definition of an IIS while others do not. This information was not available for Austria as they completed the short version of the survey where this question was not asked.Four countries, Greece, Hungary, Latvia and Slovakia are piloting a national system. Latvia had planned to pilot its system in 2017. France is piloting more than one subnational IIS. Bulgaria is piloting one subnational IIS. Among the countries piloting an IIS, whether at sub-national or national level, how the IIS was defined was only provided by Hungary and Latvia, as these two countries participated in the comprehensive survey. Both countries had an IIS fitting the US CDC IIS definition.Six countries, including Croatia, Cyprus, Czech Republic, Estonia, Luxembourg and Slovenia have no IIS in operation or being piloted. Cyprus, Estonia, Luxembourg and Slovenia all have concrete plans to implement an IIS in the future.The results discussed in the following sections are based on questions only included in the comprehensive survey, hence only the 16 countries that responded to this survey  are inclOf 16 countries, which participated in the comprehensive survey, 13 have systems fitting the US CDC definition of an IIS [IIS in two countries (Germany and Sweden) do not fulfil the criteria of the US CDC definition of an IIS. The subnational systems in the UK (England) are varied, with some fulfilling the US CDC definition and others not. In Germany the system is based on insurance claims data from all physicians providing medical services (including vaccinations) to the statutory health ensured population in Germany . Physicians or vaccination providers  do not have access to this database. In Sweden the objective of the national vaccination register is to improve monitoring of the national vaccination programmes and is not used by vaccination providers in determining appropriate client vaccinations at the point of clinical care. In the UK (England) availability of vaccination history at point of clinical care is variable. In primary care, it is dependent on the supplier of the General Practice Information Technology (GP IT) system and the local Child Health Information System while in secondary care it is not available.In 15 of the 16 countries, the government authority is the owner of the IIS software; whereas in the UK (England), there are five major private sector software suppliers. Fifteen of 16 countries provided information on software source code development, this information was missing for Hungary. Seven countries used a private company and six countries used programmers from the government authority. Two countries systems were developed by a mix of private and government programmers. Fourteen of the 16 countries provided information on the type of software used. Seven countries used commercial software. Three countries, Germany, Latvia and Spain  had both a partially open and partially commercial source. In Finland and Malta, it was open source with no license required, whereas in Romania, it was a free to use software, but a license was necessary. In Portugal (mainland), the software was developed specifically by the MoH. Information on the type of software was missing for Belgium and Hungary.The survey did not collect elements related to data hosting, applied standards and system architecture.Information included in the IIS is fed by a population registry in 13 of 16 countries. Of these 13, seven used the civil population registry, three used the healthcare registry, Denmark and Iceland used both the civil and the healthcare registries and Finland’s system is fed by patient data system records. In Germany, Ireland and Romania personal data are entered manually when the patient comes for their first vaccinations. Ten of 16 countries reported that an individual vaccination record was created automatically in the IIS database when a live birth is registered (or a time later). In seven countries vaccination records were also set-up automatically at the time of immigration to the country. Ten countries record life-long vaccination data in the IIS with no restriction of age or vaccination setting . The IISAll 16 systems use a unique personal identifier for each immunised individual recorded in the IIS . In 11 cFourteen of 16 countries can record vaccinations administered in the past and 13 systems can record vaccinations administered abroad . This isFor 15 countries, to ensure that a vaccination entry is valid, vaccine providers are able to select the vaccination to be administered from a list included in the system. For seven countries the data captured in the IIS is validated automatically by the system through pre-set rules and similar. The measures that countries use to audit the quality of the data in the IIS was not captured in this survey.When a vaccine is administered, vaccination information is entered into the IIS in real-time in Denmark, Iceland, Malta, Norway, Portugal (mainland), Spain , Sweden and the UK (England).In order to estimate vaccination coverage nine countries of 15 use the civil population registry as the source of denominator data for the IIS. Germany, Hungary, Portugal (mainland) and Spain  use healthcare registries as the denominator. In Ireland the number is manually obtained from the school census and Romania uses the number of newborn children from maternity hospitals. Information on vaccine coverage denominator was missing for Latvia.In order to compute aggregated vaccination uptake on the smallest administrative area, eight countries of 16 used nomenclature of territorial units for statistics (NUTS) 3  and HungSix countries of 16 can use their systems to record adverse events following immunisation (AEFI). In Belgium (Flanders), AEFIs can be added and marked in colour, so at the time of future vaccination when the provider goes online this can clearly be seen. In two countries (Ireland and Latvia) the system is used for routine passive reporting of AEFIs to health authorities. In Portugal (mainland), the system allows recording of AEFIs, however reporting to fulfil regulatory requirements is done through another system.Eleven countries can link their IIS with various health outcome registers. For five countries some of these registers are integrated within the IIS and for the other six countries linkage with other health outcome registers is either routinely carried out or performed for specific purposes. Thirteen of 14 countries allow public health organisations to use IIS data for research, such as in vaccine effectiveness studies and safety studies. Latvia has not yet defined this and there was no information from Spain  for this question. In five of these 14, other non-public health organisations can have access to the IIS data for research.Ten countries can use their IIS to identify unvaccinated individuals in the event of an outbreak.Five of 16 countries  and the UK (England)), have automated systems that can send reminders to people who are due to get vaccinated. The systems in Latvia, Spain , Portugal (mainland) and the UK (England) can send automatic reminders to the vaccine provider to call a patient for the next vaccination.In five of 15 countries ), the vaccine recipient or guardian has access to the IIS. There was no information available for Hungary for this question. These five countries, plus Belgium (Flanders), also provide vaccine recipients with the ability to independently obtain an individual immunisation history that is accepted as an official immunisation record directly through the IIS or through an exchange platform.Regarding outputs from IIS systems, five of 16 countries have a system that allows vaccine providers to identify which vaccines to administer based on the recipient’s age, previous vaccination, allergies, travel and risk factors. In Belgium (Flanders), Portugal (mainland) and Spain , the IIS can be used to communicate information on new vaccines, updated policies, safety concerns and out-of-stock situations to vaccine providers. Thirteen countries can use it to identify individuals who are incompletely vaccinated according to age and ten countries can use it to record reasons for refusing vaccination.Countries had encountered a number of challenges during the different phases of IIS implementation.The most common challenges faced during the decision to set up an IIS were a lack of human resources  and a lack of funding ), followed by issues relating to data protection (9/14 – no answer from UK (England) and Spain ).During the design phase, challenges faced by most countries included defining the functions required by the system (12/15 – no answer from UK (England)) and a lack of standards to provide a point of reference for developing the system (10/15 – no answer from UK (England)), and defining the core dataset of information to be collected (10/15 – no answer from UK (England)).During the early use phase (those countries that were piloting IIS were asked to leave this section blank), the main issues encountered included training vaccine providers to use the system ), validation of data entered by different users ) and quality control of data completeness ).For the nine countries with no IIS in place or in the initial stages of implementation and who answered the brief survey, the main challenges were a lack of standards (7/8 – no answer from Austria), data protection issues (7/9) and issues relating to governance and ownership of the system (6/8 – no answer from Austria).The findings of the survey provide information on the extent of IIS implementation and systems functionalities in 27 EU/EEA countries. Most EU/EEA countries either have an operational IIS or are piloting one. Of the countries who have no systems in operation, Estonia, Luxembourg and Slovenia all have concrete plans to implement an IIS as part of their larger eHealth strategies in the coming years and Cyprus plans to implement a system as part of the new National Health System . This wiMonitoring vaccination programmes relies not only on accurate and complete denominators and numerators for calculating vaccination coverage but also ensuring that the data captured in the system is reliable. The quality of data contained in each of the IIS in operation was not assessed through this survey. However, in regards to the source used for denominator data, an IIS that is populated automatically from birth and civil population registers, from national health insurance schemes or school registration is more likely to be complete. The countries who responded to the survey were advanced in this area. All countries used either the civil population registry, healthcare registries, school census or number of newborn children from maternity hospitals as data sources. All countries were also able to estimate coverage at subnational levels. In Finland and Portugal (mainland) for example, coverage can be assessed for populations with the same postal code and for populations using the same healthcare centre. At a population level, it is particularly important to be able to assess coverage in areas that are at high risk for low vaccination uptake. For example, in the Netherlands, the IIS can monitor coverage in areas of known low vaccination coverage, such as the ‘Bible Belt’ area, so as to adapt interventions .For the numerator, the recording of vaccinations and vaccine details are also critical pieces of information required for coverage calculation. To minimise errors, manual data entry of vaccine details should be avoided. All the countries can validate the data entered into the IIS through methods such as bar code readers ), drop-down menus to select from a pre-defined list of vaccines (in 15 countries), linking to a product database (e.g. in Finland and Hungary) or uploading from electronic medical records by web services (e.g. in Belgium (Flanders)). This is another major strength of the systems operating in the EU/EEA in that they do not rely on manual data entry to capture information on vaccinations received.In regards to the characteristics of an IIS it is desired that the data captured in the IIS are complete, timely and of high quality. To ensure completeness, the IIS should ideally be populated with data from all vaccine delivery sites (whether public or private providers), they should cover the entire population and hold information on all vaccines recommended by health authorities regardless of funding. Many countries’ systems only capture vaccines provided in public health services and for those vaccines that are recommended and funded under the national immunisation schedule. To ensure timeliness and reduce underreporting it is essential that the time between vaccination and the information being entered into the IIS is minimised so that the information is in real-time. This is particularly relevant during emergency situations  or outbrThe IIS can also be used as a tool for informing public health decisions and research beyond vaccination coverage. The IIS constitutes large datasets that can be used in pharmaco-epidemiological studies to assess vaccine safety and effectiveness. Interoperability of the IIS with other health information systems has been used in studies such as the investigation of narcolepsy with pandemic influenza vaccination in Finland ; and simOther important features of an IIS include automated reminder/recall, access and education. At present, systems in Latvia, Malta, the Netherlands, Portugal (mainland) and the UK (England) can send reminders to people who are due to get vaccinated and the systems in Latvia, Portugal (mainland), Spain  and the UK (England) can send automatic reminders to the vaccine provider to call a patient for the next vaccination. Providing public access to the IIS and allowing vaccine recipients to print immunisation records are valuable features. Vaccine recipients can view their records in the IIS in six countries ). Six countries allow recipients to directly access an official immunisation record through the IIS. By providing vaccine recipients with some level of ownership over their records and having online access to information on particular vaccines and the disease they protect against may be beneficial to the uptake of vaccination. Such systems also provide the opportunity for being used as educational tools for both vaccine providers and recipients. This can be done by including an easily accessible platform that provides clear information and visualisation of data, using, for example, dashboards. The systems in Denmark and Norway are linked to a web-based application that allows users to visualise in real-time the coverage at communal level with a graphical snapshot of current or historical vaccination coverage trends. This can be useful for informing interventions and raising community awareness.The implementation of an IIS is a significant commitment at national and subnational levels in terms of financial investment to cover both human resources and technology developments as well as ensuring supportive legislation to allow for personal data to be recorded and used. Some of the challenges identified through the survey include the need for human resources and funding. Other challenges brought forward included the lack of standards. ECDC is well-placed to facilitate such exchange and collaboration in a more systematic way such as supporting EU countries in developing and agreeing to a minimal set of functionalities for an IIS, as a reference to help countries with IIS in the development phase. ECDC could also help in identifying lessons to be learned from other countries outside the EU/EEA. In the US, individuals and organisations with an interest in IIS have formed the American Immunization Register Association (AIRA), which in collaboration with the US CDC, has published platform neutral IIS best practices and standards . Also thThe survey had some limitations. First, the survey did not include interviews with immunisation programme managers or other key stakeholders, such as decision-makers, programme and IT staff, which would have been useful to provide a more detailed overall picture of the IIS in countries surveyed. Second, the survey did not cover the transition period from paper-based to electronic registries. Last, the survey did not cover in detail the measures that countries use to audit the quality of the data in the IIS, such as the use of a paper-based questionnaire to compare with the data captured in the IIS. Despite these limitations this survey has provided critical information about systems across the EU/EEA and can be used as a further step for in-depth assessment of system performance. The survey also provided key information about the challenges and barriers that countries faced at different stages of implementation of the IIS. Sharing this knowledge and lessons learnt can potentially assist countries to overcome these issues especially those countries that are in the early stages of developing/using an IIS or are planning to implement a system in the future.Within the EU/EEA, countries vary considerably with respect to recommended vaccines, organisation of health services, mandate of public health agencies, legislation on confidentiality and other relevant factors. Despite this, the exchange of information and experience between national programmes has been useful in the development of IIS in many EU/EEA countries.The setting up of an IIS is an important commitment for countries and requires careful planning of resources and time. ECDC can play an important role in bringing together key stakeholders, defining common areas of work and challenges, and facilitating exchange of knowledge and experience in order to support countries to implement or upgrade an IIS. The current focus on eHealth in the EU and at national level provides the perfect opportunity for IIS to become an integral part of electronic health systems.


Realgar as a potentially toxic element is contained. Our previous experiments revealed that it was less toxic for combined Realgar in NJT. However, the active fraction of this prescription with toxicity alleviation effect on Realgar was still obscure. NJT was divided into five different polar fractions , and we explored the toxicity alleviation effect on Realgar. Based on 1H NMR spectra of urine and serum from rats, PCA and PLS-DA were performed to identify different metabolic profiles. Liver and kidney histopathology examinations and serum clinical chemistry analysis were also performed. With pattern recognition analysis of metabolites in urine and serum, Realgar group showed a clear separation from control group, while the metabolic profiles of NJT-PET, NJT-25, NJT-50, and NJT-95 groups were similar to Realgar group, and the metabolic profiles of NJT and NJT-75 groups were very close to control group. Statistics results were confirmed by the histopathological examination and biochemical assay. The present work indicated that 75% EtOH fraction of NJT was the most valid fraction with the toxicity alleviation effect on Realgar.Niuhuang Jiedu Tablet (NJT) is a classical formula in treating acute tonsillitis, pharyngitis, and so on. In the formula, significant level of Realgar (As2S2), as a type of mineral drug that contains arsenic, has been used in traditional Chinese medicines (TCMs) for thousands of years. It has been used for the treatment of carbuncles, boils, insect and snake bites, intestinal parasitosis, convulsive epilepsy, and psoriasis [ Realgar is commonly prescribed with other herbal medicines or minerals in Chinese compound formulae. Niuhuang Jiedu Tablet (NJT) composed of Realgar and seven other TCMs is one of the most commonly used over-the-counter TCMs for the treatment of acute tonsillitis, pharyngitis, periodontitis, and mouth ulcer. Each tablet contains 3.33 mg of Bovis Calculus Artifactus, 33.3 mg of Realgar, 133.2 mg of Gypsum Fibrosum, 16.7 mg of Borneolum Syntheticum, 133.2 mg of Rhei Radix et Rhizoma, 99.9 mg of Scutellariae Radix, 66.6 mg of Platycodonis Radix, and 33.3 mg of Glycyrrhizae Radix et Rhizoma [ Realgar is poorly soluble in water and considered to be less poisonous, upon ingestion of Realgar-containing TCMs, the potential of arsenic poisoning cannot be ignored [ Realgar have been reported [soriasis . Rather ealgar AsS2, as a  ignored . Recentlreported . The saf1H NMR, which is noninvasive and nondestructive, reveals the overall metabolic profile of biofluids or tissue extracts and offers vital information on metabolite structure [Metabonomics is defined as “the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification” . It can tructure . NMR-bastructure . Realgar combined with other TCMs in NJT was investigated, and the results indicated that it was more secure and less toxic for combined Realgar in NJT [ Realgar was still obscure. The objective of the present work was to evaluate the toxicity alleviation effect of five different polar fractions of NJT on Realgar using metabonomics method based on NMR.In our previous study, the toxicity ofr in NJT . However2O  and sodium-3-(trimethylsilyl) propionate-2, 2, 3, 3-d4 (TSP) were purchased from Norell Inc. (USA). All the other chemicals were of analytical grade and commercially available. Realgar, Bovis Calculus Artifactus, Borneolum Syntheticum, Gypsum Fibrosum, Rhei Radix et Rhizoma, Scutellariae Radix, Platycodonis Radix, and Glycyrrhizae Radix et Rhizoma were purchased from Anguo Changda Chinese Herbal Pieces Ltd.  and identified by Professor Yuehu Pei . The voucher specimens were deposited in the School of Traditional Chinese Materia Medica of Shenyang Pharmaceutical University.D Realgar), that is, Bovis Calculus Artifactus, Borneolum Syntheticum, Gypsum Fibrosum, Rhei Radix et Rhizoma, Scutellariae Radix, Platycodonis Radix, and Glycyrrhizae Radix et Rhizoma in the weight ratio of 0.1 : 0.5 : 4 : 4 : 3 : 2 : 1, was extracted with water (4 L) under reflux for 2 h, a process that was repeated twice. After filtration, the extracts were combined and concentrated by vacuum evaporation to syrup, which was suspended in water (800 mL). The suspension was extracted with equal volumes of petroleum ether, and the petroleum ether fraction (NJT-PET) was obtained (8.8 g). The water layer was further chromatographed over a D101 macroporous resin column, eluted with 25%, 50%, 75%, and 95% ethanol gradually and the fractions NJT-25, NJT-50, NJT-75, and NJT-95 were obtained , respectively. Preparation of the extract and derived fractions is illustrated in A 730 g amount of mixed herbs  were purchased from Experimental Animal Center of Shenyang Pharmaceutical University (no. SCXK (Liao) 2010-0001). During the whole experiment procedure, the rats were maintained in a 12 h/12 h light/dark cycle at a constant temperature of 22 ± 3°C with relative humidity of 45–60% and allowed free access to food and water. Animal studies were conducted under approved guidelines of the Animal Ethics Committee of Shenyang Pharmaceutical University.Realgar 2 g/kg), (3) NJT group (Niuhuang Jiedu Tablet 10 g/kg), (4) NJT-PET group , (5) NJT-25 group , (6) NJT-50 group , (7) NJT-75 group , and (8) NJT-95 group . Animals were given the calculated amounts of materials via gastric intubation one time each day for consecutive seven days with the administration of volume of 20 mL/kg (rat body weight).All drugs were dispersed in 1% CMC-Na. After one-week acclimatization, the rats were randomly distributed into eight groups of six each: (1) control group , (2) Realgar group  over ice packs from 8:00 p.m. to 8:00 a.m. on predose day −1 and on postdose days one to eight. The collected urine samples were stored at −20°C until analysis. Rats were sacrificed on day eight, and blood samples drawn from abdominal aorta were centrifuged at 15000 ×g for 10 min at 4°C to obtain serum samples. All serum samples were frozen at −80°C for further analysis. Livers and kidneys were rapidly isolated, washed in sterile 0.9% (w/v) sodium chloride solution, and fixed in 10% formalin solution for histopathological examination.Urine samples were collected in tubes containing 50 Clinical biochemical parameters of serum included aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (CREA), triglyceride (TG), and total cholesterol (TC) were performed with an automated Hitachi Analyzer .μm thickness, and stained with hematoxylin and eosin (H + E) for histopathological evaluation.Liver and kidney tissues were embedded in paraffin blocks, sectioned to 5 μL urine and 200 μL phosphate buffer  were mixed to minimize chemical shift variation. Then 500 μL supernatants were obtained by centrifugation at 3500 ×g for 5 min and were transferred into 5 mm NMR tubes to which 40 μL TSP (1 mg/mL) and 20 μL D2O were added. TSP served as an internal chemical shift reference (δ 0.0) and D2O provided a field frequency lock-solvent for the NMR spectrometer. 1H NMR measurements of urine samples were recorded on a Bruker AV 600 MHz spectrometer at 298 K using one-dimensional (1D) Nuclear Overhauser Effect Spectroscopy (NOESY) pulse sequence with presaturation during relaxation delay (RD = 3.0 s) and mixing time (tm = 0.1 s) to suppress the water signal. A total of 64 transients were collected into 64K data points with a spectral width of 12 kHz and an acquisition time of 2.73 s.After thawing at room temperature, an aliquot of 400 μL of the supernatant was transferred into 5 mm NMR tubes containing 100 μL TSP (1 mg/ml) and 200 μL D2O. NMR spectra of serum samples were also measured on a Bruker AV 600 MHz spectrometer. Pulse program was one-dimensional Carr-Purcell-Meiboom-Gill (CPMG) sequence. 64 free induction decays (FIDs) were collected into 64k data points using a spectral width of 12 kHz and an acquisition time of 2.73 s.Serum samples were thawed at room temperature and centrifuged at 15000 ×g for 10 min. 300 1H NMR spectra were manually phased, baseline adjusted, and referenced to TSP at δ 0.00 using MestReNova 9.0.1 software (Mestrelab Research SL). Every spectrum was subsequently segmented into regions at 0.04 ppm intervals across the chemical shift δ 9.60–0.20. The regions of δ 5.20–4.72 in urine spectra and δ 5.20–4.60 in serum spectra were excluded to avoid any baseline distortion caused by imperfect water suppression. The regions of δ 6.20–5.48 in urine spectra were also removed to eliminate the influence of broad resonance from urea. The regions contributed to citrate (δ 2.70–2.64 and δ 2.58–2.52) were combined into two signals δ 2.66 and δ 2.54, respectively. The integrated data were normalized to the total integrals of each spectrum to compensate for the concentration differences.All R2 and Q2 values. R2 is used to evaluate the fitting condition of the models and Q2 is used to assess the predictive ability. Data were visualized with the scores plots and loadings plots. Each point on scores plots represents an individual spectrum of a sample and each point on loadings plots represents a single spectral region or chemical shifts. Classification of samples and the endogenous metabolites responsible for the classification can be shown from the scores and loadings plots, respectively.The mean-centered NMR data were submitted to SIMCA-P 11.5 software package  for pattern recognition analysis. Principal component analysis (PCA) was initially used to obtain a general overview of the metabolic pattern. Then partial least square discriminant analysis (PLS-DA) was performed to reveal the different metabolic alterations between the dosed and control group. The quality of these models can be explained by t-test using SPSS 16.0 software . Values of P < 0.05 were considered to be statistically significant.All numerical data are presented as mean ± SD. The data were statistically analyzed through one-way analysis of variance (one-way ANOVA) and Dunnett  Realgar did not exert significant effects on serum clinical chemistry parameters. Serum enzyme values of the rest groups were elevated in varying degrees compared to the control group.As shown in  Realgar. Rats of NJT and NJT-75 group showed recovery trends for liver and kidney injuries induced by Realgar.Histopathological findings of livers and kidneys of rats from the eight groups were displayed in Figures 1H NMR spectra of urine samples from each group were shown in β-glucose, α-glucose, allantoin, phenylalanine, 3-indoxylsulfate, hippurate, and formate were detected.Typical PCA-based profiling of the NMR spectra of the urine samples from the eight groups was employed to explore the intrinsic differences in the metabolisms of these rats . The samIn order to maximize the separation between experimental groups and to focus on metabolic variations significantly contributing to classifications, the PLS-DA models were subsequently performed. As shown in the scores plot , the sepThe PLS-DA scores plot reveals that Realgar group could be easily distinguished from control, NJT, and NJT-75 groups . From ex1H NMR-detected relative integral levels of metabolites in urine samples of different groups are shown in The 1H NMR spectra of serum from controls and treated groups. Assignments of endogenous metabolites were based on the literatures [3, leucine/isoleucine, valine, VLDL/LDL-CH2, lactate, alanine, acetate, N-acetyl glycoprotein, methionine, acetoacetate, pyruvate, 2-ketoglutarate, citrate, creatine, choline, TMAO, glucose, and unsaturated lipid.eratures , 16 and PCA was firstly conducted for the eight groups, and all the spots are located inside of the confidence interval . An obviTo further identify the distinction of the metabolic profiles and discriminating metabolites, PLS-DA was applied to the data. The scores plot  shows th1H NMR spectra are increased leucine, isoleucine, valine, alanine, 2-oxoglutarate, and TMAO with decreased lactate, acetate, pyruvate, creatine, and choline in NJT and NJT-75 groups compared with Realgar group.The performance of scores plot reflects discrimination where NJT and NJT-75 groups lie apart from Realgar group . The maj1H NMR-detected relative integral levels of metabolites in serum samples of different groups are shown in The  Realgar was verified [ Realgar was not yet clear. In the present tests, NJT was therefore divided into five fractions: NJT-PET, NJT-25, NJT-50, NJT-75, and NJT-95, to find the active fraction. Based on histopathology examinations, serum clinical chemistry analysis, and metabolomics method, NJT-75 was ascertained to be the active fraction of NJT that had toxicity alleviation effect on Realgar.In our previous experiments, the toxicity alleviation effect of other TCMs in NJT onverified , but theALT and AST in liver cells can leak into serum during hepatic injury , and the Realgar-dosed rats, which indicated that Realgar caused suppression of the TCA cycle and could further induce organ failure [ Realgar-exposed rats.Tricarboxylic acid (TCA) cycle is a significant biological metabolic pathway that not only involves glucose aerobic oxidation but also participates in the major pathways of lipid oxidation and amino acid metabolisms . One of  failure , 22, 23. failure . This no Realgar-dosed rats suggested that Realgar caused stimulated glycogenolysis and glycolysis, which makes contribution to the elevated levels of pyruvate [Marked elevated level of plasma glucose found in thepyruvate . Meanwhipyruvate , 26. Realgar-dosed rats. These ketone bodies are the products of β-oxidation for fatty acid in mitochondria [β-oxidation of fatty acid and the reduced utilization of acetyl-CoA into the TCA cycle or an increase in anaerobic cell respiration, which was a kind of energy metabolism process [Furthermore, levels of ketone bodies, such as acetate and 3-hydroxybutyrate, were increased in urine and serum samples ofchondria . Elevati process . Realgar, which suggested that NJT-75 had the same accommodation effects on the energy metabolism disorder as NJT.NJT-75 could recover the lower metabolic levels of citrate and 2-oxoglutarate, and the higher levels of pyruvate, lactate, acetate, and 3-hydroxybutyrate induced by Realgar exposure [Choline is a major membrane constituent and is important to the integrity of cell membranes and lipid metabolism . Consistexposure , and the Realgar treatment was combined with the administration of NJT and NJT-75, the concentration of choline got close to its normal level, which indicated that NJT and NJT-75 have protective effects on membrane impairment induced by Realgar treatment.However, when Realgar treated group, which indicated the alteration of intestinal flora. Betaine, which is a metabolite of choline, serves as organic osmolytes in biological organisms. Significant raised levels of betaine in urine and serum reflect the enhanced bioavailability of choline to form betaine as a consequence of inhibited degradation pathway from choline to TMAO [The choline metabolism is one of the metabolic pathways that is affected by gut microbiota that leads to the formation of trimethylamine (TMA) through host microbial interactions . TMA is  to TMAO . In case to TMAO . Realgar-exposed rats also indicated that the balance of intestinal microbial environment was influenced by Realgar.Hippurate is formed in hepatic tissue by the conjugation of glycine and benzoic acid, and the formation depends on the favorable supply of ATP . The cha Realgar was cotreated with NJT and NJT-75, the levels of TMAO, betaine, and hippurate in urine and serum got to the normal standard, which suggested a potential recovery of intestinal environment regulated by NJT and NJT-75.In this study, when Realgar treated rats.Liver is the main metabolic regulatory organ and deeply involved in amino acid absorption, degradation, and metabolism , and any Realgar treated animals implied a Realgar induced slowdown of glucose-alanine cycle [ Realgar group. The increase of urinary amino acids might mean disruption to hepatic amino acid metabolism as well as decline to reabsorb low molecular weight compounds as a typical manifestation of nonspecific proximal tubular damage [Alanine plays a key role in glucose-alanine cycle between tissues and liver, in which alanine is formed by transamination of glucose-derived pyruvate and is transported to the liver, where its carbon skeleton is reconverted to glucose . The decne cycle . It has ne cycle , 42. Ther damage . Realgar.Taurine, which is one of the end-products of cysteine catabolism and synthesized mainly in the liver , is one Creatine is a nitrogenous organic acid, which is synthesized primarily in liver from the methylation of glycocyamine by S-adenosyl methionine . It is t Realgar combined with NJT and NJT-75, respectively, these situations mentioned above could be effectively improved.However, when rats were treated with2S2 is insoluble in aqueous solutions, whereas only elements dissolved in gastrointestinal tracts could be absorbed by the body [III) and arsenate (AsV), were reported to be very toxic, with AsIII being even more toxic than AsV. Monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) exhibit only about 1/400 toxicity of inorganic forms, while arsenobetaine (AsB) and arsenocholine (AsC) are almost nontoxic [Asnontoxic . Realgar is commonly prescribed in Chinese compound formulae, the amount of arsenic leached from the Realgar-containing TCM prescriptions might be influenced by the coexisting components and therefore the arsenic absorption and metabolism might be altered [ Rhei Radix et Rhizoma, Scutellariae Radix, Platycodonis Radix, and Glycyrrhizae Radix et Rhizoma could reduce the solubility of arsenic to varying degrees in artificial gastric juice or artificial intestine juice [ Realgar alone or NJT showed increased transformation from AsV to DMA and faster elimination rate of DMA in NJT, which might be attributed to the drug-drug interactions among the chemical constituents in the compound preparation of NJT [ Realgar in NJT.Since altered . In vitrne juice , 51. A cn of NJT . The main of NJT , 53, flan of NJT –56, and n of NJT –59, whicn of NJT . All of  Realgar. Our results indicate that 75% EtOH fraction of NJT has the toxicity alleviation effect on Realgar. The obtained results might be useful for scientific understanding of the compatibility mechanisms of NJT. In addition, our work also illustrates that metabonomic approach is a useful tool in the investigation of toxicological effects of traditional Chinese medicines and other xenobiotics.In the present study, a combination of NMR spectroscopic metabonomic analysis, histopathology, and clinical biochemistry assays were used to investigate the active fraction of NJT with toxicity alleviation effect on


Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine with an important role in the pathogenesis of several diseases. Its encoding gene is located in the short arm of chromosome 6 in the major histocompatibility complex class III region. Most of the TNF-α gene polymorphisms are located in its promoter region and they are thought to affect the susceptibility and/or severity of different human diseases. This review summarizes the data related to the association between TNF-α gene and its receptor polymorphisms, and the development of autoimmune diseases. Among these polymorphisms the −308G/A TNF-α promotor polymorphism has been associated several times with the the development of autoimmune diseases, however some discrepant results have been recorded. The other TNF-α gene polymorphisms had little or no association with autoimmune diseases. Current results about the molecules controlling TNF-α expression are also presented. The discrepancy between different records could be related partly to either the differences in the ethnic origin or number of the studied individuals, or the abundance and activation of other molecules that interact with the TNF-α promotor region or other elements. Tumor necrosis factor (TNF), first termed by O’Malley et al. , was iniThe roles that TNF-α play seem to be contradictory and this was related to the genetic polymorphisms in the genes regulating its production and effect , −863 (C/A), −857 (C/A), −851 (C/T), −419 (G/C), −376 (G/A), −308 (G/A), −238 (G/A), −162 (G/A), and −49 (G/A)  in Dutch and UK Caucasian population as approved by Bayley et al.  and Bartα drugs when compared to 676TG  showed conflicting results. For example, the −238 G/A polymorphism did not have an effect on the patients’ outcome in either Turkish or Czech patients while the −308 G/A polymorphism was significantly associated with a poor outcome in the Turkish group but not in the Czech patients   , was thought to play a role in up regulation of TNF-α gene expression as Ang II activates two transcriptional factors that are important in mediating TNF-α gene expression, nuclear factor (NF)-Oxidized low density lipoprotein (LDL) and LDL were demonstrated to decrease TNF mRNA expression in NK cells  has been also reported to play a key role in the induction of TNF-α (Barnes et al. In conclusion, the expression of TNF-α seems to be controlled by the activation of other cellular molecules including signal transducer molecules, nuclear factors and second messenger molecules. More studies to address the interaction between these molecules and TNF-α are required to finally map a TNF-α pathway. Profiling the expression of the key molecules inside such pathway can open an avenue to control TNF-α over or downregulation.TNF-α plays an important role in the pathogenesis of autoimmune diseases. Several studies show that TNF-α gene promoter polymorphisms affect the susceptibility and/or severity of autoimmune diseases. Polymorphisms in the TNF receptors does not seem to be associated with the development of autoimmune diseases. The −308 G/A TNF-α promotor polymorphism seems to be highly associated with the development of these diseases, however some discrepant results have been recorded. Other TNF-α gene polymorphisms had little or no association with autoimmune diseases. This discrepancy might be explained by the differences in the ethnic orgin or number of the studied individuals. TNF-α gene expression is controlled by the presence of some polymorphisms in its promoter region and by several types of signalling molecules or nuclear factors that interact with the TNF-α promotor region or other elements.


Astrobiol Outreach1, 105 (doi:10.4172/2332-2519.1000105)). Evidence from two studies indicates that the nucleated Planctomycetes–Verrucomicrobia–Chlamydia superphylum members are the most ancient Bacteria known ; Jun et al. 2010 Proc. Natl Acad. Sci. USA107, 133–138 (doi:10.1073/pnas.0913033107)). This review summarizes the evidence for the NuCom hypothesis and discusses how simple the NuCom hypothesis is in explaining eukaryote evolution relative to the other hypotheses. The philosophical importance of simplicity and its relationship to truth in hypotheses such as NuCom and Domain Cell Theory is presented. Domain Cell Theory is also proposed herein, which contends that each of the three cellular lineages of life, the Archaea, Bacteria and Eukarya domains, evolved independently, in support of the NuCom hypothesis. All other proposed hypotheses violate Domain Cell Theory because they posit the evolution of different cellular descendants from ancestral cellular types.In 2015, the Royal Society of London held a meeting to discuss the various hypotheses regarding the origin of the Eukarya. Although not all participants supported a hypothesis, the proposals that did fit into two broad categories: one group favoured ‘Prokaryotes First’ hypotheses and another addressed ‘Eukaryotes First’ hypotheses. Those who proposed Prokaryotes First hypotheses advocated either a fusion event between a bacterium and an archaeon that produced the first eukaryote or the direct evolution of the Eukarya from the Archaea. The Eukaryotes First proponents posit that the eukaryotes evolved initially and then, by reductive evolution, produced the Bacteria and Archaea. No mention was made of another previously published hypothesis termed the Nuclear Compartment Commonality (NuCom) hypothesis, which proposed the evolution of the Eukarya and Bacteria from nucleated ancestors (Staley 2013  This phThat the origin of the Eukarya is still a hotly debated subject is attested to by the contributions to a recent meeting of the Royal Society in London in 2015 [before nucleus) organisms are simpler and evolution leads to greater complexity, the prokaryotes, i.e. the Bacteria and Archaea, must have been the first organisms.Those who favoured ‘Prokaryotes First’ hypotheses trace their ideas most recently to the Ring Theory of Life . A basicMariscal & Doolittle  summarizUnfortunately, an entirely different hypothesis termed the Nuclear Compartment Commonality (NuCom) hypothesis  was not Finally, cell theory will be discussed. Current cell theory holds that every cell comes from a cell. Domain Cell Theory, proposed below, states that when the domains of life evolved, each of the three domains evolved from separate and unique cellular lineages.2.PVC Bacteria are the PVC superphylum [sn-1,2 stereochemistry linked to the fatty acid side chains by ester bonds (G3P PLFA). Some species such as Gemmata obscuriglobus have cellular compartments with nuclei [The erphylum , some meh nuclei . In addiEnucleation is the process whereby a nucleated organism loses its nuclear compartment through reductive evolution. For example, nucleated ancestors of the Verrucomicrobia may have evolved to produce the Proteobacteria because both contain methanotrophic bacteria, use the Calvin–Benson carbon dioxide fixation process and contain prosthecate bacteria [bacteria ,8.Common bacteria are defined as typical Bacteria, like Escherichia coli, a proteobacterium whose DNA is not contained in a nuclear compartment.Protokaryote (Greek proto meaning ‘first’ and karyon ‘nucleus’) refers to the pre-domain ancestral cell state of the last universal common ancestor (LUCA). NuCom proposes that the ancestor of the PVC Bacteria and the Eukarya were two emergent phylogeneticaly distinct protokaryotic lineages with a simple nuclear compartment  are homologous proteins currently found as remnants in certain representatives of the PVC superphylum as well as almost all representatives of the Eukarya (some have termed these ‘eukaryotic signature proteins').Eukaryogenesis is defined as the continuous evolution of eukaryote cellular complexity and organization in this unique domain.3.3.1.A popular view held by many is that the Archaea are the ancestors of the Eukarya either by evolving directly to produce the Eukarya  or via aAdvocates of Prokaryotes First hypotheses at the 2015 Royal Society meeting fell into two groups:Group A: Fusion event occurred between a bacterium and an archaeon that led to the evolution of the EukaryaFusion advocates have invoked the synthesis of the eukaryotic cell from the biological merger between a bacterium with an archaeon. The most challenging issue facing the ‘Prokaryotes First’ fusion advocates is that they need to explain how two highly divergent cell types produced a protoeukaryote. This is difficult to explain from three primary standpoints.Firstly, and perhaps most importantly, if fusion occurred it was a ‘once-only’ or singular event. This singular event cannot be reproduced in the laboratory or subjected to rigorous scientific study. From a philosophical standpoint, a hypothesis that is not verifiable or falsifiable is unscientific  and therSecondly, several questions remain unanswered. For example, how did the resulting eukaryote retain one cell membrane type rather than another? Several groups question, for example, the ability of an archaeon to engulf a bacterium, a necessary step in the entrainment of a mitochondrion in eukaryotic evolution ,12 and fThirdly, this hypothesis is in violation of Domain Cell Theory because one cellular lineage was created by the fusion of two different cell types (see below).Group B: The Archaea were the direct ancestors of the EukaryaOther ‘Prokaryotes First’ advocates propose that the Archaea, alone, evolved to produce the Eukarya . This cuet al. [Williams & Embley  proposedet al.  raise doBoth groups of the Prokaryotes First school of thought have retained an early view of organism evolution that believes eukaryotes must have evolved from simpler organisms. There is virtually no evidence that this is what actually happened.et al. [et al. [Prosthecobacter dejongeii) and a Planctomycete (Gemmata strain Wa1-1). That study concluded it was unlikely that the PVC superphylum gave rise to the Eukarya.Also, some microbiologists proposed that the PVC superphylum is ancestral to the Eukarya. More recently, McInerny et al.  rebutted [et al. , who conet al., this author believes they wrongly regarded the ‘ESPs’ (eukaryotic signature proteins), such as bacterial tubulin and serine threonine kinase (STK) genes as horizontal gene transfers (HGTs) from Eukarya [Although the latter reference agreed with most of the conclusions of McInerny  Eukarya ,19. By rSignificantly, although most of these ancient proteins have been explained as more recent HGT events by some , the enz3.2.Hypotheses of the Eukaryotes First proponents were also presented at the Royal Society meeting , with vaThis author agrees with one very important point that the Eukaryotes First proponents espouse, namely that it is simpler to produce a prokaryote from a eukaryote (i.e. a nucleated organism) than to produce a eukaryote from one or two prokaryotes. This point of view is consistent with the NuCom hypothesis that explains the evolution of the Bacteria and Eukarya from nucleated ancestors.4.The NuCom hypothesis ,19 stateet al. [According to NuCom, the Bacteria are also descended from nucleated organisms. Phylogenetic evidence supporting NuCom comes from two independent groups. One group provided phylogenetic information from highly conserved regions of 16S rDNA  that indet al.  arrived The Common Bacteria are regarded as having become enucleate by reductive evolution from PVC superphylum ancestors. The rationale for this is that by maintaining a smaller and less complex genome, they could compete more efficiently for their niches. The example given in the original NuCom hypothesis is that of the Verrucomicrobia giving rise to the Proteobacteria . Both grFurther phylogenetic support for NuCom comes from ancient PSPs such as α- and β-homologues of tubulin that have been found in the PVC superphylum. These proteins have been called ESPs by many because they are found in phylogenetic trees with eukaryote homologues. By contrast, these proteins, which are found in a few representatives of the PVC superphylum, are regarded as PSPs of LUCA by the NuCom hypothesis because of their ancient phylogeny. They occur as remnants in certain species indicating that reductive evolution has occurred in PVC phyla as well as in the enucleate Common Bacteria but their presence in the PVC reveals their deep ancestry from LUCA.Finally, and perhaps most importantly, the NuCom hypothesis is the simplest hypothesis to explain the origin of the Eukarya. The Simplicity analysis, which regards the simplest hypotheses and theories to be more likely true philosophically, is used in physics and in chemistry, although much less often in biology. Hypotheses and theories that are the most simple are considered not only most likely to be true, but are also aesthetically more favourable . This ar5.Prokaryotes First advocates believe that the Eukarya arose later in time because fossil evidence for them is non-existent until about 1.5–2.0 Ga bp. However, NuCom regards that eukaryotes evolved during the time that DNA replication evolved in the PVC Bacteria. The NuCom hypothesis explains the later appearance of the Eukarya in the fossil record by a series of stages of a long process of complexification termed eukaryogenesis (dates below are approximate).Stage A. Evolution of DNA replication in LUCA.When DNA replication evolved in LUCA it gave rise to two disparate lineages, the Bacteria and the Eukarya. Therefore, both lineages date to about 3.0 Ga bp.Stage B. Early period for the Eukarya: detection of Eukarya was difficult because:(1) Cells were unicellular with only a membrane enveloping them—therefore they did not leave identifiable fossil traces.(2) Few cells were formed because they had a poor energy source—they probably were fermentative and lived off available sugars.12C-fractionated methane or Bacteria such as the Cyanobacteria that produced oxygen.(3) No exceptional, unique products were produced by eukaryal metabolism—unlike methanogenic Archaea which give rise to (4) Actin evolution began about 2.5 Ga bp. This led to the ability of Eukarya to engulf foodstuffs, the singular early means that still characterizes the unique eukaryal feeding mode, phagocytosis. Notably, we as human omnivores still use it and make a big fuss about it, too!Stage C. Mitochondrial entrainment—ca 2.5 to 2.0 Ga bp .(1) The mitochondrion evolved from an aerobic member of the Alphaproteobacteria which, after engulfment, was entrained by symbiosis within the ancestor of all Eukarya. This enormously enhanced their ability to make ATP.(2) These early Eukarya were still unicellular and difficult to detect because they lacked cell walls.(3) The Cellular Compatibility argument (see below) provides a rationale for how a bacterium became the mitochondrion.Stage D. Period of evolution of mitosis, meiosis and sexuality and larger, more complex multicellular organisms. About 2.0–l.5Ga bp until the present.Eukaryogenesis occurred over many millions of years, but it was not until they had fully evolved that the Eukarya as we know them today could be readily detected in the fossil record.Although the early stages (A – early stage D) could not have been easily detected in the fossil record, by about 1.5–2.0 Ga bp, the evolution of the Eukarya eventually gave rise to the more readily detectable contemporary single and multicellular organisms including certain protists, algae, plants and animals.6.Several examples of ancient highly conserved proteins (PSPs) are found in the PVC superphylum as well as the Eukarya. A summary of this information is provided below that supports the common origin of these proteins in LUCA and the nucleated descendants of the PVC Bacteria and Eukarya.6.1.prima facie evidence for the ancient common ancestry of these PSPs in both Bacteria and Eukarya completely in accord with NuCom.As noted previously, the cell membranes (and hence nuclear membranes of the nucleated organisms) of the Bacteria and Eukarya are identical so far as is known. They both comprise G3P PLFA. The pathway for their synthesis is also identical so far as is known, including homologous enzymes for each of the steps . This isSome have proposed these cell membrane proteins (enzymes) represent HGTs between the Bacteria and Eukarya. For example, in their review Poole & Penny  discuss Interestingly, although cell and nuclear membranes represent early commonalities between the Bacteria and Eukarya, their genomes diverged from one another enormously and gave rise to the two most plentiful and diverse forms of life on Earth.all Bacteria, not simply the PVC superphylum. These ancient membrane proteins from LUCA provide irrevocable testimony to a common origin of the Bacteria and the Eukarya that is consistent with NuCom.Most importantly, these cell membrane proteins are found in The following ancient PSPs found in the PVC superphylum have been regarded by others as due to HGT from a eukaryote. However, there is no basis for this other than that they are found in phylogenetic trees close to those of the Eukarya. NuCom rightly reclaims them as PSPs derived from LUCA and not examples of HGT events.6.2.Prosthecobacter genus contain the highly conserved tubulin proteins bacterial tubulin A (BtubA) and bacterial tubulin B (BtubB) which are homologous to α- and β- eukaryotic tubulin, respectively [Perhaps the most remarkable protein reported in the PVC superphylum is tubulin. The α- and β-homologues of tubulin have been found in all members of the Eukarya. Lynn Margulis, an early proponent of Prokaryotes First concepts, hypothesized that tubulins came from the spirochetes, which was consistent with her view that the spirochetes evolved tubulin that provided motility to some immotile protists. However, no spirochete genome has ever been reported to contain tubulin genes whereas some of the Verrocomicrobia do. Several species of the ectively .Prosthecobacter species [FtsZ is a much smaller homologue of tubulin that is found in all Bacteria and some of the Archaea and is required for cell division. Importantly, FtsZ is also found in the tubulinate  species , suggest6.3.The ubiquitin system contains enzymes that are responsible for the degradation of proteins and is found in all eukaryotes. Eukaryote-like serine/threonine kinases (STKs) and E2-ubiquitin-conjugating enzymes are also found in the PVC superphylum, including members of the Planctomycetes , Chlamyd6.4.Gemmata genus contains deeply rooted enzymes (PSPs) consistent with their origin in LUCA [Pirellula staleyi provides strong support for the pathway in extant members of the PVC superphylum [Some members of the Planctomycetes contain sterols in their cell membranes which are also found in the Eukarya as well as some Common Bacteria. Importantly, the sterol synthesis pathway in the  in LUCA , althoug in LUCA . The exterphylum .7.Studies of the phylogeny of ancient protein folding families (FF) are also consistent with NuCom . These aThis perspective article also proposes the Domain Cell Theory of Life which supports the NuCom hypothesis because Woese's three domains of life comprise three independent cellular lineages. Fusion between two cellular types does not occur. The entrainment of an alpha-proteobacterium to become a mitochondrion and a cyanobacterium to become a chloroplast in the Eukarya do not change the fundamental cellular type of the Eukarya in which they became endosymbionts.8.omnis cellula e cellula).Schleiden and Schwann proposed the cell theory of life which states that all living organisms are cellular. All cells are derived from pre-existing cells. As stated by Virchow in 1859 , ‘every different cellular types: Archaea, Bacteria and Eukarya.The Tree of Life provides a further elaboration of the meaning of cell theory. This is herein named Domain Cell Theory, which posits that the domains in Carl Woese's Tree of Life comprise three sn-1,2 stereochemistry linked to the fatty acid side chains by ester bonds (G3P PLFA).— All Bacteria have cell membranes containing glycerol 3-phosphate with — All Bacteria have or have had peptidoglycan cell walls during their evolution.— The PVC superphylum contains the most ancient members of the Bacteria, some of which are nucleated.1. Only Bacteria can give rise to other Bacteria with their unique genetic composition and evolutionary trajectory.sn-1,2 stereochemistry linked to the fatty acid side chains by ester bonds (G3P PLFA).— All Eukarya have cell membranes containing glycerol 3-phosphate with — All members of the Eukarya contain a nucleus with nuclear membranes.— Eukaryogenesis describes the process by which Eukaryotic cells evolved engulfment of particulate materials (phagocytosis), mitosis, meiosis and sexuality.2. Only Eukarya can give rise to other Eukarya with their unique genetic composition and evolutionary trajectory.— All Archaea have glycerol 1-phosphate (G1P) ether linkages in their cell membranes.3. Only Archaea can give rise to other Archaea with their unique genetic composition and evolutionary trajectory.Domain Cell Theory  contains the following tenets:In particular, each of the three cellular lineages is distinct from the other two on the basis of cellular evolution, genetic composition and cell envelope types, which include cell and nuclear membrane and cell wall, if present. This is logical because cell membranes must have existed at the time LUCA gave rise to the three separate domain lineages. Domain Cell Theory states that the descendants of each of the three domains retained its identity throughout its own unique evolutionary pathway.A primary feature or tenet of Domain Cell Theory is that it is not possible to produce a different cell type from the fusion of two other cellular types as proposed by ‘Prokaryotes First’ proponents. Another characteristic is that it is impossible for one cellular type to become a different cellular type, such as the direct evolution of the Eukarya from the Archaea . AccordiLikewise, the various ‘Eukaryotes First’ proposals that the Archaea and Bacteria evolved from the Eukarya are also contrary to the Tree of Life, which clearly indicates a separate evolution of all three cellular lineages. In particular, the Eukaryotes First proponents propose that the Eukarya evolved by reductive evolution to produce the Bacteria and Archaea. This is also a violation of Domain Cell Theory. Thus, none of the Prokaryotes First or the Eukaryotes First hypotheses is valid.Further support for Domain Cell Theory is that, of all the thousands of Bacteria, Archaea and Eukarya that have been studied, each of them can be placed into one of the three domains of life. If cellular types are freely able to change, then one should ask, ‘Where are the intermediate types or species among these three different domains?’ To my knowledge, none exist.Regarding Domain Cell Theory discussed above, two mitigating factors need to be mentioned. The first is that viruses are known to play a very important role in transferring genes from one organism to another, which could lead to the introduction of genes into different lineages. An especially interesting proposal is that the three domains of life originated in an RNA world in which, during the transition to DNA, three different founder DNA viruses gave rise to the three domains of organisms .In addition, HGT is also known to be a mechanism to transfer genes from one group of organisms to another. However, it is noteworthy that these events, including viral transfer, typically occur between closely related taxa. Unfortunately, HGT has been sometimes misused to support hypotheses that are otherwise unsupportable.9.It is interesting to note that modifications of cells do occur within at least two of the domains, the Bacteria and the Eukarya. Regarding the Bacteria, according to NuCom, the original nucleated ancestors of the PVC superphylum gave rise to the enucleate Common Bacteria which lost their nuclei through reductive evolution . This prWith respect to the Eukarya, all species have a mitochondrion or did have a mitochondrion during their evolution. Those that no longer have mitochondria lost them through reductive evolution. This is explained by Cellular Compatibility as follows. The proto-eukaryotic cells lacked mitochondria because the Alphaproteobacteria lineage that gave rise to the mitochondrion had not evolved until approximately 2 Ga bp. Because cell membranes of the Bacteria and Eukarya are highly similar, the two cellular types are compatible. The engulfed pre-mitochondrion lost its peptidoglycan layer during the entrainment process making it compatible within eukaryotic cells. A similar argument can be made for chloroplasts that are derived from Cyanobacteria that were entrained in algae following ingestion by single-celled protists enabling them to become photosynthetic. Interestingly, these entrained cells also lost their peptidoglycan. The plants that also have cell walls evolved from the algae with cyanobacterial chloroplasts.By contrast, there is no evidence that the Archaea produced intracellular organelles of or in the Bacteria or Eukarya. Cellular incompatibility may account for this because the archaeal cell envelopes are markedly different from the Bacteria or Eukarya. Although the evolution of the Archaea is not discussed here, their evolution was independent from that of the Bacteria and Eukarya because of their unique cell envelope features and generally smaller genomes. This is in agreement with the Tree of Life and the NuCom hypothesis.10.Carl Woese's Tree of Life was a seminal event in biology because it was the first scientific Tree of all organisms on Earth. His Tree of Life actually revealed that the Archaea comprise a separate, third branch of life that was previously unknown. What is so surprising is that the interpretation of the Tree of Life has been so controversial.Three main lines of thought have contributed to the confusion about what the Tree of Life means:Life evolved from prokaryotic organisms, the Bacteria and Archaea, because they are simpler than the Eukarya and evolution leads to greater complexityStudents of microbiology are taught about prokaryotic organisms. Since ‘pro’ means ‘before’ this implies that the Bacteria and Archaea were the first organisms, i.e. prior to the Eukarya. Secondly, many regard evolution as a process that always leads to complexity, so simple organisms, i.e. prokaryotes, probably evolved to produce nucleated organisms. Proponents of ‘Prokaryotes First’ hypotheses frequently believe that the Bacteria and Archaea contain all the genetic information necessary to produce a eukaryotic organism.All members of the Bacteria including the PVC superphylum are prokaryoticAlthough members of the PVC superphylum contain nuclei, many microbiologists either do not know this or they expect these cannot be nuclei because they are not identical to those of the Eukarya despite the marked divergence of the Bacteria from the Eukarya as shown by the Tree of Life. They then further deny the PVC superphylum of their remarkable phylogenetic characteristics such as the PSP proteins which are among the most complex and significant phylogenetic markers confirming their ancient heritage in LUCA. Their explanation for their occurrence in the PVC superphylum is, without evidence, that they have been transferred by HGT from the Eukarya.The Eukarya did not evolve until about 1.5–2.0Ga bpBecause the Eukarya did not leave a fossil record until about 1.5 Ga bp, many believe it was because they evolved more recently than the Bacteria and Archaea. This view also promotes the idea of Prokaryotes First hypotheses.As discussed here, this author believes that all three of these views are misconceptions that have led to incorrect interpretations of the meaning of the Tree of Life. A simple examination of Woese's Tree shows there are three major, independent lines of descent, the Bacteria, Eukarya and Archaea, leading to the concept of three domains. Moreover, the late appearance of the Eukarya which is clearly indicated by the long branch of this lineage in Woese's Tree of Life is not because they evolved last, but because their complete evolution required much more time and they did not leave detectable evidence in the fossil record until much later. Present-day eukaryotes required hundreds of millions of years of evolution to attain the hallmarks of the present-day Eukarya: complex genomes with mitochondria that could carry out mitosis and meiosis, sexuality and could develop noticeable traces of their existence in the fossil record.Of course those who believe that the Eukarya evolved first do not accept the Prokaryotes First interpretation either. However, their idea that the Eukarya evolved to produce the Bacteria and Archaea does not fit with Woese's Tree either because each of life's domains is independent from the others.This paper also posits the Domain Cell Theory, which is an extension of Cell Theory. As clearly shown in Carl Woese's Tree, there are three independent cellular lineages of life. The product of each led to a descendant cell type of its own kind: Bacteria from Bacteria, Archaea from Archaea and Eukarya from Eukarya.The NuCom hypothesis is the only one that complies with Domain Cell Theory. All Prokaryotes First and Eukaryotes First hypotheses violate Domain Cell Theory.


Studies have demonstrated that the solute carrier family 11 member 1 (SLC11A1) is heavily glycosylated and phosphorylated in macrophages. However, the mechanisms of SLC11A1 phosphorylation, and the effects of phosphorylation on SLC11A1 activity remain largely unknown. Here, the tyrosine phosphorylation of SLC11A1 is observed in SLC11A1-expressing U937 cells when differentiated into macrophages by phorbol myristate acetate (PMA). The phosphorylation of SLC11A1 is almost completely blocked by treatment with PP2, a selective inhibitor of Src family kinases. Furthermore, we found that SLC11A1 is a direct substrate for active c-Src kinase and siRNA-mediated knockdown of cellular Src (c-Src) expression results in a significant decrease in tyrosine phosphorylation. We found that PMA induces the interaction of SLC11A1 with c-Src kinase. We demonstrated that SLC11A1 is phosphorylated by Src family kinases at tyrosine 15 and this type of phosphorylation is required for SLC11A1-mediated modulation of NF-κB activation and nitric oxide (NO) production induced by LPS. Our results demonstrate important roles for c-Src tyrosine kinase in phosphorylation and activation of SLC11A1 in macrophages. Mycobacteria-containing phagosomes appears to impair the ability of Mycobacterium to block phagolysosomal fusion and acidification. It also affects the bacteriostasis or bactericidal activity of phagocytes and the survival of intracellular pathogens. Indeed, it has been reported that SLC11A1-positive mycobacterial phagosomes show enhanced fusion to vacuolar H+-ATPase-positive vesicles  ATP using DNA 5’ End-Labeling System  according to the manufacture’s instruction. For EMSA assay, 10μg of nuclear protein was incubated with 32P-labelled NF-κB oligonucleotides  in 1× EMSA buffer ) at room temperature for 20 min. DNA-protein complexes were resolved by electrophoresis in native 4% non-denaturing polyacrylamide gels. The gels were transferred to Whatman 3M paper, dried and autoradiographed.Cells were treated with PMA for different time points and then the nuclear extracts were prepared as described previously . The NF-TM kit C according to the manufacturer’s instructions. Briefly, 1×106 log-growth cells were suspended in 100μl of Cell Line NucleofectorTM Solution C and mixed with 1.5 μg of pGL4.32[luc2P/NF-κB-RE/Hygro] vector and 0.5 μg pRL-TK vector. Transfection was performed with a NucleofectorTM II device using the program W-001. At 6 hrs after transfection, cells were treated or left untreated with PMA for various time and cell lysates were prepared. Luciferase reporter assays were performed using the Dual-Luciferase® reporter assay system , and the luminescence was measured with a Turner Designs model TD-20/20 luminometer. Firefly luciferase activity was normalized to Renilla luciferase activity.Cells were transiently transfected with pGL4.32[luc2P/NF-κB-RE/Hygro] vector using the Cell Line NucleofectorTM kit C according to the manufacturer’s instructions. Briefly, 1×106 log-growth cells were suspended in 100μl of Cell Line NucleofectorTM Solution C and mixed with 2μg of c-Src siRNA (20μM in siRNA buffer). The siRNA duplexes used in the experiment were SMARTpool® SRC. Transfection was performed with a NucleofectorTM II device using the program W-001. Transfected cells were cultured for 48 hrs at 37°C in 5%CO2.U937-SLC11A1 cells were transiently transfected with the siRNA targeting c-Src using the Cell Line NucleofectorCell were seeded in 12-well plates and were differentiated with PMA (10 ng/ml) for 72 hrs. Then, the medium was replaced with fresh medium containing 0, 20, 40 or 80 ng/ml LPS and the cells were incubated for another 24 hrs. NO production was evaluated by measuring the accumulation of nitrite in the culture medium by the Griess reaction, as previously described 6 U937 or U937-SLC11A1 cells were first washed twice with ice-cold 1X PBS to eliminate serum from culture media. The cells were incubated with Fixable Viability Dye eFluorTM 780 (eBioscienceTM) in ice-cold 1X PBS for 30 minutes at 4°C to label dead cells and washed once with ice-cold 1X PBS containing 0.2% BSA. The cells were subsequently incubated with BUV395-conjugated anti-CD11b (BD HorizonTM) and PerCP/Cy5.5-conjugated anti-CD14 (Biolegend®) for 30 minutes at 4°C to stain for macrophage surface markers and then washed once with ice-cold 1X PBS containing 0.2% BSA. The cells were resuspended in ice cold 1X PBS containing 0.2% BSA and analyzed on LSRFortessaTM X-20 (BD BiosciencesTM). The data were analyzed using FlowJo version 10.4.1 (FlowJo®).Fluorochrome-conjugated monoclonal antibodies specific for macrophage surface markers were used. Specifically, after differentiation with 10ng/ml PMA for 48 hours, a total of 0.5x10P<0.05.Data are presented as mean ± S.E. Comparisons between groups were performed using one-way analysis of variance (ANOVA) followed by Tukey's Multiple Comparison Test (TMCT) when ANOVA indicated a statistical significance existed. Significance was established at in vitro using Sam68 as a phosphorylation substrate for this kinase. As shown in The U937 cells can be differentiated into macrophages by PMA . After tP<0.001). The expression of total SLC11A1 protein was not significantly changed with PP2 treatment. PP3, as an inactive analogue of PP2, did not affect the phosphorylation of the SLC11A1 protein. These results demonstrate that Src family kinase activities are required for tyrosine phosphorylation of SLC11A1.Tyrosine phosphorylation of SLC11A1 has not been previously reported. In order to determine if Src family kinases are involved in PMA-induced tyrosine phosphorylation of SLC11A1, U937-SLC11A1 cells were first treated with PMA for 2 days, followed by treatment with either the broad spectrum Src family kinase inhibitor PP2, PP3 (an inactive derivative of PP2) or left untreated. Initially, we examined the effect of PP2 on c-Src kinase activity. We used a phospho-specific antibody to detect the active p-c-Src that is phosphorylated on tyrosine 418. This residue, located in the Src tyrosine kinase domain, is autophosphorylated when c-Src is activated and its phosphorylation state is correlated with the kinase activity . As showP<0.01), whereas the expression of total SLC11A1 was not significantly changed . As shown in  changed . Accordiin vivo, U937-SLC11A1 cells were transiently transfected with c-Src and the interaction between SLC11A1 and c-Src was analyzed by co-immunoprecipitation followed by protein immunoblotting with specific antibodies to SLC11A1 and c-Src. As shown in To determine whether SLC11A1 and c-Src are associated in vitro Src binding assays were performed with synthetic peptides. As shown in The SLC11A1 protein contains three PXXP motifs that resemble SH3 binding domains. To test if the PXXP motifs are involved in the interaction of SLC11A1 with c-Src, we constructed three vectors for expression of c-Myc-tagged SLC11A1 mutants as either deletions or amino acid replacements in the three different PXXP motifs of SLC11A1, as depicted in P<0.001). Conversely, the phosphorylation level of the Y38F mutant was almost the same as for the wild-type SLC11A1. Overall, these results demonstrate that the SLC11A1 protein is primarily phosphorylated by c-Src kinase on tyrosine 15.The N-terminal region of the SLC11A1 protein contains two tyrosine residues, Y15 and Y38, which could potentially be phosphorylated by c-Src kinase. To identify the specific phospho-tyrosine residue(s), we generated SLC11A1 constructs bearing tyrosine-to-phenylalanine substitutions at Y15 or Y38 . c-Myc-tTo assess if tyrosine phosphorylation affects the function of SLC11A1, U937 cells stably expressing myc-tagged Wt SLC11A1,Y15F or Y38F mutant were differentiated into macrophages by treatment with PMA for 72 hrs. Then, the cells were treated with various concentrations of LPS  for another 24 hrs. NO concentrations were detected using Griess reagents. Compared with U937 cells expressing Wt SLC11A1, LPS-induced NO production was significantly decreased in the cells expressing the Y15F mutant but not in the cells expressing the Y38F mutant  that exhThe expression level of SLC11A1 Y15F mutant was lower than that of Wt SLC11A1 in stably transfected cell lines (around 72–83% of Wt SLC11A1), therefore, lower LPS-induced NO production and NF-κB activation in Y15F mutant cells could be caused by the less expression of the SLC11A1 mutant. To exclude this possibility, the two mutant cell lines that stably express SLC11A1 Y15F or Y38F mutant were re-generated. We selected two stable cell clones that express mutant SLC11A1 (Y15F or Y38F) at almost the same level as the cell clone expressing Wt SLC11A1  and repeMycobacterium [Toxoplasma [leishmania [Listeria [Mycobacterium tuberculosis by macrophages carrying functional Slc11a1 can be abrogated by the addition of iNOS inhibitors, which suggests the importance of NO generation and its modulation by Slc11a1 for host defense against intracellular pathogens [Macrophages execute their antimicrobial activities by producing different biochemical mediators, including NO. When stimulated with lipopolysaccharide, interferon-γ, tumor necrosis factor-α or infected with several intracellular microorganisms, macrophages release NO due to the expression of inducible NO synthase (iNOS) . The expacterium ,66, Toxoxoplasma ,68, leisishmania ,70 and LListeria ,72 speciListeria ,74. MutaListeria ,76 and mListeria ,77. The athogens . Our resathogens ,79. It w15YGSI18 motif resembles the SH2 domain binding motif pYXX (I/L/V), which can be recognized and bound by a class of SH2 domain-containing proteins. The SH2 domain was first identified in the oncoproteins Src and Fps and this domain is about 100 amino-acid residues long. Human genomic analysis revealed 121 SH2 domains that are distributed in 115 individual proteins involved in a wide range of signaling events [Phosphorylation of tyrosine residues by c-Src and/or other members of the SFKs has been found in a variety of proteins that are implicated in many important cellular processes, such as cell growth, differentiation, adhesion, transcription, ion channels, ROS production, as well as synaptic transmission and plasticity –84. In tg events . SH2 domg events . It is pg events , it willP<0.05), which might be caused by two reasons: (1) transfection of c-Src siRNA was unable to completely inhibit the c-Src expression; (2) c-Src, although important, may not be the only one of SFKs that is involved in the tyrosine phosphorylation of SLC11A1. So far, the Src family of protein-tyrosine kinases is composed of eleven members, which share major structural and regulatory features. Along with Src, Hck, Fyn and Fgr are also expressed in macrophages [In this study, we found that knockdown of c-Src kinase by siRNA results in a significant reduction in tyrosine phosphorylation of SLC11A1 compared with untreated cells. Further analysis showed that the level of tyrosine phosphorylation of SLC11A1 in c-Src knockdown cells was higher than that in the cells treated with PP2 Click here for additional data file.S2 FigU937 cells were stably transfected with constructed pCB6 vectors expressing c-Myc-tagged wild-type (U937-SLC11A1 Wt) or mutated SLC11A1 (U937-SLC11A1Y15F and U937-SLC11A1Y38F). (A) Cell lysates were prepared, and expression of Wt and mutant SLC11A1 was detected by Western blot analysis. β-actin was used as a loading control. (B) Same as (TIF)Click here for additional data file.