Persistent infection of high-risk human papillomaviruses 16 (HPV16) has been considered as the leading cause of cervical cancer. In this study we assessed HPV16 sequence variation and genetic diversity of HPV16 variants in cervical cancer in Uigur women in Xinjiang, China. We analyzed the nucleotide sequences of the open reading frames of E6 and E7, and part of the open reading frames of L1 of HPV16 in Uigur women.Biopsies of histologically confirmed HPV16 infections with cervical cancer were obtained from 43 Uigur women in Xinjiang, China. E6, E7 and L1 genes of HPV16 of all samples were amplified and sequenced; the sequences were used in phylogenetic analysis of HPV16 variants.Our analysis revealed nine nucleotide changes in E6 (five changes), E7 (one change) and L1 (three changes) gene. The most frequently observed variations were T350G (79.1 %). One variation T295G (D64E) at E6 were detected in 6 cases . Deletion (464Asp) along with insertion (448Ser) were observed in L1 (100 %). Most variants were European lineage (97.7 %); only one belongs to Asia variants with common T178G (D25E) in E6 and A647G (N29S) in E7.The most prevalent HPV16 variants in the Uigur women we studied were of the European lineage. Our results indicate that HPV16 European lineage may serve as a harmful factor associated with the development and progression of cervical cancer.The online version of this article (doi:10.1186/s13027-016-0089-2) contains supplementary material, which is available to authorized users. Cervical cancer was the third most common cancer among women in the world, with 527,624 new cases and 265,653 deaths in 2012 [Persistent infection by high-risk HPV16, has been recognized as a critical etiological factor for cervical cancer , which iMultiple factors are involved in the development and progression of cervical cancer and most HPV16 infection can be removed by the immune system but a small proportion can progress to cervical cancer. Previous studies demonstrated that HPV16 variants increased the risk for progression to cervical cancer –10, but Based on the genomic analysis of HPV16, seven major lineages of HPV16 variants have been detected and are related to geographic areas: European (E), Asian (As), Asian American (AA), African 1 (Af1), African 2 (Af2), North American (NA)  and a reSeveral mutations in E6 and E7, L1 genes may have great influence on the efficiency of infection, viral antigenicity and immunogenicity. A number of studies have suggested that the non-European variants have an increased risk for progression to high-grade squamous intraepithelial lesions (HSIL) when compared with European variants , 10, 17.In Xinjiang, previous research showed that HPV16 was the most prevalent HPV type in Uigur women . We analBiopsies of histologically confirmed HPV16 infections with cervical cancer were obtained from 43 Uigur women, who attended the People Hospital of Kashi (southern Xinjiang) and the People Hospital of Autonomous region (northern Xinjiang) during the years 2011 to 2014. 20 of the 43 women were residents of the southern Xinjiang, and the other 23 were in the northern Xinjiang. All of the cases were identified as squamous carcinomas. The diagnosis of histopathological grades was examined independently by two gynecologic pathologists. Tissues were stored at 4 °C no more than 24 h after surgical removal and subsequently cut into small fragments and stored in liquid nitrogen for genomic DNA extraction. Informed consent was obtained from all patients and the study protocol was reviewed and approved by the ethics committees of the hospitals.DNA was extracted from the 43 cervical samples with the SK1252 Genomic DNA Isolation kit  according to the manufacturer’s instruction. HPV16 DNA was identified by polymerase chain reaction (PCR) using HPV16-specific primers  and 5 μl template DNA. The cycling conditions were: 94 °C for 5 min; 30 cycles of 55 °C for 45 s, 72 °C for 60 s, 94 °C for 15 s, 55 °C for 45 s, 72 °C for 5 min. Sequencing primers were listed in Table Fragments of HPV16 E6, E7 and L1 were amplified by PCR (Table PCR products were purified using SAP (Promega) and Exo I (Epicentre) and sequenced directly using ABI BigDye Terminator v3.1 Cycle Sequencing Kit on a DNA analyzer (ABI3130XL) at the Genesky Biotechnologies Inc . Single Nucleotide Polymorphisms (SNP) were analyzed by software Polyphred and were aligned with the prototype (GenBank: NC_001526.2)  and otheThe sequences of E6, E7 and L1 genes were subsequently assembled together using Sequence Matrixv 1.7.8 (http://dx.doi.org/10.1111/j.1096-0031.2010.00329.x). Phylogenetic trees were built by MEGA 6  The sequences generated in this study were deposited in GenBank with accession numbers KT959524 to KT959566 for E6, KT966608 to KT966650 for E7 and KT966651 to KT966689 for L1 genes.Forty-three full-length E6, E7 and 39 partial L1 genes (nt 6659–7023) were successfully amplified and sequenced. Sequences obtained were compared to the HPV16 prototype reference . Gene sequence variation in E6, E7 and partial L1 genes of HPV16 were shown in Table Six nucleotide changes were observed in E6 and E7 Table , which cOur sequence analysis showed that three base pair (ATC/GTC) were inserted into nt 6901–6902 along with three base pair (GAT) deleted at nt 6951–6953 in all samples (100 %); thus, a serine was inserted in amino acid (aa) position 449 whereas an asparagine was deleted from aa position 464. The sequence insertions and deletions of HPV16 L1 gene have not been reported before. The variant with the nucleotide insertion (GTC) at nt 6901–6902 was only observed in one sample. In addition, a silent change at nt 6989 (A to G) was observed in one sample  , and 1 case of Asia variant. HPV16 E6 L83V variant is prevalent in high-grade lesions and is associated with progression of cervical malignancy in Moroccan ; this vaIn good agreement with previous reports , 33, 34,Surprisingly, at the L1 gene, three nucleotide variations were found. One silent and two novel nucleotide variations are found; three base pair (ATC/GTC) were inserted at nt 6901–6902 along with 3 base pair (GAT) deleted at nt 6951–6953 in all cases (Table We investigated the genetic variation of HPV16 in Uigur women in Xinjiang. Our results show that HPV16 variant of European lineage is the most common type in Uigur women in Xinjiang, which is markedly different from elsewhere in China. Future studies should expand into larger population of patients to evaluate the association between HPV16 variants and the risk for cervical cancer, and to understand the evolution of HPV16 variants in Uigur women in Xinjiang.AA, Asian American; Af1, African 1; Af2, African 2; As, Asian; D, aspartic acid; DNA, deoxyribonucleic acid; E, European; E, glutamic acid; HPV, human papillomaviruses; HPV16, human papillomaviruses 16; HSIL, high-grade squamous intraepithelial lesions; Java, Javanese variant; L, leucine; N, asparagine; NA, North American; NE, Non-European; PCR, polymerase chain reaction; S, serine; V, valine; VLPs, virus-like particles


In this research, an improved psychrometer is developed to solve practical issues arising in the relative humidity measurement of challenging drying environments for meat manufacturing in agricultural and agri-food industries. The design in this research focused on the structure of the improved psychrometer, signal conversion, and calculation methods. The experimental results showed the effect of varying psychrometer structure on relative humidity measurement accuracy. An industrial application to dry-cured meat products demonstrated the effective performance of the improved psychrometer being used as a relative humidity measurement sensor in meat-drying rooms. In a drying environment for meat manufacturing, the achieved measurement accuracy for relative humidity using the improved psychrometer was ±0.6%. The system test results showed that the improved psychrometer can provide reliable and long-term stable relative humidity measurements with high accuracy in the drying system of meat products. Relative humidity is at all temperature and pressures defined as the ratio of the water vapour pressure to the saturation water vapour pressure (over water) at the gas temperature . There aThe psychrometric method of relative humidity measurement has been known for a very long time. The psychrometer measures the relative humidity based on evaporative cooling. The common structure of an aspirated psychrometer is shown in A prominent advantage of a psychrometer is its adaptability to measurement environments. Compared to modern electronic relative humidity sensors such as capacitive relative humidity sensors or resistive relative humidity sensors, the psychrometer is one of the relative humidity measurement devices that can be used in a dirty environment, can withstand state changes of water, and can provide a large range of measurement values. The psychrometer is used in different measurement environment in many current applications ,11,12,13However, as a relative humidity sensor in application system, the weakness of the psychrometer is its low measurement accuracy compared to electronic sensors ,15,16. TThe wet bulb temperature should be not a thermodynamic property, but, under certain conditions it well approximates the thermodynamic wet bulb temperature . The senet al. calculated the correlation coefficients of psychrometer with varying wind speeds. An aspirated psychrometer was designed and tested for broiler chicken houses [A number of prior works studied the psychrometer measurement method in different environments and the effect of different factors influencing measurement accuracy. Yin n houses . The tesn houses . Duuren n houses .et al. designed an intelligent dry and wet bulb relative humidity sensor, with simulation and test results demonstrating the accuracy to within 2% RH [Several researchers discuss the measurement accuracies of psychrometer. Ustymczuk and Giner examined the errors of psychrometric measurement of relative humidity and used an error propagation formula to convert temperature errors to relative humidity errors . Mittal in 2% RH . The accin 2% RH . The stain 2% RH . The tesin 2% RH . Nicholsin 2% RH . Nakahamin 2% RH . When thetc. Moreover, the accuracy of conversion formula, measurement circuits, and calculation equipment are important in the psychrometer measurement process. These factors combine to affect the measurement result and limit the widespread usage of the psychrometer.A psychrometer measures the relative humidity indirectly from two temperature probes. By the structure of the dry and wet bulb probes, the temperature sensing element is encased within a stainless steel tube. The sensing element is not in direct contact with the environment. The psychrometer avoids directly exposing the relative humidity sensing element to the measurement environment. This key difference allows the psychrometer to be used in difficult measurement environments. However, the relative humidity measurement process of the psychrometer is a complex physical process. Many factors can influence the measurement result such as barometric pressure, ambient temperature, velocity of the air flow, accuracy of temperature probes, location of the probes, wet cloth size and quality, et al. introduced a parameterized psychrometer coefficient with wind speed in their paper [Nakahama investigated the psychrometer construction for performance testing in temperature and humidity chambers and the accuracy of humidity measurements meeting the challenge of quality engineering . Nakahamir paper .From the above discussion and the literature, it is clear that the psychrometer has been widely used as a method for relative humidity measurement over a wide range of values in a harsh environment, with varying degrees of measurement accuracy. Therefore, it is important to study the interrelated elements in the measurement environment and optimize the measurement accuracy of the psychrometer. This paper introduces an improved measurement device of aspirating psychrometer with specific construction parameters and provided real test results that are conducted in a meat-drying room in a food company. A specific psychrometric measurement device was developed and tested, including the structure of the improved psychrometer, signal conversion method, calculating unit, and the real-time test results. The influences of the psychrometer structure and interrelated factors of the measurement environment were demonstrated. The research analyzed the accuracy, stability, and reliability of the specific improved psychrometric measurement method based on a real-time temperature and relative humidity process control system.In this section, a common psychrometer implementation is briefly given. Then, a method of signal conversion between the dry and wet temperature probe measurements and relative humidity that is suitable for digital processors is presented.We tested several temperature sensors, including the thermocouple temperature sensor and the resistance thermometer. The PT100 is a resistance thermometer showed better accuracy and consistency than the thermocouple temperature sensors. Furthermore, the thermocouple temperature and the resistance temperature have a strong hull of stainless steel and its ingress protection number is 67. Thus, we selected the PT100 as the dry and wet temperature probes for the improved aspirated psychrometer. In order to fit the special measurement environment, the probes were chosen to be stainless steel with waterproof cable connections like IP67 ingress protection. The wet bulb temperature probe was soaked in water via a piece of cotton cloth. A fan was used to allow consistent and controllable airflow over the probes. The dry bulb and wet bulb temperature are denote by As an indirect measurement method, signal conversion between temperature and relative humidity is an important factor that directly affects the measurement accuracy of the psychrometer. Prior references exist on different conversion methods ,36 and tThe signal conversion is based on the Sprung primary formula . The masThe ratio of the partial pressure of water vapour in the mixture to the saturated pressure of water vapour at a prescribed temperature is defined as relative humidity, normally expressed as a percentage byituation . Ps is tP is the station pressure, C is a constant. The value of C differs according to the measurement environment. When the ambient temperature is above freezing, C = 0.5; and when the ambient temperature is below freezing, C = 0.43 [The partial pressure of water vapour in the mixture can be calculated using the Sprung formula  by(3)PdC = 0.43 . In thisC = 0.43  is validC = 0.43 . In a NAC = 0.43 . The uniThe These two equations are tested to valid for meat drying processes when the temperature is the in range of 4–18 °C. Finally, the relative humidity Based on the measurement methods for relative humidity, the humidity sensors can be classified into two categories: direct and indirect measurement methods. The measurement principles of capacitive humidity sensor and resistive humidity sensor belong to the category of direct measurement methods. The indirect measurement methods of humidity do not expose the humidity sensitive device to the measurement environment. The psychrometric measurement method is a typical indirect relative humidity measurement method, which measure the temperatures and then convert them into RH values.This research focuses on the structural parameter’s improvement of the aspirated psychrometer and specifically analyzes the influence of several factors in the measurement accuracy of the aspirated psychrometer based on the practical measurement. The experiments that were conducted included: the air flow speed test that air flow is passing through the dry and wet bulb temperature probes; the cloth size test that is put on the wet bulb probe; and the distance test between the wet bulb probe and water level. A specific improved psychrometer device was implemented in order to perform these experiments. There are different styles of psychrometers, and for the same style of psychrometer, the construction sizes are mostly different. The motivation to select two different distances to demonstrate that the effect of the distance between the water level and the wet bulb cannot be ignored. The first experiment was set up with two different distances between the wet bulb probe and the water level, at 23 mm and 71 mm. The variable was the air flow speed, which was changed from 0 m/s (stationary) to 4 m/s at 0.5 m/s intervals. The size of the wet cloth was 63 mm × 122 mm. The test results are shown in The second experiment was designed to examine the influence of different cloth size to wet bulb temperature. In the experiment, the cloth size was changed to 126 mm × 122 mm and other variables were same as in the first experiment. The test result is shown in The influence of environmental factors is shown in Based on the above results, we estimated the sensitivity of wet bulb temperature to the variation of various environmental factors. At a constant air flow speed of 2 m/s, different cloth sizes caused 0.2 °C uncertainty in the wet bulb temperature. Different distances between wet bulb temperature probe to water level added 0.3 °C uncertainty, and if the air flow speed changed from 2 m/s to 0.5 m/s then the uncertainties would increase approximately 0.3 °C. The total change was approximately 0.8 °C of the measured wet bulb temperature value.From Based on the analysis in The dry bulb and wet bulb temperature signals were measured in a three-wire circuit using a specific integrated circuit-ASIC 7030 A/D for analog to digital conversion. The profile controller operated with a micro-controller to acquire the measurement data. The test instruments include A/D converter and micro controller. The ASIC 7030 was used as the A/D converter. The two styles of micro controllers used were Neuron chip 3150 BF by Toshiba Corporation Semiconductor Company  and 89 C52 by ATMEL Ltd. . Several accurate instruments were used to calibrate the specific improved psychrometer measurement device and measure its accuracy. The instruments included: VAISALA humidity and temperature probe (HMP75) and measurement indicator (MI70), PACER digital anemometer (DA400), Ebro precision thermometer (TFX 430), MadgeTech accurate data logger (RHTemp 101A) for temperature and relative humidity, and Fluke instrument dry-well (9102S).In these experiments, the dry-well (Fluke 9102S) and Ebro precision thermometer (TFX 430) were used for dry bulb and wet bulb temperature probe calibration. The accuracy of Ebro precision thermometer (TFX 430) is ±0.05 °C and resolution is 0.01 °C. The humidity and temperature probe (HMP75) and measurement indicator (MI70) were used as a confirmation instrument. The improved psychrometer and the humidity and temperature probe (HMP75) with measurement indicator (MI70) were tested in a drying room environment. The test results of the improved psychrometer and the humidity measurement instrument (MI70 with HMP75) are shown in The test data shows that the measurement value of the improved psychrometer is very close to the measurement value of the humidity measurement instrument (MI70 with HMP75). The deviation between the two instruments was less than ±0.5% RH. According to the certified accuracy of the humidity measurement instrument (MI70 with HMP75), the measurement accuracy of the improved psychrometer was ±0.6% RH in a drying room measurement environment.A comparison test was made between the original psychrometer and the improved psychrometer in a functioning meat drying room control environment. The original psychrometer device was removed from the same general drying room control system.The original psychrometer device and the improved psychrometer device used the same probe and instrument. The calibration instrument was the same as in the previous section, and the atmosphere pressure was 964 hPa at the time of the experiment.The test results for temperature and relative humidity are shown in The measurement accuracy of the original psychrometer and the improved psychrometer are clearly shown in Another issue of a relative humidity sensor is the stability of measurement signal. Most relative humidity sensors have zero drift issue which depends on the properties of the relative humidity sensitive element. This issue is more evident in a harsh environment such as the drying room for meat production. Measurement stability of the prototype psychrometer was quantitatively analyzed in a real-time temperature and relative humidity control system. The experiments in a drying room control system using the prototype psychrometer as the relative humidity sensor was conducted. The testing environment was challenging since the air contained smoke particles, which did not allow capacitive relative humidity sensors to work at any extended period of time. It is a big challenge to have a sensor work in meat-drying room environments to measure the relative humidity. Since the humidity sensitive device of capacitive humidity sensors or resistance humidity sensors is exposed in a measurement area, it cannot perform properly in a harsh environment. The real tests show that some relative humidity sensors, such as capacitive humidity sensors or resistance humidity sensor, do not work properly for a long period of a time. A psychrometer does not use the humidity sensitive device for relative humidity measurement. It uses two temperature probes, named the dry bulb and wet bulb probes, to measure the relative humidity indirectly. This feature of measurement demonstrates that the modified psychrometer cam work stably in a harsh measurement environment for a very long period of time.3 at −7 °C, and heating source of the system was low pressure steam at 15 PSI. The humidifier was a dry fog generator with water droplet size of ≤4.2 microns. The drying room control system was two independent closed-loop control systems for temperature and relative humidity using the proportional, integral, and differential control algorithm. The relative humidity sensor was a prototype psychrometer.The dimension of the test drying room was 28 m × 9 m × 3.8 m. The control variables were the room temperature and relative humidity. The cooling source of the system was NHTwo temperature and relative humidity data loggers (MadgeTech TempRetriever-RH and RHTemp101A) were used as the recording measurement equipment. Among them, the TempRetriever-RH had a relative humidity resolution of 0.1% RH and a temperature resolution of 0.1 °C. The RHTemp101A had a relative humidity resolution of 0.1% RH and a temperature resolution of 0.01 °C. The set points of temperature and relative humidity for the drying room control system were 13 °C and 74% RH. The test results of temperature and relative humidity under closed-loop control are shown in The 65 h recording of temperature and relative humidity in a drying room under closed-loop control is shown in Temperature directly influences relative humidity measurement accuracy and stability. The effect of the varying factors in the relative humidity measurement can be evaluated using Based on The test results showed the temperature control accuracy was achieved ±0.05 °C for over seven hours in the control system. In The above test results distinctly revealed the influence of temperature control accuracy to the relative humidity control accuracy in the drying room control system. When the temperature control accuracy was ±0.1 °C, the relative humidity control accuracy was ±0.8% RH , and wheThis paper focused on a discussion of the application for the relative humidity measurement device in a current control system in challenging environments, in particular, the drying system for dry-cured meat products. Test results revealed the effects of different parameters of the psychrometer in the measurement accuracy and stability of relative humidity.The relative humidity measurement accuracy of the specific improved psychrometer that can be achieved is ±0.6% RH in a meat manufacturing environment.The specific improved psychrometer can perform long-term, stable, and reliable work in a severe environment of a meat manufacturing drying room.Based on the test results of a drying system for dry-cured meat products using a specific improved psychrometer, the control accuracy of a relative humidity system that can be achieved is ±0.8% RH.Based on experimental results, the characteristics of the specific improved psychrometer are summarized as follows.In summary, these characteristics of the improved psychrometer, including its higher measurement accuracy and stability, make the psychrometer especially suitable as a relative humidity sensor for applications in challenging measurement environments.


Accurate and complete identification of mobile elements is a challenging task in the current era of sequencing, given their large numbers and frequent truncations. Group II intron retroelements, which consist of a ribozyme and an intron-encoded protein (IEP), are usually identified in bacterial genomes through their IEP; however, the RNA component that defines the intron boundaries is often difficult to identify because of a lack of strong sequence conservation corresponding to the RNA structure. Compounding the problem of boundary definition is the fact that a majority of group II intron copies in bacteria are truncated.Here we present a pipeline of 11 programs that collect and analyze group II intron sequences from GenBank. The pipeline begins with a BLAST search of GenBank using a set of representative group II IEPs as queries. Subsequent steps download the corresponding genomic sequences and flanks, filter out non-group II introns, assign introns to phylogenetic subclasses, filter out incomplete and/or non-functional introns, and assign IEP sequences and RNA boundaries to the full-length introns. In the final step, the redundancy in the data set is reduced by grouping introns into sets of ≥95% identity, with one example sequence chosen to be the representative.These programs should be useful for comprehensive identification of group II introns in sequence databases as data continue to rapidly accumulate. Although not numerous, group II introns are found widely across the domains of life, being present in eubacteria, archaebacteria, and eukaryotic organelles -3. The cThe retromobility mechanism of group II introns has been well-studied biochemically and genetically, and thorough descriptions can be found elsewhere ,7,12,13.Approximately 25% of eubacterial genomes and 10% of archaeal genomes contain at least one group II intron or a fragment of an intron ,17. NearGroup II introns can be classified either phylogenetically according to their IEPs, or by features of their RNA secondary structures. Based on the IEPs, eight lineages of group II introns have been identified  ,19. By cThe IEPs of group II introns are easily identified through BLAST searches, because of their conservation. Incomplete IEPs are identified as truncations, in cases where RT subdomains or the X domain are missing or have premature stop codons. By contrast, the RNA component is not easily identified due to insufficient sequence conservation. For many introns, the RNA must be folded into a secondary structure in order to confidently predict the correct boundaries. Identifying the RNA boundaries is critical given that they specify the splice sites and define the protein translation product.Across all group II introns, only domain V is highly conserved and reliably identified, while the remainder of the RNA has short and scattered segments of conservation . For exaA useful fact in identifying RNA boundaries is that the ribozyme and IEP co-evolved  . Thus, tIn the past we have compiled and catalogued group II introns in bacterial genomes in order to define structural types, distribution and spread, and to collect data for evolutionary analyses . HoweverThe pipeline consists of eleven programs listed in Table To briefly summarize the process, the 22 representative IEPs are used as queries to search GenBank for candidate IEPs. Detected DNA sequences are downloaded along with 3 kb of flanks upstream and downstream of the IEP. The putative IEPs are screened to retain only bacterial and archaeal sequences, and to exclude RTs that are not group II introns. Each candidate intron is tentatively assigned to a class . The ORF domains present in each IEP are determined and IEPs that are judged to be intact are assigned an amino acid sequence. Intron RNA boundaries are predicted based on class-specific sequence profiles for 5′ and 3′ boundaries. To eliminate redundancy in the final set of introns, introns are grouped into sets of ~95% identity and one intron in each set is selected as the representative, or prototype.The output of the programs is in two folders, one containing data for the complete set of full-length and presumably functional introns, and the second containing data for a reduced set of non-redundant introns (<95% identity); however, if an intron in a 95% group is from a different species, it is included as a unique intron despite the sequence redundancy. In addition to this final output, data can be accessed at each step of the pipeline, and it is possible to write the accumulated data to more reader-friendly files using flags.http://hmmer.janelia.org/software/archive; the path to the HMMER2 programs should be an environment variable $HMMER2), HMMER3 ; the BLAST + suite (2.2.26) from NCBI (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.2.26/); CLUSTALW ; and PHYLIP (3.69/ http://evolution.genetics.washington.edu/phylip/getme.html). Other modules such as BioPerl utilities are embedded within the executable files.The programs run on a 64-bit LINUX system with internet access. Programs that must be installed on the computer include the following: HMMER2  where updates will become available.The program runs by command lines. Typical commands are specified in Additional file -20. The use of TBLASTN is important because it detects unannotated IEPs and the output format gives genomic coordinates of the ORF matches. If desired, the user can choose to use another set of ORFs as queries, or search another GenBank database , or specify a different E value cut-off . For each ORF, the program connects to NCBI and searches GenBank for sequence matches using TBLASTN (protein query vs. translated DNA database), and using the non-redundant database with an E value cut-off of 1x10For each accession number and coordinates, the DNA sequence is downloaded from GenBank in standard GenBank format along with 3 kb of flanking sequence both upstream and downstream of the HSP match . The DNA sequence is downloaded in the orientation that encodes the IEP on the forward strand. Taxonomic information is collected from the GenBank entry, which allows the files to be divided into five sets: “bacteria & archaebacteria”, “eukaryotes”, “mitochondria”, “chloroplasts”, and “other.” For each of the five sets, the texts of all of the GenBank entries are concatenated and saved as a single text file. The “bacteria_and_archaea” set is used by default in subsequent programs, but it is possible to input other folders or to introduce other folders as input of data in subsequent programs.A folder is created, “storage_new”, which over the course of the pipeline will contain a series of accumulated information. At this point, the folder “0” is created, but in subsequent programs storage folders 1–7 are generated, each containing progressively more data as further analyses are done Figure . Folder A significant challenge in assembling a comprehensive set of group II introns is that there are RTs in bacteria that give BLAST matches to IEPs but are not group II introns. One way to eliminate them is to use a more stringent E value cut-off in the initial BLAST search; however, the stringency also eliminates some true group II introns that are not closely related to those already identified. Complicating this scenario is the fact that some RT types are very closely related to group II introns and presumably derived from them, yet do not have associated RNA structures  . Therefo-20 in the BLAST search; relaxing the stringency mainly increases the number of short fragments of group II introns. Previously, we compiled a list of RT types in bacteria, including group II introns, retrons, diversity-generating retroelements and a variety of additional uncharacterized RTs [Our strategy to deal with this issue is to do multiple BLAST searches with relatively low stringency, using as queries a variety of intron subtypes, and then combining the results and subtracting out the non-group II introns. We use an E value cut-off of 1x10ized RTs . Using tThe phylogenetic class for each candidate IEP is next assigned, using a BLASTX search (translated nucleotide query vs. protein database) in which a local database of 475 group II intron IEPs is searched using as a query each candidate DNA sequence. If the three best matches are of the same class, the candidate is taken to belong to that class as well. Classes assigned are ML, CL, A, B, C, D, E, F, undefined, or unclassified. “Undefined” denotes that the top three hits are not of the same class, whereas “unclassified” means that the top three hits are to introns designated “unclassified”). The BLAST results are also used to identify the closest intron relative of each candidate among the 475 curated group II introns, which will be used later in the pipeline.To determine which ORF domains are present in each candidate sequence, a set of nine reference IEPs is used, with one IEP per class . For each reference IEP, one conserved amino acid in each domain  is chosen as a proxy for the presence of that domain. A BLASTP alignment is made between each candidate sequence and the reference intron for its class. If a given amino acid proxy is present in the pair-wise alignment, then the IEP is judged to contain that domain . This step determines whether the candidate contains all expected IEP domains, or is truncated and missing expected domains. Candidate sequences are sorted into folders according to the ORF characteristics. The IEP is assigned as “normal” if all domains are present once in the sequence, “missing domains” if one or more expected domains are absent, “extra domains” if one or more domains are present more than once , and “missing and extra domains” if one or more domains are missing and one or more domains are present more than once . By default, only complete IEPs continue to the next step in the pipeline.The presence of all domains does not necessarily mean that the IEP is intact, because there may be frame shifts, premature stops, or other problems within the ORF. While predicting IEP function is inherently speculative, we use the criterion of a single, uninterrupted alignment between a candidate IEP and its closest, curated IEP relative. To identify such proteins, a pairwise BLASTX alignment is done between each candidate DNA sequence and its closest IEP relative. A putatively intact ORF is indicated by the absence of stop codons, by the absence of insertions (gaps), and also by the alignment consisting of a single HSP . If no problems are identified, then the amino acid sequence of the IEP is assigned. Because many GenBank sequence files are not annotated, and also because N-termini are often misannotated in GenBank, the IEP sequences are assigned based on the amino acid sequences of the closest relative in the set of 475 curated introns. The start codon is chosen according to the start codon of the closest annotated group II intron sequence and the presence of an upstream Shine-Dalgarno sequence.As described above, group II intron boundaries are not identified reliably on the basis of a generalized consensus sequence; however, class-specific sequence patterns give greater accuracy. Taking this into account, hidden Markov model (HMM) profiles have been made for the boundary sequences of each class, and are used to search for potential 5′ and 3′ termini. Simple consensus sequences of the boundary sequences for the classes are shown in Additional file The profile matches for each intron candidate are evaluated in order to judge the most probable intron boundaries out of the compiled possibilities. A full-length, intact intron is indicated when there is only one plausible 5′ and one 3′ boundary, and they are in the correct order and separated by a reasonable distance. If these conditions are met, then the full intron sequence is generated based on those boundaries. Candidate introns are sorted into folders of “have boundaries” and “ambiguous intron boundaries”. By default, only introns with “have boundaries” continue to the following programs.Because group II introns are mobile DNAs, there are sometimes many identical or nearly identical intron sequences in a genome or in different GenBank entries. In order to eliminate the redundancy, introns are grouped into sets of introns of >95% identity. To do this, each phylogenetic class of IEPs is analyzed separately. The ORF sequences are aligned across the region corresponding to domains 0 to X, using ClustalW . The aliFinally, one intron in each group of 95% identity is chosen to be the representative, or prototype, unless there are multiple species within the group, in which case each species is assigned a prototype as well. The final output goes to a series of folders and files listed in Additional file The described programs successfully collect and download sequence information from GenBank, sort them into classes, identify the ORF and ribozyme components and boundaries, and create a non-redundant list with <95% identities. At each step of the pipeline, an aspect of the introns is examined and introns appearing not to be full-length and/or functional are set aside, with only intact sequences going forward. This produces a steadily smaller set of introns, with more information accumulated at each step for the introns that remain. As of July 2013, 3,191 non-redundant HSPs were identified initially as candidate group II introns in the “blast_and_parse” step, while at the end of the pipeline 572 of these were identified as prototypes. A detailed account of the segregation of the 3,191 sequence files into different categories over the course of the programs is listed in Additional file To measure the accuracy and completeness of the pipeline, a set of 513 introns present in the initial set of 3,191 downloaded sequences was followed through the process. At the end of the pipeline, 451 of 513 (88%) were included in the set of prototypes, which by default includes only “high probability” introns. If introns in the categories of medium and low probability are included as well, then 479 of 513 (93%) were identified. If one considers only the single step that identifies boundaries for introns using the HMM profiles (“find_intron_boundaries”), and take into account the best predictions regardless of the probability estimates , then 477 out of 492 (97%) introns analyzed by the program have the correct boundaries.A substantial portion of the missed introns  corresponds to the set of twintrons (introns nested within other introns) and other candidate DNA sequences containing more than one intron copy. These sequences were excluded at the “find_orf” stage of the pipeline. Their removal serves to prevent errors in subsequent steps due to multiple introns being present in a sequence being analyzed; however, it has the consequence of excluding sequences with more than one intron in the 8–10 kb of downloaded sequence. These introns, however, can be recovered from the “multiple ORF locations” folder generated by the “find_orf” program, and manually examined to determine their exact organizations.A major reason for the remaining missed introns is the specificity of some of the HMM profiles. Sequences considered “unclassified” were screened with the profiles of other classes, which not surprisingly reduced specificity and success. In addition, boundary profiles for Classes E and F were constructed from a relatively small number of introns and the prediction success was notably lower than for profiles of other classes. Overall, the boundaries for Class E, F and unclassified introns were correctly predicted with “high probability” for only 36 of 67 introns (54%) by the program “find_intron_boundaries.” However, including the single, best “low” or “medium” probability prediction resulted in 58 of 67 introns (87%) with correct boundaries.In the future, the HMM profiles can be improved substantially by increasing the number of E and F introns in the alignments, and by identifying additional classes from the “unclassified” introns, when enough examples are available to define a group and corresponding sequence pattern. An alternative strategy for identifying introns not belonging to established classes is to manually examine the sets of failed sequences after the “find_intron_boundaries” program. These sequences have intact IEP sequences but lack predicted 5′ and/or 3′ boundaries. Several of them appear to be bona fide introns that belong to new classes (unpublished data), which will be reported elsewhere.Another limitation of the boundary prediction algorithm is illustrated by two sets of introns that have insertions or extensions at either the 5′ or 3′ termini. An unusual subset of CL1 (IIB1) introns has a 5′ insertion near the start of the intron, which can be hundreds of nucleotides long . A seconThiomargarita namibiensis. However, these introns do not appear to be common in bacteria [Finally, it should be noted that introns without IEPs will be missed by this algorithm, as well as introns that encode an IEP not belonging to the RT family, such as the LAGLIDADG-encoding intron in bacteria .This suite of programs allows for comprehensive, automated detection of group II introns from GenBank, and provides an alternative to manual curation of group II introns amidst the rapidly expanding sequence databases. While not without limitations, the programs give effective tools for handling group II intron sequences and determining the scope and diversity of group II intron sequences present in bacterial genomes. Future updates to the programs, as well as larger libraries of curated group II introns, will improve the performance of the pipeline over time.• Project Name: Group II intron identification pipelinehttp://webapps2.ucalgary.ca/~groupii/index.html• Project Home Page: • Operating System: Linux 64-bit• Other requirements: HMMER2 2.3.2, HMMER3 3.0, BLAST + Suite 2.2.26, ClustalW 2.1, PHYLIP 3.69• License: None• Restrictions of use by non-academics: NoneD: DNA binding domain; En: Endonuclease domain; HMM: Hidden Markov model; IEP: Intron-encoded protein; RNP: Ribonucleoprotein; RT: Reverse transcriptase.The authors declare that they have no competing interests.Programs were written by MA, MAC, AD, KSH, TL, RAEN, AS, and RS, and were devised by these authors and SZ. Output generated by the programs were evaluated by LW and AMJ. The manuscript was written by SZ and CJS with input from all authors. All authors read and approved the final manuscript.Class-specific consensus sequences for 5′ and 3′ intron boundaries.Click here for fileTable S2. Output of each program.Input commands and options for each program. Click here for file


Acquisition of the M1 or M2 phenotypes by microglia has been shown to occur during the development of pathological conditions, with M1 activation being widely involved in neurotoxicity in relation with the anatomical localization and the reactivity of subtypes of microglia cells. On the contrary, little is known on the ability of microglia to undergo M2 polarization by interleukin-4 (IL4), the typical M2a polarization signal for peripheral macrophages.Fizz1, Arg1, and Ym1 genes, known to be up-regulated by IL4 in peripheral macrophages, together with additional polarization markers, were evaluated in the striatum and frontal cortex at different time intervals after central administration of IL4; in parallel, M2a protein expression was evaluated in tissue extracts and at the cellular level.Recombinant mouse IL4 was injected in the third cerebral ventricle of mice to induce brain alternative polarization. The mRNA levels of Fizz1 mRNA induction reached 100-fold level, underscoring the potency of this specific IL4 signaling pathway in the brain. In addition, immunochemistry assays demonstrated the localization of the M2 response specifically to microglia cells and, more interestingly, the existence of a subpopulation of microglia cells amenable to undergoing M2a polarization in the healthy mouse brain.Our results show that the potency and temporal profile of IL4-mediated M2a gene induction vary depending on the gene analyzed and according to the specific brain area analyzed, with the striatum showing a reduced M2a response compared with the frontal cortex, as further substantiated by assays of polarization protein levels. Of notice, These results show that the responsiveness of brain macrophages to centrally administered IL4 may vary depending on the gene and brain area analyzed, and that M2a polarization can be ascribed to a subpopulation of IL4-responsive microglia cells. The biochemical pathways that enable microglia to undergo M2a activation represent key aspects for understanding the physiopathology of neuroinflammation and for developing novel therapeutic and diagnostic agents.The online version of this article (doi:10.1186/s12974-014-0211-6) contains supplementary material, which is available to authorized users. Microglia are myeloid cells that populate the parenchyma of the central nervous system (CNS); their physiological activity includes most of the biological properties that are typical for peripheral macrophages, although their developmental origin and anatomical distribution allows these cells to perform distinctive immune and neuromodulatory functions in the CNS. Through their physical and biochemical interaction with neurons, microglia are able to sense and remodel neuronal activity, support neurogenesis, and maintain CNS homeostasis .in vivo [Microglia also possess the striking ability to rapidly react to endogenous or exogenous signals with a variety of physiological responses . Like pein vivo -6. The nin vivo ,8. Furthin vivo . This fuin vivo , neural-in vivo -14, and in vivo  and lipoin vivo ,17.in vitro or ex vivo studies, showing the responsiveness of microglia to IL4 and the subsequent activation of gene expression programs related to neuroprotection, tissue remodeling, and angiogenesis [Conversely, macrophages and microglia undergo an alternative ‘M2’ phenotype as a consequence of parasite invasion and in response to the endogenous immune signals, interleukin-4 (IL4) and interleukin-13 (IL13), in order to provide tissue repair and resolution of inflammation. A more detailed analysis led to the identification of two distinct alternative activation states, M2a and M2c, which reflect the actions of IL4 and IL13, on the one hand, and interleukin-10 (IL10) and TGFβ, on the other hand; importantly, these responses are associated with specific panels of regulated genes and a distinct range of effects . Howeverogenesis . As a coogenesis . Furtherogenesis .in vivo and evaluate the responsiveness and functional heterogeneity of microglia to undergo M2 polarization among and within selected brain areas. We thus stimulated microglia by central administration of IL4 in the third cerebral ventricle of the mouse brain. Interestingly, our data show that the induction of microglial M2a phenotype varies among the two brain areas analyzed, with M2a gene expression being more potent in the frontal cortex than in the striatum, and that only a subpopulation of microglia cells within these areas is amenable to undergoing an M2a response. Finally, we show that, among the M2a genes tested, Fizz1 reached the highest induction levels, underscoring the potency of this IL4 signaling pathway in the brain.The aim of this study was thus to investigate microglia M2 activation Recombinant mouse IL4 was obtained from PeproTech . Unless otherwise specified, chemicals were purchased from Merck .C57BL/6− male mice 4 months of age were supplied by Charles River Laboratories . All animals were allowed free access to food and water and were kept in temperature-controlled facilities on a 12-hour light and dark cycle. Animals were housed in the animal care facility of the Department of Pharmacological and Biomolecular Sciences at the University of Milan, and experiments were performed in compliance with regulations approved by the Institutional Animal Care and Use Committee of the University of Milan and in accordance with European legislation.Bone-marrow-derived macrophages were prepared as previously described . BrieflyIntracerebroventricular (icv) injections were made as previously described . BrieflyThe striatum and frontal cortex were homogenized using steel beads and tissue Lyser  at 28 Hz, for three cycles of 20 s followed by 30 s, on ice and in RLT buffer and total RNA was purified using RNeasy minikit (QIAGEN) according to the manufacturer’s instructions including a step with deoxyribonuclease incubation. One μg RNA was used for cDNA preparation using 8 U/μl of Moloney murine leukemia virus reverse transcriptase  in a final volume of 25 μl; the reaction was performed at 37°C for 1 h, and the enzyme inactivated at 75°C for 5 min. Control reactions without the addition of the reverse transcription enzyme were performed (data not shown).arginase-1 , chitinase-like 3 , Found-in-the inflammatory zone , IL4 receptor-α , CD206 , TGFβ , IL1β , TNFα , and for the reference genes 36B4  and complement component 1qA . The reactions were carried out according to the manufacturer's protocol using a 7900HT fast real time PCR system , and the data were analyzed using the 2-ΔΔCt method.A 1:16 cDNA dilution was amplified using SYBR technology. The PCR was carried out in triplicate or duplicate on a 96-well plate using GoTaq®qPCR Master Mix technology (Promega) according to the manufacturer’s protocol using 7900HT fast real time PCR system  with the following thermal profile: 2 min at 95°C; 40 cycles, 15 s at 95°C, 1 min at 60°C. Gene expression of target genes was assessed for 2, 20 mM HEPES (pH 7.9), 420 mM NaCl, 0.1 mM EDTA, 20% glycerol, 0.1% Triton, 5 mM β-mercaptoethanol, 0.1 mM PMSF, 10 μg/ml aprotinin, and 1 μg/ml leupeptin. The lysates were frozen on dry ice for 5 min and then thawed at 37°C for 5 min for three times. The samples were centrifuged at 13,000 rpm at 4°C for 20 min and the supernatants were collected and stored at −20°C. Protein concentration was estimated by Bradford protein assay using BSA as standard. Equal amounts of protein (20 μg) were dissolved in Laemmli’s sample buffer, boiled for 5 min, and separated with a SDS-polyacrylamide minigel  and then transferred overnight at 15 mA into 0.45 μm Hybond-ECL membrane . Membranes were incubated for 1 h with blocking solution containing 5% (w/v) nonfat milk in Tris-buffered saline (TBS) and subsequently probed for 1 h at room temperature with a rabbit anti-mouse YM1 antibody  in incubation solution (TBS containing 5% (w/v) nonfat milk and 0.1% Tween 20. After extensive washing in TBST (TBS + 0.1% Tween 20), or goat anti-mouse MMR/CD206 polyclonal antibody . Blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG  or horse anti-goat IgG  in incubation solution, for 1 h at room temperature. After extensive washing in TBST, immunoreactive bands were visualized using a chemiluminescence assay detection system according to the manufacturer’s instructions . To ascertain that blots were loaded with equal amounts of protein lysates, they were also incubated in the presence of the antibody against β-actin protein . Subsequently, for semiquantitative analyses, the densities of the protein bands of YM1 (45 kDa), CD206 (175 kDa), and β-actin (42 kDa) were measured by densitometric scanning of the membrane with Gel Doc™ XR Imaging Densitometer  and a computer program . Western blotting images were arranged in the final figures using Microsoft software.Brain tissues were homogenized using steel beads with a tissue Lyser (QIAGEN) at 28 Hz, for 3 cycles of 20 s followed by 30 s, on ice and in a buffer for total cellular extracts, containing 5 mM MgClAll immunohistochemical analyses were performed on animals treated for 16 h with IL4. Right brain hemispheres were removed and fixed overnight at 4°C by immersion in 4% formalin solution.n = 3).Brains were immersed at 4°C in 30% sucrose solution until they sank, embedded in optimal cutting temperature compound, and stored at −80°C until analysis. Coronal sections of brain 20 μm thick were collected using a cryostat . Free-floating sections were washed five times with TBS + 0.01% Triton and incubated with blocking solution (TBS + 10% goat serum + 0.4% Triton) for 1 hour at room temperature. Next, sections were incubated overnight with the following antibodies, diluted in TBS with 1% goat serum: rabbit anti-mouse antibody against Ym1 , rat anti-mouse antibody against macrophage antigen complex-1 Mac-1 , mouse anti-mouse antibody against feminizing locus on X-3 , mouse anti-mouse glial fibrillary acidic protein . Sections were washed five times with TBS + 0.01% Triton and incubated for 2 h with secondary antibodies  at room temperature. Sections were washed five times with TBS + 0.01% Triton and then incubated for 15 minutes with Hoechst stain (Sigma-Aldrich). In parallel, some sections were tested for antibody specificity by omitting primary or secondary antibodies. After five washings in TBS + 0.01% Triton, sections were mounted on slides and observed using a Zeiss Axioskop microscope equipped with a digital camera ; images were captured at 40,000× and 63,000× magnification. Quantification of Ym1-positive microglia cells was performed by counting the number of cells showing a red-labeled Ym1 signal and a green-Mac-1 signal; cells were scored as positive on the basis of nuclear DAPI staining in close proximity. Four counting fields of 50 × 50 μm were analyzed in two sections from three different levels, at least 120 μm apart, of the striatum and frontal cortex (n = 3).Brains were trimmed using a brain matrix  and sections were routinely processed, paraffin embedded, and sectioned in 4 μm serial sections. For Iba1 and Arg1 immunohistochemistry, sections were immunostained with rabbit polyclonal anti-Iba1 antibody , and goat polyclonal anti-Arg1 antibody ; sections were than incubated with biotinylated goat anti-rabbit (Iba1) and rabbit anti-goat (Arg1) secondary antibodies , labeled by the avidin-biotin-peroxidase procedure with a commercial immunoperoxidase kit . The immunoreaction was visualized with DAB  substrate and sections were counterstained with Mayer’s hematoxylin . Digital image analysis was performed by scoring the number of Iba1- and Arg1-positive cells (microglia) in three 400× microscopic fields in the frontal cortex of both vehicle and IL4 treated mice  for 15 min at 4°C; after washings, cells were suspended in 500 μl of PBS + 0.05% BSA and applied to a magnetic column to purify CD11b+ cells, namely microglia. Immediately after isolation of microglia, cells were fixed with 4% paraformaldehyde, extensively washed with 125 mM glycine in PBS and permeabilized overnight in PBS containing 0,1% Triton X-100, 5% normal goat serum, and 2% BSA, at 4°C. Cells were incubated with rabbit anti-mouse Ym1 antibody (Stem Cell Technologies) diluted 1:50 in Incubation Solution  at room temperature for 1 h. After extensive washes in incubation solution, cells were incubated with Alexa633-conjugated anti-rabbit secondary antibody  for 1 h at room temperature. Cells were extensively washed with PBS and then analyzed with a flow cytometry system . Isotype IgG controls were also used to evaluate nonspecific signals. Incubations with FITC-antiCD11b antibody (Miltenyi Biotec) and flow cytometry analyses were performed separately on dissociated brain cells as well as on CD11b-immunosorted cells to calculate recovery and purity; our protocol allowed us to obtain 80 to 85% recovery of CD11b-positive microglia cells, that is, 12% of the total brain cells population after tissue dissociation and myelin removal, with 90 to 95% purity after magnetic immunosorting.After 16 h of IL4 or vehicle treatment, brains were dissected and washed in Hank’s Balanced Salt Solution ; after removing the meninges, cortices from five mice were pooled as a single experimental group. Enzymatic cell dissociation was performed using Neural Tissue Dissociation Kit P , following a modified version of the protocol supplied by the manufacturer. Briefly, after enzymatic digestion with papain, samples were dissociated mechanically, homogenized, and filtered through a 40-μm cell strainer. After extensive washes in HBSS, myelin was removed by centrifuging the dissociated brain cells, which had previously been suspended in 10 ml of cold 0.9 M sucrose solution, at 850 n observations. The results were analyzed by one-way ANOVA followed by a Bonferroni post-hoc test for multiple comparisons using GraphPad Prism 5 software [P <0.05 was considered significant.Unless otherwise stated, all values are expressed as mean ± standard error of the mean (SEM) of software . A valueFizz1, Arg1, and Ym1 genes, known to be induced by IL4 in peripheral macrophages, were analyzed in a time course experiment following icv IL4 treatment. As shown in Figure Fizz1 mRNA levels in the frontal cortex were significantly elevated following 8 h treatment and further increased after 16 h, while after more prolonged time intervals the effect of IL4, although not statistically significant, could still be observed and remained around 20-fold higher than vehicle-injected mice 48 h after the injection of IL4. Analogously, a significant induction of Fizz1 mRNA levels was observed in the striatum at the earliest time points analyzed, with a temporal profile of mRNA induction that is superimposable to that observed in the cortex, except for the 8 h treatment; interestingly, at this time point the effect of IL4 is significantly different in the two brain areas, with the cortex resulting in a significantly higher gene induction than the striatum. The increased variability in Fizz1 mRNA levels that is observed in both brain areas along with treatment duration could probably account for loss of statistical significance in the IL4 effect observed at 30 and 48 h after injection.To evaluate whether microglia populating different brain regions are able to attain a similar M2 response, we analyzed M2 gene expression in the frontal cortex and striatum, in which M2 polarization was shown to occur under neurodegenerative conditions ,24. The Arg1 mRNA levels in the brain; also, for this M2 gene, the IL4 activity is significantly more pronounced in the frontal cortex than in the striatum following 16 and 30 h treatment, although the response after 8 h treatment is statistically significant only in the striatum; unlike Fizz1 induction, no difference between vehicle or IL4-treated mice was observed at the last time point analyzed. Analogously, the temporal profile of Ym1 induction also supports diversity in the response to IL4 among the frontal cortex and striatum. In fact, Ym1 mRNA levels are increased for up to 30 h in the frontal cortex, although never reaching statistical significance, while the effect of IL4 is weaker and faster in the striatum  mRNA levels are significantly increased in the frontal cortex following 8 and 16 h treatment, while no effect is observed after longer treatments. This effect was expected based on the fact that CD206 is also associated with the M2a phenotype induced by IL4; however, the induction level of this gene is much lower than those observed for the M2a genes, reported in Figure CD206 mRNA induction is observed in the striatum following 8 h, while IL4 activity is lost afterwards; this is consistent with a reduced IL4 response of this brain area as compared to the frontal cortex. The mRNA levels of TGFβ, as an M2c marker, as well as IL1β and TNFα, which are M1 response genes associated with inflammation, showed no alteration at any points of our time course experiment, as shown in Figure Based on recent knowledge suggesting that microglia or macrophage activation exists as a spectrum of combinations of activation markers that are strictly dependent on experimental conditions , we evalCD206 Mrc mRNA levFizz1 is a more reliable and targetable marker of IL4-induced activation in the brain.Altogether, these data show that activation of the M2 response in the striatum is less efficient when compared with the frontal cortex in the experimental conditions utilized. Furthermore, our data show that the response to icv IL4 is gene-specific, suggesting that per se does not alter Ym1 expression, at least in the experimental condition assayed. The potency of the IL4 induction of Ym1 protein is in agreement with that related with Ym1 mRNA levels, which are shown in Figure We next analyzed whether IL4 activity in the brain correlated with an increase in M2 protein levels and whether the temporal profile of protein expression reconciled with that observed for the mRNA levels of M2 genes. To this aim, M2 protein levels were evaluated by Western blot analyses in the striatum and frontal cortex of mice injected with IL4; we were unable to assay Fizz1 and Arg1 proteins, owing to low specificity of the antibodies tested. As shown in Figure Importantly, the induction of CD206 protein by IL4 is also restricted to the frontal cortex, with no effect occurring in the striatum , mRNA levels of IL4Rα were evaluated in the striatum and frontal cortex of control and IL4-treated mice following 48 h treatment. Similar levels of IL4Rα mRNA were detected in these experimental groups (see Additional file Altogether, the difference in the Ym1 and CD206 levels between the striatum and frontal cortex demonstrates a region-specific difference in M2 polarization in response to IL4.We then asked whether the observed induction of M2 gene expression by icv IL4 could be ascribed specifically to microglia. Double-labeling immunohistochemistry was used to localize Ym1 protein expression within specific brain cell types using antibodies against cell type-specific proteins, such as Mac-1, NeuN, and GFAP, known to be exclusively expressed by microglia, neurons, and astrocytes, respectively, each one was assayed together with an antibody against Ym1. As expected, the Ym1 signal was detected in microglial cells of the frontal cortex and striatum following 16 h of IL4 treatment, as shown in Figure in vivo.To assess more precisely the percentage of IL4 responsive microglia cells, fluorescence-activated cell sorting analyses were performed on microglia purified from the cortex of adult mice 16 h after icv IL4 injection. Data show that the dissociated brain cells population before immunosorting contains CD11b-positive microglia that are about 12% of the total cell number and show a specific morphological and scattering profile, as expected (see Figure To extend our observation on the existence of subsets of IL4 responsive microglia, we analyzed the percentage of microglia expressing Arg1. As shown in Figure Altogether these results demonstrate that the M2 polarization induced by IL4 in brain can be ascribed specifically to a subset microglia cells.in vivo. We thus developed a method that makes use of centrally administered IL4 to induce the M2a phenotype in brain. This experimental model allowed us to estimate the different ability of microglia populating the striatum and cerebral cortex to induce M2a gene expression and to identify the existence of subpopulations of microglia that support the IL4 response in brain.The aim of this study was to evaluate the extent to which microglia undergo M2 polarization Phenotype and functional plasticity is a characteristic of microglia; the presence of M1 and M2 phenotypes and a wide spectrum of intermediate and concomitant activation programs has been documented in animal models of cerebral diseases although with different ratios, temporal involvement and functional outcomes ,24,27-30per se, might be exaggerated or unrestrained as a consequence of both acute CNS damage, such as after traumatic and ischemic reperfusion injuries, or under chronic neuroinflammation, as in Parkinson’s and Alzheimer’s diseases or multiple sclerosis; under these pathologic conditions several studies have shown that the anti-inflammatory and reparative response driven by alternative microglia polarization is dampened, as a consequence of the increased, self-propagating proinflammatory phenotype, revealing that the loss of an appropriate M2 response harms the dynamic and heterogeneous nature of microglia response and thus contributes to neurodegeneration [Activation of the brain M2 phenotype has been previously observed in animal models of neurological diseases and associated with suppression of inflammation, tissue remodeling and matrix deposition in a time- and environment-specific manner ,24,27-30neration . HoweverIntriguingly, our data show that the M2a response in the striatum is less efficient than that observed in frontal cortex, in terms of M2a gene expression both at the mRNA and protein levels (see Figures Besides the nature, intensity, and persistency of the trigger, the responsiveness of microglia at a given time and within a specific anatomical location plays a central role in neuroinflammation. Activation of a specific phenotype is dictated by the number of responsive microglia and the ability of these responder cells to undergo polarization; it is believed that such properties are influenced by the specific environment in which microglia reside ,9. AddinFizz1 reached the highest induction levels in both the striatum and the frontal cortex. Expression of all M2a genes analyzed in this study has been shown to be dependent upon the IL4 signaling and coordinate action of IL4-inducible transcription factors [Fizz1 expression in the brain is highly efficient and, although its role in neuroinflammation is not clear [Fizz1 may be considered as a reliable marker of IL4-induced M2a activation in brain.In this perspective, icv IL4 administration represents a valuable experimental procedure to study the signaling pathways that control microglia M2 polarization. The use of this experimental model allows induction of the M2a response all over the brain, with no signs of inflammatory reactions in vehicle-treated subjects. Along with distribution, the rapidity, potency, and persistency of the M2a response observed following central administration of IL4 represent advantageous features in estimating the capability of microglia cells to acquire the M2a phenotype among and within different brain areas, in physiological as well as pathological conditions. Among the genes analyzed,  factors -38; yet,ot clear ,40, suggFizz1 being the most inducible gene among those tested. Our results show that the responsiveness of brain macrophages to centrally administered IL4 might change among brain areas and that microglia M2a polarization can be ascribed to a subpopulation of IL4 responsive cells. Therefore, the biochemical pathways that instruct and enable microglia to undergo M2a activation represent key aspects in the physiopathology of microglia and challenging opportunities for the development of novel therapeutic and diagnostic agents.The results presented here show that central administration of IL4 induces a specific temporal pattern of M2a gene expression in striatum and frontal cortex, with


Cold-acclimation genes in woody dicots without winter-dormancy, e.g., olive-tree, need investigation. Positive relationships betweenOeFAD8, OeOSM, andOeLIP19and olive-tree cold-acclimation exist, and couple with increased lipid unsaturation and cutinisation.FAD8, coding a desaturase producing α-linolenic acid, is activated by temperature-lowering, concomitantly with bZIP-LIP19 genes. The research was focussed on finding OeLIP19 gene(s) in olive-tree genome, and analyze it/their expression, and that of OeFAD8 and OeOSM, in drupes and leaves under different cold-conditions/developmental stages/genotypes, in comparison with changes in unsaturated lipids and cell wall cutinisation. Cold-induced cytosolic calcium transients always occurred in leaves/drupes of some genotypes, e.g., Moraiolo, but ceased in others, e.g., Canino, at specific drupe stages/cold-treatments, suggesting cold-acclimation acquisition only in the latter genotypes. Canino and Moraiolo were selected for further analyses. Cold-acclimation in Canino was confirmed by an electrolyte leakage from leaf/drupe membranes highly reduced in comparison with Moraiolo. Strong increases in fruit-epicarp/leaf-epidermis cutinisation characterized cold-acclimated Canino, and positively coupled with OeOSM expression, and immunolocalization of the coded protein. OeFAD8 expression increased with cold-acclimation, as the production of α-linolenic acid, and related compounds. An OeLIP19 gene was isolated. Its levels changed with a trend similar to OeFAD8. All together, results sustain a positive relationship between OeFAD8, OeOSM and OeLIP19 expression in olive-tree cold-acclimation. The parallel changes in unsaturated lipids and cutinisation concur to suggest orchestrated roles of the coded proteins in the process.Olive-tree is a woody species with no winter-dormancy and low frost-tolerance. However, cold-tolerant genotypes were empirically selected, highlighting that cold-acclimation might be acquired. Proteins needed for olive-tree cold-acclimation are unknown, even if roles for osmotin (OeOSM) as leaf cryoprotectant, and seed lipid-transfer protein for endosperm cutinisation under cold, were demonstrated. In other species, The online version of this article (doi:10.1007/s00425-016-2490-x) contains supplementary material, which is available to authorized users. Arabidopsis and winter cereals  are among the early responses to low temperatures, ceasing when cold acclimation is acquired  subfamily members of the basic–domain leucine zipper (bZIP) transcription factors  in olive-tree genome, and analyze it/their expression, and those of OeFAD8 and OeOSM, under different cold-conditions/drupe-developmental stages, in comparison with possible changes in unsaturated lipids and cell wall cutinisation, in drupes and leaves of genotypes traditionally known for a different cold-tolerance, with the goal of establishing whether and how these genes were involved in cold-acclimation.The present research aimed to find OeLIP19 gene was present in olive tree genome, and that, together with OeFAD8 and OeOSM, was involved in cold-acclimation starting from its induction. Changes in unsaturated lipids and cutinisation paralleled the changes in the transcription of these genes, suggesting orchestrated roles of the coded proteins in the cold acclimation process.Results showed that an Olea europaea L. cv. Canino, endemic of the volcanic areas of Northern Lazio, cv. Leccino from Northern Tuscany, cv. Frantoio from Southern Tuscany, cv. Moraiolo from Central Italy, and cv. Taggiasca from Ligury . The experiments were planned in the way that each year 6-year-old specimens were used per genotype. Each year, the genotypes synchronously showed full blooming (last week of May), and drupe development during the weeks after flowering (WAFs).Trees of In the first 2 years (2009–2010), specimens from all genotypes were used, whereas in the lasting 3 years (2011–2013) only those from cv. Canino, cv. Moraiolo and cv. Frantoio.−2 s−1), and exposed to cold-treatments of various intensity/duration. In detail, plants at the beginning of oleogenesis in the drupes, i.e. at WAF 10 , or for 24 h (A2-type cold treatment), or for 72 h (A3-type cold treatment). Alternatively, the plants were exposed at 0 °C for 2 h, and then at 10 °C for 14 days (B-type cold treatment). The controls were kept at 25 °C. In some cases A3-type cold treatment was also applied at WAF19 , under 16 h/8 h (light/dark) photoperiod (300 μmol photons mFull winter plants (WAF26) were also put in the growth-chamber, and progressively exposed to a temperature decrease from 7 °C (open-air temperature) to −10 °C into 35 h, maintained at −10 °C for 2 h, and then exposed to a temperature increase up to 7 °C into other 35 h (C-type cold treatment). The controls were kept at 7 °C. WAF 26 was chosen because it was several weeks after the end of oleogenesis  . Other three WAF19-plants per genotype, coming from the open air stocks, were used for the test. Discs (5-mm-in-diameter) were excised from the leaf lamina and the drupe epi-mesocarp, and one gram/fresh weight per organ type was frozen at a rate of 2 °C/h up to −10 °C, and maintained at −10 °C for 30 min  were selected randomly at WAF19, and 50 mg/dry weight (DW) of each sample extracted. The samples were split in two aliquots. One aliquot was used for quantitative analysis of oxylipins by LC–MS/MS, as reported by Ludovici et al. , and theTen drupes and leaves per year and genotype  were randomly collected at WAF19 and WAF22, hand-cut, and oil bodies and cutin detected by SUDAN IV staining. Epi-mesocarp and leaf samples were either fixed, dehydrated, embedded, sectioned, and stained with toluidine blue, or attached on a scanning electron microscopy stub, and treated as in Matteucci et al. . MoreoveTotal RNA was isolated from ten drupes (epi-mesocarp) and leaves of Canino and Moraiolo, collected from specimens either grown under open-air, or exposed to A- to C-types of cold-treatments, as specified in the Results. Moreover, at WAF26 ten leaves per genotype were excised from open-air grown plants and incubated in 5 μM solution of A23187 calcium ionophore . For the construction of the phylogenetic tree, various accessions of plant b-ZIP proteins (including LIPs) were obtained from the GenBank database (http://www.ncbi.nlm.nih.gov/nuccore/). The multiple sequence alignment was performed by ClustalW2.1 program  and the relative similarity matrix visualized, according to Neighbor–Joining method , according to Matteucci et al.  and D’Ani et al. , 2013. OCry2 was used as housekeeping gene for qPCR based on the stability of its mRNA expression in different plant tissues and developmental stages, and in presence of temperature variations, in accordance with previously reported housekeeping gene selection in the same species . Data from the third year shown.P < 0.05), followed by Tukey’s post test , used to compare mean values (±SE) of the same organ, either within the same genotype or between different genotypes. Alternatively, means (±SE) in pairs were compared by Student’s t test. Results were similar in all the crop years.A normality test was applied before analysis of variance , and one-way/two-way ANOVA , showing acclimation acquisition during the interval between WAF19 and WAF22, in accordance with previous results for the drupes  lower than from the drupes in both genotypes, however it was significantly (P < 0.0001) reduced in Canino in comparison with Moraiolo independently of the organ.Based on these results, the following analyses were focussed on Canino and Moraiolo because representative of the cold responsiveness of the other genotypes, after having confirmed the acquisition (Canino), and the non-acquisition (Moraiolo) of cold acclimation at WAF19 by the application of the ion leakage test see “”. In fac1- and A2-types of cold treatment resulted into the persistence of [Ca2+]cyt-rises, i.e., presence of cold-responsiveness, in both genotypes . An A3-type cold treatment to the plant was still not-sufficient to cause cold-acclimation in Moraiolo, but sufficient in Canino, because, under the cold-shock, only the drupe and leaf protoplasts of Moraiolo continued to show calcium rises   reduced in comparison with Moraiolo under the same A3-type cold treatment to the plant . Also when the plants were exposed to the B-type cold treatment before protoplast cold-shocking, Moraiolo remained cold-sensitive, differently from Canino, because only the cold-shocked protoplasts of the former showed the [Ca2+]cyt-rises  higher in Moraiolo than in Canino . Collectively, results from cytosolic calcium response in the protoplasts, and ion leakage from cell membranes after the B-type cold treatment to WAF10-plants, demonstrate that the sudden exposure of Canino plants to 0 °C for 2 h, coming from an open-air temperature of 25 °C, followed by a long exposure to a higher temperature, which however was fifteen degrees lower than the open-air temperature, is sufficient to induce and maintain a sudden cold acclimation in the genotype. In accordance, perennial plants without winter dormancy, as olive tree, are known to acclimate rapidly and maintain cold hardiness even under periods of unseasonably higher temperatures  mean value, Fig. To verify whether Moraiolo might become cold-acclimated later than WAF19, the cytosolic-calcium response induced by the cold-shock in the leaf protoplasts  was evaluated at WAF26 (full winter) in the open-air grown specimens. Calcium rises continued to be present, sustaining that Moraiolo had remained cold-sensitive . When, at WAF26, the plants were exposed to a C-type cold treatment before protoplast isolation, the inability of Moraiolo to acquire cold-acclimation was confirmed, because its protoplasts still showed presence of .The presence of the oxylipin 13S-hydroxy-9Z,11E,15Z-octadecatrienoic acid (13-HoTre), a C18:3 oxidation-product, was also evaluated, because it is a stress-induced compound and a possible cutin component , a significant (P < 0.01) reduction in OeFAD8 transcripts occurred in the drupes of both genotypes , increase in OeFAD8 levels at day 2 in comparison with day 0, there was a P < 0.0001 rise at day 7, but no further significant variation  increase at day 2, a strong decrease at day 7 , and a new increase (P < 0.01) at the treatment-end , and stable, in Canino  increased in Canino, and decreased in Moraiolo, as in the drupes . However, the gene transcripts were highly lowered in comparison with WAF19 , whereas it did not change significantly in Canino .The application of the Caino Fig. c. The pob-Zip LIP genes of other species was investigated. Total cDNA, synthesized starting from RNA samples of Canino, was used as template for PCR amplifications of partial regions of OeLIP, as described in “OeLIP nucleotide succession  registered in GenBank. Multiple sequence alignment clearly indicated that OeLIP presented the typical features of b-ZIP proteins: the basic domain , and the leucine zipper  . The similarity matrix produced by the comparisons of the b-ZIP protein successions (Table S1) revealed that OeLIP showed a percentage identity of its sequence with the other b-ZIP proteins ranging between 63.37 %  and 30.39 % . According to this matrix, a phylogenetic radial tree was also constructed; OeLIP branch was perfectly inserted in the genetic structure among the dicot b-ZIP proteins  occurred at WAF12/16, and were followed, at WAF19, by a slight decrease in level, which however, remained higher than WAF10 (P < 0.001)  Fig. a.Fig. 7E2-type cold-treatment, a P < 0.01 reduction in OeLIP transcripts occurred in the drupes of both genotypes in comparison with the control plants  OeLIP levels were reached in Moraiolo at days 2–7, but were followed by a strong (P < 0.0001) decrease (day 14)  at WAF19 in comparison with WAF10, whereas decreased (P < 0.01) in Moraiolo , and 72 h (P < 0.0001 difference with 0 h), whereas did not change significantly in Canino  and cold- (winter) drought , known to be triggered by the cold stress  were present in Canino in comparison with Moraiolo, when acclimation occurred in the former and not in the latter (WAF19). Because the expression of the OeFAD8 exhibited lower levels during advanced acclimation in comparison with early acclimation, suggesting a lower necessity of OeFAD8-mediated C18:3 production to maintain acclimation during advanced winter. Conversely, in Moraiolo, OeFAD8 levels continued to be low and fluctuating, but increased in full-winter leaves under the calcium-ionophore-treatment. Because Ca2+-ionophores induce [Ca2+]cyt-transients similarly to cold-shocks in the absence of acclimation  present on OeLIP-target genes, and that should be involved in the interactions with this TF during cold acclimation, remain to be determined. In Arabidopsis, the activation of Rab18, a Cor (cold-responsive)/Lea (Late embryogenesis abundant) gene, coding a dehydrin, is strongly associated to low-temperature stimuli and cold acclimation  were similar under prolonged cold-exposure  is cleaved and goes to the nucleus to induce PR-5 genes .In OeFAD8, OeOSM, and OeLIP19, participate to olive tree cold acclimation starting from its induction. They code proteins with different activities, i.e., a FAD, a PR-5, and a TF, respectively, but orchestrated for causing and maintaining cold acclimation, possibly through an increase in dehydration tolerance.In conclusion, as proposed in Fig. All Authors contributed to the writing of the manuscript and approved it.Supplementary material 1 (DOCX 559 kb)Below is the link to the electronic supplementary material.


Human endogenous retroviruses HERV-W encode a pro-inflammatory protein, named MSRV-Env from its original identification in Multiple Sclerosis. Though not detected in various neurological controls, MSRV-Env was found in patients with chronic inflammatory demyelinating polyradiculoneuropathies (CIDPs). This study investigated the expression of MSRV in CIDP and evaluated relevant MSRV-Env pathogenic effects.50 CIDP patients, 19 other neurological controls (ONDs) and 65 healthy blood donors (HBDs) were recruited from two different countries. MSRV-env and -pol transcripts, IL6 and CXCL10 levels were quantified from blood samples. MSRV-Env immunohistology was performed in distal sensory nerves from CIDP and neurological controls biopsies. MSRV-Env pathogenic effects and mode of action were assayed in cultured primary human Schwann cells (HSCs).In both cohorts, MSRV-env and -pol transcripts, IL6 positivity prevalence and CXCL10 levels were significantly elevated in CIDP patients when compared to HBDs and ONDs . MSRV-Env protein was detected in Schwann cells in 5/7 CIDP biopsies. HSC exposed to or transfected with MSRV-env presented a strong increase of IL6 and CXCL10 transcripts and protein secretion. These pathogenic effects on HSC were inhibited by GNbAC1, a highly specific and neutralizing humanized monoclonal antibody targeting MSRV-Env.The present study showed that MSRV-Env may trigger the release of critical immune mediators proposed as instrumental factors involved in the pathophysiology of CIDP. Significant MSRV-Env expression was detected in a significant proportion of patients with CIDP, in which it may play a role according to its presently observed effects on Schwann cells along with previously known effects on immune cells.Experimental results also suggest that a biomarker-driven therapeutic strategy targeting this protein with a neutralizing antibody such as GNbAC1 may offer new perspectives for treating CIDP patients with positive detection of MSRV-Env expression.Geneuro-Innovation, France. •A protein from endogenous retroviruses (8% of human genome) is found in chronic inflammatory demyelinating polyradiculoneuropathies (CIDP).•Identified in multiple sclerosis, MSRV-Env induces inflammation and autoimmunity.•It is now shown in peripheral nerve lesions, serum and blood cells of CIDP patients.•In Schwann cells, it induces pro-inflammatory factors; the same is detected in serum.•Antibody neutralization of MSRV-Env effects raises therapeutic perspectives in CIDP.Since the 1980's, a new horizon in the search for pathogenic players in complex human disease arose from studies in which human endogenous retrovirus families (HERV) were identified. HERVs represent 8% of the human genome and may express pathogenic proteins as shown here in a chronic inflammatory demyelinating polyradiculoneuropathies (CIDPs). The present study shows that MSRV-Env, an HERV protein, is detected in CIDP lesions and blood. It triggers inflammation in immune and nervous cells. A specific antibody revealed efficient in experimental conditions. This offers new therapeutic and diagnostic perspectives for this disabling disease without curative treatment. HoweverBeyond this fundamental research context, independent studies confirmed an association of MSRV expression with MS . Its envChronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a rare immune disease of the peripheral nervous system (PNS), with multifocal inflammatory and demyelinating lesions in nerve roots also expanding to distal regions . Its cliIn a previous study on MS, MSRV-Env was not detected in healthy controls and in various other Neurological Diseases except for CIDP cases (5/8) . This ob22.1The overall study group consisted of 51 CIDP patients, from the Neurology Departments of Henri Mondor University Hospital  and of Vaudois University Hospital . CIDP patients fulfilled criteria of the EFNS and PNS Joint Task Force guidelines . The majIn a first cohort (Study 1), 20 CIDP patients from Creteil and 21 HBDs from Annemasse, were enrolled. A second cohort (Study 2) included 11 additional CIDP patients and 19 OND patients from Créteil, 20 CIDP patients and 18 HBDs from Lausanne, and 26 HBDs from Annemasse, for a total of 31 CIDPs, 19 ONDs and 44 HBDs in study 2. MSRV transcript levels in PBMC were assessed separately in Studies 1 and 2. In the light of results obtained in human Schwann cells,IL6 and CXCL10 serum levels were determined retrospectively, at the same time for both cohorts .For serum, 6 mL of blood was collected on a dry tube and 500 μL of serum aliquots were frozen at − 80 °C. For peripheral blood mononuclear cells (PBMCs), 4 mL of blood was collected in a Cell Preparation Tube  and treated according to the manufacturer's instructions. PBMCs in heat-inactivated Fetal Calf Serum with 10% dimethyl-sulfoxide were stored at − 80 °C.2.2− 1 with RNase-free water. 50 ng of cDNA was used with iQ supermix  and corresponding sets of primers/probes for qRT-PCR. The internal control was glucuronidase beta gene, GUS B  and specific sets of primers and probes for MSRV-env as described (Thawed PBMCs were washed with PBS (1700 g for 20 min at 10 °C). Total RNA was isolated with QIAamp RNeasy Mini Kit  and treated with Turbo DNA-Free™  according to the manufacturer's instructions. RNA concentration was assessed with a Nanodrop 2000  before adjustment to 10 ng/μL with RNase-free water. First-strand cDNA was synthesized with i-script select cDNA-synthesis kit using oligonucleotide dT (18)  at 42 °C for 60 min, inactivated at 85 °C for 5 min and adjusted to 10 ng μLescribed . MSRV-po2.3IL6 and CXCL10 protein levels were respectively quantified with Human IL6 ELISA Ready-SET-Go!® and BD OptEIA™ Human IP-10 ELISA Set according to the manufacturer's instructions . The absorbance was read at 450 nm with Biotek EL800 device .2.4Frozen superficial peroneal nerve biopsies from 7 patients with CIDP and 2 ONDs, one axonal inflammatory neuropathy with perivascular inflammatory changes and one diabetic neuropathy, were collected from the Biological Resource Platform of Henri Mondor University Hospital (to French Ministry of Research #DC-2009-930). Biopsy samples were obtained independently from prospective studies 1 and 2, and used for research purposes according to French regulation (#AC-2014-2056). 15% PFA fixed and paraffin embedded 3 μm-sections were prepared. After paraffin removal, they were washed with Tween-TBS solution. Endogenous peroxidase activity and avidin/biotin proteins were quenched by Biotin blocking system  according to the manufacturer's instructions. Sections were then washed in Tween-TBS solution, incubated with anti-MSRV-Env mouse monoclonal antibody GN-mAb_04  or an isotype control  diluted at 5 μg/mL in PBS with 10% normal human serum (NHS) for 1 h at room temperature. After washing with Tween-TBS, sections were incubated with a secondary biotinylated antibody  diluted at 10 μg/mL in PBS with 10% NHS for 30 min at room temperature. Sections were washed again in Tween-TBS, streptavidin complex was added for 20 min at room temperature, followed by DAB for 7 min . Then, after washing with water and counterstaining with Hemalun, MSRV-Env specific and isotype control staining were examined by light microscopy on two successive sections of each biopsy.2.5HSCs were purchased from ScienCell Research Laboratories  and cultivated according to the manufacturer's instructions.2.5.1l-lysine coated  labtek , fixed in PBS with 4% paraformaldehyde, washed 3 times with PBS and incubated in PBS with 0.01% Triton X-100 for Schwann cell markers , or in PBS alone for extracellular TLR4. Nonspecific binding sites were saturated by incubation in PBS with 10% Fetal Bovine Serum for 1 h at 37 °C. They were then incubated with primary antibodies diluted in Fetal Bovine Serum 10% in PBS overnight at 4 °C . After 3 PBS washes, HSCs were incubated with secondary antibody solutions for 1 h at 37 °C . Following 3 PBS washes, plastic chambers were separated from the slides and mounted with Vectashield® mounting medium containing DAPI  before examination by fluorescence microscopy.HSCs were grown for 48 h on poly-2.5.2HSCs were cultured for 24 h in 6-well plates. At 60% confluency, HSC medium was replaced by serum-free OPTIMEM medium . Lipofectamine 2000 was used according to the manufacturer's instructions  to transfect HSC with empty pHBB control plasmid or pHHB plasmids expressing either the full length envelope protein (MSRV-Env-T) or its extracellular domain only (MSRV-Env-SU). After 4 h, transfection medium was replaced by HSC medium and cells were cultured for 48 h with GNbAC1 or GNbAC1 vehicle before collecting culture supernatant and cells for analyses.2.5.3Escherichiacoli and purified as endotoxin-free protein by PX'Therapeutics  from plasmid pV14 encompassing the complete env orf cloned from MSRV virion RNA . MSRV Env solubilization buffer  was provided in parallel.HSCs were grown on 6-wels plates and incubated with endotoxin-free recombinant MSRV-Env or its solubilization buffer (negative control), together with LPS-RS , GNbAC1 or GNbAC1 vehicle, at 37 °C for 1 h (IL6) or 4 h (CXCL10) before RNA isolation from cells. Recombinant MSRV-Env was produced in 2.5.4After appropriate treatments, HSCs were washed with PBS and total RNA extracted with QIAamp RNeasy Mini Kit. Relative expression of IL6 and CXCL10 to GUS B was performed with Taqman gene expression assays for IL6, CXCL10, and GUS B  according to the manufacturer's instructions.2.5.596-well microplates were coated overnight at 4 °C with an anti-MSRV-Env capture antibody  diluted at 5 μg/mL in 50 mM bicarbonate buffer, with a 0.05% Tween in PBS, saturated with 1% BSA PBS and washed 4 times. Culture supernatants diluted 1/2 in PBS were then incubated for 2 h at 37 °C, plates washed 4 times and incubated with HRP-coupled anti-MSRV-Env detection antibody  for 1 h at 37 °C. After 6 washes, revelation of antigen-bound HRP-antibody used 3,3′,5,5′-tétraméthylbenzidine  and absorbance at 450 nm wavelength was measured with Biotek EL800 device .2.6post hoc test were used when data passed the normality test, otherwise Spearman rank order correlation test, Mann–Whitney rank sum test, and Kruskal–Wallis one-way analysis of variance on ranks followed by Dunn's post hoc test were used. Chi-square or Fisher Exact tests were used to compare rates and proportions. Statistical analyses were performed with SigmaStat 3.5  and data plotted with Prism 5.04 .Kolmogorov–Smirnov normality test was applied to all data sets. Pearson product moment correlation test, Student's t-test, and one-way analysis of variance followed by Bonferroni's 2.6.1The sample collection and the experimental study were financially supported by Geneuro-Innovation, France. The funders had no role in the study design, nor the data collection and analysis, nor in their interpretation or in the writing of the manuscript.33.1Demographical and clinical characteristics are presented in 3.2Results are illustrated in In Study 1, PBMC mRNA from 20 HBD and 15 CIDP patients passed quality criteria (Cf. Methods). MSRV-env  was calculated (Cf. Methods). In Study 1, 6 CIDP patients had HE for MSRV-env (40%) but none in HBDs . 4 s (27%) and 1 HBD (6%) had HE for MSRV-pol (p = 0.161). 7/15 CIDPs  had HE for at least one MSRV transcript. A dual MSRV env/pol HE profile was observed in 0/17 HBDs and 3/15 CIDPs .In Study 2, 2 HBDs (7%) versus 6 CIDP (53%) presented HE for MSRV-env (p < 0.001). 3 ONDs (25%) had MSRV-env HE, which was not significantly different from HBD or CIDP groups. 8 CIDP (57%) had HE for MSRV-pol, which was significantly different from both HBD  and OND  groups. 9/14 CIDP  presented a HE profile for at least one MSRV transcript. A dual HE profile was observed in 2/26 HBDs (8%), in 0/6 OND and in 6/14 CIDPs  and -pol  expressions in CIDP patients are inversely correlated to the disease duration in Study 2 .3.3We analyzed IL6 cytokine and CXCL10 chemokine levels in sera from Studies 1 and 2. Statistics of comparisons between groups are presented in IL6 values above the limit of detection were more frequent in CIDP (30%) than in ONDs (11%) and HBDs (8%), which was statistically significant (p < 0.01). 2 CIDP patients presented highly elevated IL6 (230 and 387 pg/mL) whereas the highest IL6 levels in ONDs and HBDS were 10 and 14 pg/mL respectively. Similarly, CXCL10 levels were significantly higher in serum of CIDP patients (115 ± 10 pg/mL) than in HBDS  and ONDs . No correlation existed between IL6 and CXCL10 expressions, nor with the age of subjects, the age at disease onset, the gender, the treatment regimen, nor even with MSRV-env and -pol mRNA levels.3.4Distal sensory peripheral nerve (PN) biopsies were examined with an MSRV-Env specific monoclonal antibody or an isotype control antibody. MSRV-Env was detected in biopsies from five CIDP patients out of seven tested (71%), in the absence of staining with the isotype control. As shown in 3.5We then studied the effects of MSRV-Env expression on in vitro transfected HSC, mimicking MSRV-Env expression in HSC as observed in CIDP nerve biopsies. Primary cultured HSCs were transfected with plasmids encoding the complete MSRV-env protein (Env-T) or encoding its extracellular domain (Env-SU). Immunocytofluorescence analyses showed that HSCs strongly express toll-like receptor 4 (TLR4), the pharmacological target of MSRV-Env, at their surface . HSC canAs presented in 3.6We developed experimental conditions appropriate for a pharmacological evaluation of MSRV-Env and GNbAC1 by stimulating HSC for 4 h with purified and endotoxin-free recombinant MSRV-Env protein before HSC transcript isolation. A low concentration of MSRV-Env (3 nM) induced a significant increase of IL6  and CXCL10  transcript levels in HSC A–B. The 4After initial detection of HERV-W protein, MSRV-Env, in CIDP cases within a control group of inflammatory neurological diseases for a study on MS , the preMSRV-Env has for long been shown to induce innate immune dysregulation, autoimmunity and inflammation in cellular and animal models . TLR4 acUnlike available previous studies having investigated IL6 in sera of CIDP patients , we founIt therefore appears that an autoimmune reaction in CIDP may result from a TLR4-driven activation of innate immunity by MSRV-Env protein in immune and neuroglial cell types with potential downstream superantigen-like effects when T-cells are recruited and exposed to MSRV-Env . A mechaThus, MSRV-Env cannot simply represent a peripheral biomarker. Nonetheless, as MSRV-env and MSRV-pol expressions were inversely correlated to disease duration, MSRV activity may peak at early stages of the disease and its early quantification could be of value for CIDP phenotyping or diagnosis. As it may also reflect some efficacy of long-term treatments on its recirculation or expression in the bloodstream, longitudinal and/or transversal studies of accurately representative cohorts should now be envisaged.Finally, the present study also showed that the strong pro-inflammatory upregulation of IL6 and CXCL10 induced by MSRV-Env in HSC was significantly inhibited by a specific neutralizing antibody targeting MSRV-Env, GNbAC1. As this antibody is a humanized therapeutic IgG4, now in phase II clinical trials in MS , this inConcluding from these original findings, we propose that MSRV-Env is a potential therapeutic target in CIDP, at least in a significant proportion of patients, and that it may become a useful blood biomarker along with CXCL10 and IL6. This also provides arguments in favor of the humanized monoclonal antibody GNbAC1, as a potentially innovative treatment to be evaluated in CIDP.RF, AM, NG, IB, MB, CB, FC, ABL, and HP are employees of GeNeuro Innovation SAS or GeNeuro SA.AJS and TK perform consultancy work for Actelion Pharmaceuticals Switzerland.AC gave expert testimony for CSL Behring, Novartis, received grants from Biogen, Novartis, CSL Behring, Geneuro, Octapharma, and gave lectures for Genzyme.RF, AM: developed, supervised, and interpreted all protocols and experiments, except clinical and sample recruitment, interpretation of clinical data and immunohistological analysis of biopsies. They contributed to the text of the manuscript and to its successive reviews.NG, IB, MB: developed and performed all experiments except biopsies analyses, reviewed the manuscript.JA: biopsies analyses, reviewed the manuscript.FC, ABL, AJS, interpreted data and reviewed the manuscript.PAP, CL, CB, TK, AC: set-up of the clinical studies, patients' recruitment, data interpretation, reviewed the manuscript.HP, AC, FC and TK initiated and supervised the study.HP: reviewed contributions, corrected and finalized the text of manuscript as well as that of the revised version.