Noroviruses are the leading cause of acute gastroenteritis and foodborne illnesses in the United States. Enhanced methods for detecting noroviruses in food matrices are needed as current methods are complex, labor intensive and insensitive, often resulting in inhibition of downstream molecular detection and inefficient recovery. Membrane-based electro-separation (MBES) is a technique to exchange charged particles through a size-specific dialysis membrane from one solution to another using electric current as the driving force. Norovirus has a net negative surface charge in a neutrally buffered environment, so when placed in an electric field, it moves towards the anode. It can then be separated from the cathodic compartment where the sample is placed and then collected in the anodic compartment for downstream detection. In this study, a MBES-based system was designed, developed and evaluated for concentrating and recovering murine norovirus (MNV-1) from phosphate buffer. As high as 30.8% MNV-1 migrated from the 3.5 ml sample chamber to the 1.5 ml collection chamber across a 1 μm separation membrane when 20 V was applied for 30 min using 20 mM sodium phosphate with 0.01% SDS (pH 7.5) as the electrolyte. In optimization of the method, weak applied voltage (20 V), moderate duration (30 min), and low ionic strength electrolytes with SDS addition were needed to increase virus movement efficacy. The electric field strength of the system was the key factor to enhance virus movement, which could only be improved by shortening the electrodes distance, instead of increasing system applied voltage because of virus stability. This study successfully demonstrated the norovirus mobility in an electric field and migration across a size-specific membrane barrier in sodium phosphate electrolyte. With further modification and validation in food matrixes, a novel, quick, and cost-effective sample clean-up technique might be developed to separate norovirus particles from food matrices by electric force. Norovirus (NoV) is recognized as the major cause of foodborne illnesses in the U.S. each year, responsible for 58% of foodborne gastroenteritis and 95% of nonbacterial gastroenteritis , 2. The To protect public safety, sensitive, fast, and reliable methods to detect norovirus in a variety of foods and drinking water is needed. Such detection systems must also be cost-effective for industry and laboratory uses. Norovirus detection from foods can be divided into two processes, upstream sample preparation and downstream molecular detection. Upstream sample preparation involves virus elution from food matrices and further concentration and purification if needed. Downstream molecular detection involves viral RNA extraction and RT-qPCR detection. Reliable sample preparation requires efficient elution of virus from different kinds of complex food matrices without the co-elution of possible inhibitors for RT-qPCR assays . ElutionMembrane liquid-phase extraction (MLPE)  methods The objective of this study was to evaluate the feasibility of using a membrane-based electro-separation (MBES) method to evaluate norovirus mobility using an electric field as the sole driving force across a pore-size specific selective separation membrane. This is a proof-of-concept study where an ElectroPrep electrodialysis device  was adapted for our uses and murine norovirus (MNV-1) was used as the model virus to study this objective. Parameters that effect virus movement in the MBES system were evaluated and its compatibility with elution buffers commonly used in food virology was investigated. The study provides a foundation for designing a detection device to be used for separation and concentration of norovirus from food samples. Future studies should thus validate the method in the context of food matrixes.2 for 48 hrs. The infected flasks were frozen (-70°C) and thawed (room temperature) for 3 cycles, followed by centrifugation at 2,000 x g for 15 min and vacuum filtration (0.2 μm PES membrane filter) to remove large cell debris and clarify viruses. The filtrate was further concentrated by ultracentrifugation in a Beckman Coulter Ultracentrifuge (XL-80) using a Type 35 rotor at 100,000 x g for 1 hr. The pellet was dissolved in sterile PBS overnight at 4°C to prepare virus stock. Sucrose purification was performed to further concentrate the virus stock. Virus stock was loaded on top of a 30% sucrose solution for centrifugation using a NVT 90 rotor at 100,000 x g for 1 hr at 4°C, and the pellet was dissolved in PBS.Murine norovirus (MNV-1) was a gift from Dr. Herbert Virgin at Washington University. RAW 264.7 cells for MNV-1 infection were purchased from ATCC® . To prepare high titer virus stocks, confluent RAW 264.7 cells in T175 flasks were inoculated with MNV-1 and incubated at 37°C with 5% CO7 pfu/ml) and incubated for 1 hr with gentle rocking manually at 15 min intervals. The surface medium was then aspirated and an agar containing 50:50 of 1% LE agarose  and 2X minimum essential medium (MEM) was poured over the infected cells. 2X MEM consisted of Cellgro MEM powder , 10% Hyclone fetal bovine serum , 3% HEPES (Lonza), 2% penicillin/streptomycin (Lonza), 2% sodium pyruvate (Corning Mediatech), 2% L-Glutamine (Lonza), 2% non-essential amino acids (Lonza) and 3% sodium bicarbonate (Lonza). The plates were incubated at 37°C with 5% CO2 for 48 hrs. PBS containing 3.7% formaldehyde (Acros Organics) was then added to fix the cells on the plates for 2 hrs. The agar layer was removed and the plates were stained with 1% crystal violet . Visual plaques were counted to calculate the plaque forming units per ml (pfu/ml).MNV-1 virus stock titer was estimated by plaque assay. RAW 264.7 cells were infected with 100 μl of virus stock , and a union (3.5 ml) were obtained from Harvard Apparatus . As shown in The union (3.5 ml) was connected to two dialyzer chambers (0.5 ml and 1.5 ml) on each side to create a linked chamber. A pore-size selective polycarbonate membrane , was fitted into the linked chamber and two 300 kDa MWCO cellular acetate restricting membranes (Harvard Apparatus) were flanked on both sides of the linked chambers to create two membrane-separated chambers; one for sample addition (4.0 ml sample chamber) and one for sample collection (1.5 ml collection chamber) . Both ch7 genomic copies) was added to the sample chamber. The linked chambers were then submerged into the ElectroPrep tank containing 1 L of electrolyte (the same as the buffer solution used to fill the linked chamber), with the sample chamber facing the cathode and the collection chamber facing the anode. A constant voltage potential (range from 20V to 150V) was supplied by a BioRad PowerPac 300 for a period of time (range from 5 min to 60 min) at room temperature to generate an electric field. Samples for which no constant voltage (0V) was applied to the linked chambers were immersed in electrolyte for 30 min as a control for assessing simple diffusion of virus across the selective membrane. At the end of each experimental trial, 300 μl of sample was collected from both the sample and collection chambers and frozen immediately at -70°C until RNA extraction.In each experiment, 50 μl of MNV-1 . The corresponding current for each applied constant voltage was recorded and the system resistance was calculated using Ohm’s Law: Electrolyte and conductivity measurements were repeated a total of six times on different days for experiments using the 20 mM sodium phosphate buffer. For this buffer, the standard deviations for each measured parameter were: initial pH ± 0.03, final pH ± 0.13, initial conductivity ± 0.16, final conductivity ± 0.03, and current ± 1.14. Since these values did not deviate very much from the mean from day to day/experiment to experiment, we chose to only take one measurement for each experiment using the other buffers described in this study.The RNA extraction method used in all the experiments was a modification of Boom’s nucleic acid purification method using an in-house made Guanidine thiocyanate (GuSCN) lysis buffer as the chaotropic agent and silica as binding agent . To make7 genome copies per μl. All the samples and controls were tested in duplicate and the PCR efficiency for each standard curve generated ranged from 90% to 110%.The quantification of MNV-1 RNA genomic copies was carried out by real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) using a MNV-1 specific probe G54808P (CTA CCC ACC AGA ACC CCT TTG AGA CTC) and primer pair G54763F (TGA TCG TGC CAG CAT CGA) and G54863G (GTT GGG AGG GTC TCT GAGA CAT) . PCR ampin vitro transcription of MNV-1. The RNA template for in vitro transcription was produced by RT-PCR using MNV-1 pairs G54-T7 (TAA TAC GAC TCA CTA TAC GTC TTG ATC GTG CCA GC) and G54-linker (TAG TAC ATA GTG GAT CCA GCC ATT AGT TGG GAG GGT CTC). The PCR products were transcribed using a MEGAshortscript T7 kit following the manufacturer’s instructions, incubating at 37°C for 4 to 5 hrs, followed by TURBO DNase treatment to clean the transcript. Transcript concentration was measured using a NanoDrop 1000 Spectrophotometer  and confirmed by RT-qPCR using probe G54808P and primer pairs G54763F and G54863G (described above).The RNA transcripts were prepared using a MEGA shortscript high yield transcription kit  by After each experiment, recovery percentages of MNV-1 from both the sample and collection chambers were calculated by dividing the viral genomic copy number recovered in each chamber by the initial input in the chamber and multiplying by 100%.TM E-3603 laboratory tubing; United States Plastic Corp, Lima, OH). Platinum electrode wires connected to the power supply were inserted into the open ends of the syringe barrels to create an electric circuit. In one set of experiments, the device was bent into a U-shape and fixed on a rack  were connected with a 6 cm tubing  . Significant differences in % MNV-1 genomic copies recovered from collection chambers impacted by SDS addition to the electrolyte buffer and impacted by electric field strength were analyzed by one-way ANOVA. The means of % MNV-1 genomic copies recovered from collection chambers affected by different electrolyte buffers , different durations, and voltages were compared using student’s t-test for independent samples. Alpha (α) = 0.05 was used to determine significance differences between the means.In initial experiments, to test MNV-1 movement using the MBES system, tris-glycine (pH 7.5) was used as the electrolyte buffer with a 0.6 μm polycarbonate membrane as the separation membrane. Less than 1% of virus was recovered in the collection chamber with applied voltages of 20V-120V for 30 min (data not shown). Then, with the addition of 0.1% sodium dodecyl sulfate (SDS) to the electrolyte buffer (tris-glycine) and substitution with a 1 μm separation membrane, virus recovery in the collection chamber increased to 14.5% with 20V applied voltage for 30 min. The addition of 0.1% SDS to tris-glycine electrolyte increased virus recovery in the collection chamber from below 1% to 14.5%. When another electrolyte  was used, the virus recovery in the collection chamber ranged from 5.6% to 19.3% with 20V applied voltage for 30 min. Increased electrolyte concentration seemed to slightly increase virus recovery. Because virus inactivation can occur when too high of a concentration of SDS (0.1%) is used, SDS addition was decreased to 0.01%, which also resulted in a 14.2% virus recovery in the collection chamber when 20V was applied for 30 min using a 20 mM sodium phosphate electrolyte buffer (pH 7.5). The pH of the 20 mM sodium phosphate was increased to 8.5 in an attempt to increase the negative charge of norovirus to see if this would improve virus movement to the sample chamber, but this resulted in similar virus recovery (15.3%) in the collection chamber with the same voltage (20V) and duration (30 min) applied. Although the electrolyte buffer selection seemed to have an impact virus recovery in the collection chamber, increasing the strength and duration of the voltage applied did not seem to increase virus recovery in the collection chamber.TM Technology) and demonstrated the migration of charged macromolecular solutes through a dialysis membrane [In 2005, Ogle et al. patented an electrophoresis device . Therefore it was important to demonstrate this phenomenon using the newly designed system having a 6 cm distance between the electrodes. As shown in We found that the addition of 0.01% SDS to buffer electrolyte was necessary to enhance viral recovery assisted by an electric field. The MNV-1 genomic copies recovery increased from 0% (without 0.01% SDS) to 31.7% (with 0.01% SDS), as shown in Salmonella (49%); when compared to the control samples without NaPP, the recoveries were 51% and 31%, respectively [When determining particle size of type I poliovirus by syringe filtration in 1968, the addition of 1% SLS to the virus suspension enhanced virus movement through the filter membrane such that 100% of virus particles were recovered in the filtrate using a membrane with a pore-size at least twice as large as the virus. When comparing this to a virus suspension without the addition of SLS, 99% of the virus absorbed to filter membrane. Keesom et al. reported that polycarbonate membranes had a slight negative charge due to a negative zeta potential -27mV) when the membranes were placed in a buffer with a pH above 6 . AnionicmV when tectively . SDS, alAlthough norovirus viral particles have a negative charge under neutral or basic aquatic environments , the surIt is very likely that both hypotheses of the function of SDS contributed to the improvement of virus recovery in the collection chamber. Based on the findings in TM Technology [5 to 1010 cells/ml of erythrocytes (7 μm in diameter) (~80%) migrated through a 10 μm polycarbonate membrane in 2 min with an electric field of 50V/cm using a broad variety of electrolyte buffers such as Bis-Tris, HEPES, glucose, sucrose, and phosphate buffer salts  in small volume (anodic and cathodic chambers were both 420 μl). They also described how the buffer concentration and conductivity, electric field strength, and applied voltage all affected cell migration, but the selection of buffer did not seem to matter. As described in our preliminary studies, the type of buffer used in MBES system did appear to impact virus movement; therefore, we further investigated if the buffer type and concentration would affect norovirus migration across the separation membrane under the mild electric field strengths used in our system.With the modification of the Ogle et al. (2005) electrophoresis device, a fixed boundary electrophoresis method to separate living cells in a membrane-based electro-separation methodology was patented under Gradiflowchnology . They dePBS, TGBE , 31, andFor tris-glycine and 20 mM sodium phosphate buffers, the sum of the percent recovery values for the sample and collection chambers totaled approximately 90%. However, this was not the case for TGBE and PBS.With TGBE, the sum of virus recoveries in the sample chamber and collection chambers was only 72.2% with a large standard deviation for the sample chamber recovery results (65.3% ± 18.8) when 20V was applied. Likely due to the complexity of TGBE, components of this buffer could have interfered with virus movement across the membrane when voltage was applied. No virus was recovered from either the sample chamber or collection chamber when PBS was used as the electrolyte buffer. After applying 20V to the system for 30 min, the pH and conductivity of each buffer remained stable, with the exception of the PBS buffer . The pH 2) (the membrane area in our system was 4.9 X 10−4 m2). Increasing the concentration of phosphate buffer in the system did increase the current density, but because of the larger increase of buffer conductivity, the net electric field strength decreased slightly as the electrolyte buffer concentration increased  = current density (J) / conductivity (σ), where current density (J) = current (A) / membrane area , extremely high voltages ) are used to mobilize biological particles . Differences in particle sizes, particle surface amino acid residues, and charged sugars can contribute to the different particle mobility across a capillary when voltage potential is applied . A UV orWhen measuring the system resistance with an applied voltage of 20V using the 20 mM sodium phosphate with 0.01% SDS (pH 7.5) as the electrolyte buffer, the system resistance increased respectively from 385Ω to 769Ω without and with the presence of the restriction and separation membranes in the device. The increased resistance also caused a decrease of electric current from 52 mA to 26 mA (voltage = current X resistance), which might affect virus movement causing low virus mobility, and thus a decrease in virus recovery in the collection chamber. To decrease the resistance of the system in an attempt to increase virus mobility, the system was re-designed to remove all membranes. In the first re-design, a U-shaped device was constructed . HoweverTo cope with high system resistance and low electric current due to insufficient electrons flowing in the U-shaped vertical device, the system was again re-designed in a horizontal format . As showE. coli by electrochemical disinfection using a Pt (platinum) anode and a chlorine-free electrolyte [Without the shielding of the electrodes with a restriction membrane barrier, it is likely that the negatively charged virus was attracted to the anode surface by electrosorption. This would have been followed by direct electron transfer caused by oxidoreduction of the electrolysis buffer, resulting in instant virus inactivation. This phenomenon is similar to the inactivation of ctrolyte . In Rylactrolyte . A physiThis study is the first to illustrate the possibility of mobilizing viruses, and in particular, noroviruses, by electric force across a pore-size selective separation membrane in order to achieve their separation, concentration and purification. A MBES method for recovering murine norovirus from phosphate buffer was successfully designed, developed, and evaluated in this proof-of-concept study. In a subset of experiments, as high as 30.8% of MNV-1 migrated from sample chamber to collection chamber across a 1 μm separation membrane, when 20 mM sodium phosphate buffer with 0.01% SDS was used with 20V applied for 30 min. Of all variables tested, this set of parameters yielded the highest virus recovery percentage, implying that weak voltage, moderate duration, and low ionic strength electrolytes are optimal for norovirus movement across an electric field without disrupting the stability of the virus particles. Since the negatively charged surface of norovirus is not strong enough to be mobilized in a weak electric field, SDS addition to electrolyte is essential to increase norovirus mobility and recovery. The electric field strength of the system is a key factor to enhance norovirus mobility in an electric field, but from this study it was determined that this can only be achieved by decreasing the distance between the electrodes instead of increasing applied voltage because the virus is not stable at high voltages as demonstrated by RNA degradation. From this study it was also found that a horizontal electric system design is important to ensure the smooth flow of electrons and decrease the system resistance. Last but not least, although it was found that the integration of restriction membranes in the system increases system resistance, a membrane barrier separating the electrode from the collection chambers is essential to avoid virus inactivation by oxidoreduction when in contact with positive electrode (anode).This study suggests that in future designs, either increasing the separation membrane surface area, decreasing the distance between electrodes, or both can help to improve norovirus recovery beyond what was observed in this study. To accommodate larger sample input volumes, a continuous input feeding design could be explored with this system. MNV-1 infectivity was not evaluated using the MBES system because the virus concentration in each chamber was too low to be detected by plaque assay. Future studies should include a virus concentration step prior to analysis of virus infectivity in order to evaluate whether or not the system could be used to detect infectious virus.In conclusion, this study was the first to explore the movement of viruses, and in particular, noroviruses, in an electric field as a part of a sample clean-up and preparation method for norovirus detection. With further modifications of the system and optimization of its parameters, the membrane-based electro-separation method may provide a novel, quick, easy, and cost-effective method for norovirus detection. Furthermore, if improved recovery percentages can be achieved with further optimization, it will be necessary to validate the promising method in the context of food matrixes.S1 Text(DOCX)Click here for additional data file.


Flavivirus member of the Spondweni serocomplex, transmitted by Aedes mosquitoes. We report here the complete coding sequence of a Zika virus strain belonging to the Asian lineage, isolated from an infected patient returning from French Polynesia, an epidemic area in 2013/2014.Zika virus is an arthropod-borne  Flaviviridae, genus Flavivirus) is an arbovirus originally transmitted in Africa through a sylvatic cycle involving mainly Aedes vectors and nonhuman primates, with humans being occasional hosts (Aedes-human-Aedes cycle has been increasingly reported in recent years (Zika virus (ZIKV) . The only previous outbreak reported in the Pacific region was on Yap island in 2007  gene sequences, three lineages that reflect the East African, West African, or Asian geographic origins have been identified. The expanding distribution area of the virus makes Zika fever an emerging infectious disease, as confirmed by its spread to French Polynesia (FP) since October 2013    indicated that it belonged to the Asian lineage, sharing common ancestorship and ca. 99.9% nt and aa identities with isolates circulating in southern Asia and the Pacific islands in the late 2000s . This points to the spread of the Asian lineage, which has been suggested to originate from the introduction of ZIKV in Southeast Asia around 1945 , 6.In silico analysis predicted efficient detection of the FP strain genome by previously published RT-PCR systems, with no mismatch for systems 835-911c (835-911c , and ZIKKJ776791.The virus genome sequence described here has been deposited in the GenBank database under the accession no.


In higher plants, evolutionarily conserved processes playing an essential role during gene expression rely on small noncoding RNA molecules (sRNA). Within a wide range of sRNA-dependent cellular events, there is posttranscriptional gene silencing, the process that is activated in response to the presence of double-stranded RNAs (dsRNAs) in planta. The sequence-specific mechanism of silencing is based on RNase-mediated trimming of dsRNAs into translationally inactive short molecules. Viruses invading and replicating in host are also a source of dsRNAs and are recognized as such by cellular posttranscriptional silencing machinery leading to degradation of the pathogenic RNA. However, viruses are not totally defenseless. In parallel with evolving plant defense strategies, viruses have managed a wide range of multifunctional proteins that efficiently impede the posttranscriptional gene silencing. These viral counteracting factors are known as suppressors of RNA silencing. The aim of this review is to summarize the role and the mode of action of several functionally characterized RNA silencing suppressors encoded by RNA viruses directly involved in plant–pathogen interactions. Additionally, we point out that the widely diverse functions, structures, and modes of action of viral suppressors can be performed by different proteins, even in related viruses. All those adaptations have been evolved to achieve the same goal: to maximize the rate of viral genetic material replication by interrupting the evolutionary conserved plant defense mechanism of posttranscriptional gene silencing. In eukaryotes, posttranscriptional gene-silencing (PTGS) plays a pivotal role in the regulation of gene expression during development Sunkar , stress Four well-defined steps can be distinguished during PTGS: (1) detection of the dsRNAs, (2) generation and amplification of small interfering RNAs (siRNAs), (3) silencing of viral target gene, and finally, (4) spreading of the signal between plant cells and within the host through the vasculature   plays the key and specific role in the anti-Tomato bushy stunt virus (TBSV) silencing    infection in N. benthamiana  standing in the central position of the PTGS. The nucleolytic, slicing core of the RISC complex consists of, among others, Argonaute (AGO) protein  is identified by programmed RISC, it is either cleaved by AGO or it can be translationally unreadable Bartel . In factMoreover, primary siRNAs derived from direct DCL-dependent dicing of the long dsRNA templates can promote accumulation of secondary fraction of siRNAs. This process is considered to be an amplification of PTGS signal mediated by plant RNA-dependent RNA polymerases (RDR)  was described previously by various authors , transgenetically (by transformation) , is used.Most research papers describe the To determine the exact functional abilities of RSS, further analyses are required. Assessment of RSS affinity towards siRNA or long dsRNA can be done by electrophoretic mobility shift assays (EMSA), immunoprecipitation (IP), or co-immonoprecipitation (co-IP) of the ribonucleoprotein complexes. This allows to determine the siRNA–RSS affinity and the specificity of their interactions that, together with experiments based on site-directed mutagenesis of the RSS, can provide essential information on its biological role.In papers published by Pantaleo et al.  and CsorAfter the delivery of viral genetic material into the plant, PTGS machinery recognizes the pathogenic RNA, which in turn leads to its degradation. At this very stage, only immediate viral response to PTGS would enable the virus to spread systemically. In fact, this virus counteraction might be based on: (1) binding of the long dsRNA and their protection from the subsequent DCL processing, (2) sequestration and/or degradation of siRNAs, (3) inactivation of functional RISCs, (4) inhibition of short- and long-distance spread of the silencing signal. Indeed, viral suppressors can interrupt the PTGS utilizing at least one of the mentioned mechanisms.Tomato chlorosis virus coat protein (CP) and P6 virion protein of Rice yellow stunt rhabdovirus is shared with its PTGS-suppressing activity , two nonconserved regions (NONI and NONII), and helicase (HEL), each exhibiting independently both local and systemic PTGS-suppressing activities  and animal viruses (influenza A virus NS1). Conversely, Young et al. , P1 or P1b (ipomoviruses), and HC-Pro (potyviruses).RNA silencing is a common process described across all kingdoms, in plant and animal systems, and consequently, suppression of it was described for plant and animal viruses. Importantly, plant viral suppressors of PTGS retain their biological function in animals, and vice versa. For instance, VP3 protein of avian g et al.  have shoAs it will be described in the following paragraphs, the viral RSSs represent a wide biological diversity in structure, mode of action, as well as their impact on host plant. However, despite the fact that knowledge from the field of RSS is constantly verified and updated, it still seems that the fundamental mechanism of this particular virus–host interaction is yet to be discovered. New experimental data broadens and deepens the view on the RSS functional complexity. It puts rather significant emphasis on the occurrence of several levels of plant defense and virus counter-defense relationships, at least at the PTGS level , pH 8.5 , and pH 8  motifs. Therefore, the question arose whether viral RSS proteins containing GW/WG motifs can inhibit PTGS via interaction with AGO. It was shown that P1 protein of The mode of action of known viral RSSs is sophisticated: it targets very sensitive plant metabolic pathways and disrupts homeostasis of cellular regulatory signals based on distribution of small regulatory RNAs. Therefore, it is not surprising that the occurrence of RSS in plant cells might be connected with macroscopic changes manifested with disease-like symptoms, for instance leaves malformation, stem stunting or local and necrotic lesions.N. benthamiana and N. tabacum stably transformed with seven viral-silencing suppressors originated from different virus genera: P1 of Rice yellow mottle virus (Sobemovirus), P1 of Cocksfoot mottle virus (Sobemovirus), P19 of TBSV (Tombusvirus), P25 of PVX (Potexvirus), HC-Pro of Potato virus Y (Potyvirus), 2b of CMV (Cucumovirus), and AC2 of African cassava mosaic virus (Begomovirus). The authors concluded that a wide range of effects manifested differently upon expression of particular RSSs with regard to transformed tobacco species. Going further, Soitamo and colleagues asked whether and how the phenotypic effect that resulted from RSS expression in plant is connected with transcriptome and proteome changes  in A. thaliana, and revealed over-accumulation of miR168 and downregulation of AGO1. Interestingly, it was shown that this miR168 upregulation is not dependent on P19 siRNA-binding abilities , and in CPMV (Cowpea mosaic virus) expression system (Saxena et al. The knowledge of mechanisms of PTGS and its suppression is useful not only in a research on molecular biology of viruses. PTGS is frequently activated during transgenesis and manifests with lowered efficiency of transgene expression. Therefore, the appearance of a strong RSS can elevate the production efficiency of the recombinant protein (Gao et al. Conversely, virus-based expression vectors seem to be alternative for genetically modified organisms and the cheapest platform for synthesis of heterologous proteins in plants Hefferon . The higUsefulness of RSS in biotechnology is not confined only to plant engineering. Cheng et al.  found toIn summary, here we gave a brief overview on functional diversity and complexity of viral PTGS suppressors and the functions they play in plants under virus infection. However, the stream of newly described data delivered from experiments that are being constantly performed in the area of RSS, will be putting a new light on viral suppressors, the mechanism they utilize and molecular interactions during pathogenesis. In the context of the newly described results, the previously speculated molecular interactions can be interpreted differently, and in the course of time, novel biochemical and functional abilities of RSS will be characterized. This, in turn, will enrich the general knowledge about molecular biology of viruses.


To the Editor: In 2013, a 6-week-old female piglet kept in a flatdeck cage had coughing, growth retardation, and diarrhea and was taken to a local veterinarian in Hannover, Germany; the piglet was euthanized. After necropsy at the University of Veterinary Medicine in Hannover, histologic investigation found interstitial pneumonia; a mild, multifocal, lymphohistiocytic panencephalitis that affected the cerebrum and cerebellum, including brain stem and medulla oblongata; and a mild, multifocal, lymphohistiocytic panmyelitis. Results from screening for typical neurotropic viruses  were negative; Mycoplasma hyorhinis was detected by multiplex PCR  within the lung and pulmonary lymph nodes. Cerebral tissue from the pig was processed for viral metagenomics by random RNA and DNA virus screening and next-generation sequencing (NGS) with the 454 sequencing platform , as described (>97% nt identity with porcine bocavirus (PBoV) KU14. No other viral sequences were detected.http://mafft.cbrc.jp/alignment/server/), we aligned the nearly complete VP1 gene of PBoV S1142/13 with various closely related members of the genus Bocaparvovirus and built a maximum-likelihood tree by using the general time reversible plus invariable sites plus gamma distribution method, as determined by jModelTest 2.0  served as a positive control.For further substantiation of a potential cause-effect relationship of histologic  and NGS results, we performed fluorescent in situ hybridization (FISH) on formalin-fixed, paraffin-embedded central nervous system (CNS) sections of the diseased animal and of a control pig with no CNS lesions. We used an RNA probe specific for the obtained NP1 and VP1 sequences covering 1,153 nt  according to the manufacturer’s protocol, with minor variations . A probe specific for porcine ubiquitin  was first described in 2009 as porcine boca-like virus in pigs in Sweden with postweaning multisystemic wasting syndrome (M. hyorhinis on the PBoV pathogenesis cannot be ruled out. Similarly, co-infection of M. hyorhinis and porcine circovirus type 2 has been associated with enhanced inflammatory lesions in the lungs of pigs (PBoV (genus The CNS tropism of PBoV S1142/13 could result from various factors, including specific amino acid changes that enable the virus to pass the blood–brain barrier and infect neurons. Additional studies are necessary to elucidate a possible role of the amino acid differences between PBoV S1142/13 and PBoV KU14 in the tropism of these viruses.Human bocavirus has recently been found in the cerebrospinal fluid of patients having encephalitis (


West Nile virus (WNV) is a positive-sense RNA arbovirus responsible for recent outbreaks of severe neurological disease within the US and Europe. Large-scale analyses of antiviral compounds that inhibit virus replication have been limited due to the lack of an adequate WN reporter virus. Previous attempts to insert a reporter into the 3’ untranslated region of WNV generated unstable viruses, suggesting that this region does not accommodate additional nucleotides. Here, we engineered two WNV infectious clones containing insertions at the Capsid (C)/Capsid Anchor (CA) junction of the viral polyprotein. Recombinant viruses containing a TAT(1-67) or Gaussia Luciferase (GLuc) gene at this location were successfully recovered. However, rapid loss of most, if not all, of the reporter sequence occurred for both viruses, indicating that the reporter viruses were not stable. While the GLuc viruses predominantly reverted back to wild-type WNV length, the TAT viruses retained up to 75 additional nucleotides of the reporter sequence. These additional nucleotides were stable over at least five passages and did not significantly alter WNV fitness. Thus, the C/CA junction of WNV can tolerate additional nucleotides, though insertions are subject to certain constraints. Flaviviridae. The viral genome consists of a single open reading frame flanked by 5’ and 3’ untranslated regions (UTRs) (West Nile virus (WNV) is an ~11 kb, enveloped, positive-sense RNA virus belonging to the family s (UTRs) A. The inEvaluation of the efficacy of antiviral agents has traditionally relied on monitoring the effect of compounds on either viral yields, using standard plaque assays, or viral genome levels, using quantitative reverse transcription (RT)-PCR. However, these assays are time consuming, limiting their usefulness for large-scale screens of compound libraries. Therefore, multiple groups have developed subgenomic replicon systems for WNV that encode reporter genes, which serve as an easily detectible surrogate marker for viral replication ,11,12,13gfp) or Renilla luciferase into the WNV 3’ UTR , and 1X protease inhibitors [Sigma]), and subjected to SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred to nitrocellulose membranes that were then incubated with the following antiserum: monoclonal mouse α-HIV TAT (1:3000) , polyclonal mouse α-WNV (1:1000) , and monoclonal mouse α-Actin (1:400) .Cell monolayers were washed with DPBS, lysed with RIPA buffer  and placed in 1 mL of complete DMEM. After 4 h at 4 °C, the plaque-containing media was used to inoculate Vero monolayers in T75 flasks. Following 1 h of rocking at 37 °C/5% COHigh throughput screening of compounds with potential anti-WNV activity has been hindered by the lack of a WN reporter virus with wild-type replication. In this study, we generated two recombinant WNVs. As with previously generated WN reporter viruses ,15, both


The mouse vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Based on largely monogenic expression of either type 1 or 2 vomeronasal receptors (V1Rs/V2Rs) or members of the formyl peptide receptor (FPR) family, the vomeronasal sensory epithelium harbors at least three neuronal subpopulations. While various neurophysiological properties of both V1R- and V2R-expressing neurons have been described using genetically engineered mouse models, the basic biophysical characteristics of the more recently identified FPR-expressing vomeronasal neurons have not been studied. Here, we employ a transgenic mouse strain that coexpresses an enhanced variant of yellow fluorescent protein together with FPR-rs3 allowing to identify and analyze FPR-rs3-expressing neurons in acute VNO tissue slices. Single neuron electrophysiological recordings allow comparative characterization of the biophysical properties inherent to a prototypical member of the FPR-expressing subpopulation of VNO neurons. In this study, we provide an in-depth analysis of both passive and active membrane properties, including detailed characterization of several types of voltage-activated conductances and action potential discharge patterns, in fluorescently labeled vs. unmarked vomeronasal neurons. Our results reveal striking similarities in the basic (electro) physiological architecture of both transgene-expressing and non-expressing neurons, confirming the suitability of this genetically engineered mouse model for future studies addressing more specialized issues in vomeronasal FPR neurobiology. V1r  family of putative chemoreceptor genes. The Fpr-rs family comprises 7 members, 5 of which  are predominantly or exclusively expressed in subsets of VSNs  and with recommendations put forward by the Federation of European Laboratory Animal Science Associations (FELASA). Both C57BL/6 mice  and Fpr-rs3-i-Venus mice were housed in groups of both sexes at room temperature on a 12 h light/dark cycle with food and water available ® Gel extraction kit . The transgene was injected into the pronuclei of fertilized C57BL6/DBA2 mouse oocytes following standard procedures. Four founders carrying the transgene were obtained. One of these founder animals expressed the transgene in VSNs and was, thus, used to start the colony. Backcrossed to C57BL/6J, mice were kept hemizygous. Wild type and transgenic mice had no obvious differences in size, weight, fertility, life expectancy or food consumption.The transgene (Fpr-rs3-i-Venus) contains the FPR-rs3 coding sequence followed by an internal ribosome entry site (IRES), and the coding sequence for tau-Venus, a fusion between the microtubule-associated protein tau and Venus yellow fluorescent protein  4-(2-Hydroxy-ethyl)piperazine-1-ethanesulfonic acid (HEPES) buffered extracellular solution containing (in mM) 145 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES; pH = 7.3 (adjusted with NaOH); osmolarity = 300 mOsm (adjusted with glucose). (S2) Oxygenated  extracellular solution containing (in mM) 125 NaCl, 25 NaHCO3, 5 KCl, 1 CaCl2, 1 MgSO4, 5 BES; pH = 7.3; osmolarity = 300 mOsm. (S3) solution containing (in mM) 144 NaCl, 5 KCl, 1 TEACl, 1 CaCl2, 1 MgCl2, 10 HEPES, pH 7.3; osmolarity = 300 mOsm. (S4) solution containing (in mM) 134 NaCl, 5 KCl, 1 TEACl, 1 CaCl2, 1 MgCl2, 10 HEPES, 10 4-AP, pH 7.3; osmolarity = 300 mOsm. (S5) solution containing (in mM) 110 NaCl, 5 KCl, 25 TEACl, 1 CaCl2, 1 MgCl2, 10 HEPES, 10 4-AP, pH 7.3; osmolarity = 300 mOsm. (S6) solution containing (in mM) 115 NaCl, 25 TEACl, 1, 1 MgCl2, 10 HEPES, 10 4-AP, pH 7.3; osmolarity = 300 mOsm. (S7) solution containing (in mM) 105 NaCl, 25 TEACl, 5 mM BaCl2, 1 MgCl2, 10 HEPES, 10 4-AP, pH 7.3; osmolarity = 300 mOsm. (S8) Pipette solution containing (in mM) 143 KCl, 2 KOH, 1 EGTA, 0.3 CaCl2 (free Ca2+ = 110 nM), 10 HEPES, 2 MgATP, 1 NaGTP; pH = 7.1 (adjusted with KOH); osmolarity = 290 mOsm. (S9) Pipette solution containing (in mM) 133 CsCl, 10 NaCl, 2 CsOH, 1 EGTA, 0.3 CaCl2 (free Ca2+ = 110 nM), 10 HEPES, 1 MgATP, 1 NaGTP; pH = 7.1 (adjusted with CsOH); osmolarity = 290 mOsm.The following solutions were used: , decalcified overnight in 0.5 M EDTA (4°C) and cryoprotected in PBS containing 30% sucrose (4°C). The dehydrated VNO was embedded in Tissue Freezing Medium and sectioned at 20 µm on a Leica CM1950 cryostat .2 followed by decapitation using sharp surgical scissors. The lower jaw and the soft palate were removed allowing access to the vomeronasal capsule. After removal of the cartilage, the dissected VNO was embedded in 4% low-gelling temperature agarose and coronal slices (150–200 µm) were cut in ice-cold oxygenated extracellular solution (S2) using a Leica VT1000S vibratome . Sections were transferred to a submerged, oxygenated (S2) and chilled storage chamber until use.Acute vomeronasal tissue sections were prepared as previously described . Excess antibodies were removed by washing in PBS containing 0.05% Triton-X 100 . To control for nonspecific staining, experiments in which the primary antibodies were omitted were performed in parallel with each procedure.Blocking was performed for 1 h in PBS containing 2% goat serum, 1% gelatine and 0.2% Triton X-100 (blocking solution). Sections were then incubated overnight at 4°C with primary antibody sera  in blocking solution, washed in PBS containing 0.05% Triton-X 100 , and incubated for 1 h with AlexaS2 . Patch pipettes (4–7 MΩ) were pulled from borosilicate glass capillaries  on a PC-10 micropipette puller , fire-polished  and filled with pipette solution (S8 or S9) depending on experimental design. An agar bridge (150 mM KCl) connected reference electrode and bath solution. An EPC-10 amplifier controlled by Patchmaster 2.67 software  was used for data acquisition. We monitored and compensated pipette and membrane capacitance as well as series resistance. Only neurons exhibiting small and stable access resistances  were used for analysis. Liquid junction potentials were calculated using JPCalcW software ). Between recordings, holding potential (Vhold) was −60 mV. All electrophysiological data were recorded in whole-cell configuration at room temperature.Vomeronasal organ slices were transferred to a recording chamber  on an upright fixed-stage scanning confocal microscope  equipped with a 20x/1.0 NA water immersion objective  as well as a cooled a CCD-camera . Slices were continuously superfused with oxygenated e Barry,  and corrn) are denoted in figure legends. If not stated otherwise, results are presented as means ± SEM. Statistical analyses were performed using paired or unpaired t-tests or one-way ANOVA with Tukey’s HSD post hoc test. Tests and corresponding p-values that report statistical significance are individually specified in figure legends. Drug sensitivity of voltage-gated K+ (Kv) currents was examined based on an “additive” drug exposure regime, i.e., TEA (1 mM), 4-AP (10 mM), and TEA (25 mM) were sequentially applied and the inhibitor-sensitive currents were isolated by subsequent “offline” subtraction from each preceding recording.All data were obtained from independent experiments performed on at least 3 days using at least three different animals. Individual numbers of cells/experiments (1/2). Current activation time constants (τ) were calculated by fitting individual traces to monoexponential functions I(t) = I1 [exp (−t/τ)] + I0.Electrophysiological data were analyzed offline using PatchMaster 2.67 (HEKA Elektronik), IGOR Pro 6.3  and Excel  software. Activation curves were fitted by the Hill equation to calculate the membrane potential of half-maximal activation . Using standard transgenic techniques , we generated such a mouse strain in which FPR-rs3 is coexpressed with tau-Venus, an enhanced variant of yellow fluorescent protein  confirming layer-specific expression of the FPR-rs3 transgene in apical VSNs. Immunostaining with an anti-FPR-rs3 antibody , an expression level within the previously reported range between 0.4% and 0.8%  also express the FPR-rs3 protein ; Figures  Figures . Some FPThe passive membrane properties of a neuron determine its basic electrophysiological characteristics and, thus, control its individual stimulus-response function. For FPR-rs expressing vomeronasal neurons, these critical physiological parameters are unknown. Using Fpr-rs3-i-Venus mice, we performed whole-cell patch-clamp recordings from optically identified, fluorescently labeled FPR-rs3-expressing neurons in acute VNO tissue slices Figures . For quainput), membrane capacitance (Cmem), and membrane time constant (τmem)) were obtained immediately after membrane rupture. Treated, to a first approximation, as a “biological constant” with a value of ~1 µF/cm2  routine. Transgene-positive (FPR-rs3+) neurons revealed an average Cmem value of 5.96 ± 0.49 pF (n = 21), similar to data obtained from control VSNs , suggesting that FPR-rs3+ neurons share the extraordinary sensitivity of V1/2R-expressing VSNs . We obtained relatively slow τmem values of 26.79 ± 2.25 ms (n = 21) in FPR-rs3+ neurons vs. 24.29 ± 1.57 ms (n = 21) in control neurons  that FPR-rs expressing VSNs are not segregated or isolated from the “general” VSN population; and (b) that transgene expression per se does not perturb the passive biophysical properties of FPR-rs3+ neurons.Together, these results describe different passive membrane parameters of FPR-rs3+ neurons. A hallmark of VSNs is that depolarizing current injection of only a few picoamperes triggers repetitive action potential discharge  for FPRrs3+ neurons and 3.9 ± 1.08 Hz (n = 21) for control cells  or 16.54 ± 1.17 Hz ). Injection of negative current into FPR-rs3+ neurons revealed a hyperpolarization-activated rebound depolarization . In both FPR-rs3+ and control VSNs, we frequently observed rebound spikes upon repolarization  for FPR-rs3+ neurons and 73.92 ± 0.87 mV (n = 172) for control neurons  and of 2.33 ± 0.09 ms (control neurons), while FDHM was 3.65 ± 0.08 ms (FPR-rs3+ neurons) and 3.67 ± 0.12 ms (control VSNs), respectively.Next, we examined action potential discharge of FPR-rs3s Figure i. AveragThese data show that FPR-rs3 expressing VSNs exhibit rather slow action potentials and, albeit an extraordinary sensitivity, show a relatively narrow spike frequency coding range. Together, these active membrane properties are shared by both FPR-rs3 expressing and control neurons.+ (NaV) channels are primarily responsible for action potential initiation and impulse propagation. Upon membrane depolarization, NaV channels mediate the rapid Na+ influx that underlies the upstroke of the action potential. However, the electrophysiological properties of the nine homologous members of the NaV channel family (NaV1.1 to NaV1.9) are not identical and even small differences in NaV channel expression can have profound effects on electrical excitability  in FPR-rs3+ neurons. Stepwise depolarizations from −120 mV to +70 mV  in absence and presence of tetrodotoxin . Similar values were recorded from control VSNs . Figure + neurons  function demonstrates half-maximal current activation upon depolarization to approximately −50 mV . Interestingly, at voltages ranging from approximately −60 mV to −5 mV, activation and inactivation curves overlap, suggesting coexpression of multiple NaV channel isoforms and/or a substantial “window current”.Next, we examined the voltage-dependence of TTX-sensitive I Figures . Fittingv Figure iii. Steae Figure ii and re+ VSNs express one or more NaV channel isoform(s) that exhibit relatively slow activation upon membrane depolarization >−65 mV with half-maximal and complete activation at ~−50 mV and −30 mV, respectively. Moreover, the slope of the steady-state inactivation curve is relatively shallow, revealing that full channel inactivation only occurs at positive potentials. Since all measured parameters are similar to data recorded from control VSNs, our data further substantiate the notion that that FPR-rs expressing neurons do not constitute a biophysically segregated “outgroup” of VSNs.Together, these results demonstrate that FPR-rs3Kv) in FPR-rs3+ neurons.To a large extent, Kv channels control electrical signaling in excitable cells. Accordingly, the large and extended Kv channel family is functionally diversified by alternative splicing, oligomeric subunit assembly, and subcellular targeting  channels at low millimolar concentrations  triggered large outward currents that essentially showed no sign of inactivation Figure ; inset. V Figure . Linear  Figures . Somewhav channel populations shape action potential discharge in FPR-rs3+ cells. Spikes were elicited and discharge parameters were analyzed as described  channels are integral constituents of a neuron’s Ca2+ signaling toolkit  that regulates a wide variety of cellular events  in FPR-rs3+ neurons.Voltage-gated CaV channel subunits are divided into three subfamilies (CaV1 to CaV3) that differ in function and regulation  from two different prepulse potentials , values typical for T-type currents.The ten functional vertebrate Can Figure i. As expn Figure . The resn Figure ii and noe Figure iii demonV channels in FPR-rs3+ neurons. All four members of the CaV1 subfamily are characterized by both long-lasting and large (L-type) Ca2+ currents and high sensitivity to dihydropyridines, such as nifedipine  and isolated nifedipine-sensitive currents by digital subtraction  Can Figure i. As expV Figure ii. Half-V2 subfamily of HVA Ca2+ channels are selectively sensitive to peptide neurotoxins from spider and cone snail venoms , but −36.96 ± 6.50 pA/pF in control VSNs (n = 8). Moreover, half-maximal activation in controls was shifted to more positive values  and control neurons .A slight, though also significant difference between FPR-rs3+ neurons exhibit a variety of CaV currents, both LVA and HVA. Since both N- and P/Q-type currents show somewhat different properties in FPR-rs3 expressing VSNs, these two CaV2 channel isoforms might play distinct roles in FPR-rs3+ neurophysiology.In summary, the above data show that FPR-rs3For most mammals, the VNO is crucial for intra- and interspecific chemical communication. While the basic biophysical properties of both V1R- and V2R-expressing vomeronasal neurons have been described , but also for studies of axon targeting and glomerular innervation in the AOB. While, based on the experimental strategy used here, we cannot exclude that FPR-rs3+ VSNs additionally express other vomeronasal receptor genes, this appears unlikely since the negative feedback signal that ensures gene exclusion in apical VSNs is also maintained by exogenous expression of another receptor gene, even an OR  is noticeable. Frequency coding accommodates spike rates between 0 and ~15 Hz that encode receptor currents ranging to a maximum of ~25 pA (note that the “linear” dynamic range of the f-I curve is considerably more narrow). Similar values have previously been reported  we obtained for both FPR-rs3+ and control neurons ensure that brief stimulatory events will not generate significant output, in line with the idea that stimulus exchange in the VNO is relatively slow probably allowing prolonged VSN receptor-ligand interaction.The specific biophysical profile of FPR-rs3+ neurons do not segregate these neurons from the “general” VSN population.Detailed spike waveform analysis revealed rather slow and broad action potentials in line with previously published results , i.e., Nav1.1, 1.2, 1.3, 1.4, or 1.7  and spike width (FDHM/duration) where different between FPR-rs3+ neurons and control VSNs. In addition to NaV and KV channels, several types of CaV channels were identified in FPR-rs3+ neurons. T-, L-, N-, and P/Q-type ICaV was isolated, either pharmacologically  or by prepulse inactivation (T-type). While T- and L-type ICav in FPR-rs3+ VSNs did not significantly differ from control neurons, we find that both N- and P/Q-type currents show somewhat different properties in FPR-rs3 expressing VSNs. We can only speculate about the mechanisms that might link FPR-rs3 expression to altered expression and/or functionality of either N- or P/Q-type CaV channels. The scope of possible explanations ranges from altered Cacna1a/Cacna1b transcription by random transgene insertion to direct binding of Gβ/γ to the α1 subunit of either CaV2 channel (Currie, Similar pharmacological approaches were used to isolate currents mediated by K Currie, , complex Currie, , or unknThe Fpr-rs3-i-Venus mouse model we introduce and the basic electrophysiological characterization we performed provide a foundation for future functional studies of FPR-rs neurophysiology. In analogy to FPR signaling in the immune system, current concepts of FPR-rs function suggest a role as chemoreceptors for inflammation-associated and pathogen-related compounds (Rivière et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To the Editor: West Nile fever is caused by the West Nile virus (WNV), a mosquito-borne member of the genus Flavivirus. Birds are the natural reservoir of the virus, which is maintained in nature in a mosquito–bird–mosquito transmission cycle. WNV has been detected in many regions worldwide, including North America, Europe, Africa, the Near East, and Asia  became seropositive after the stay. In addition, 12.5% of military working dogs in France imported from Hungary were seropositive on initial testing. We also found that, in France, dogs are the sentinels of WNV circulation in the Var (12.0%) and Gard (9.1%) departments. All dogs we tested that were positive for IgG were negative for IgM, a finding that indicates infection by the virus did not occur recently.The results and the statistical analysis reveal notable differences in the seroprevalence rates, according to the geographic area. N’Djamena, Chad, where all native dogs tested positive for WNV, is located at the confluence of the Chari and Logone Rivers and is an area with high densities of residential and migratory birds. In contrast, the northeastern region (Haut-Ogooué) of Gabon, where no native dogs tested positive for WNV, is an ecosystem of wet forests without migratory birds, unfavorable to virus circulation. In Dakar, 18.7% of native dogs were seropositive. In these central parts of Senegal, characterized by a semi-arid climate and vegetation composed of steppe plants and bluegrass, several WNV strains have been isolated from birds and mosquitoes. The seroprevalence was lower (0%–12.5%) in the sub-Saharan area, including Côte d’Ivoire, Gabon, DRC, and Senegal , where the humid or semihumid climate is linked with tropical rain forests or woodland savannah known to favor sedentary birds , where WNV epidemics and epizootics occurred in 2000 and 2003. In addition, we observed that military working dogs purchased from Hungary, where WNV infection is common (


Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS). The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84–90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84–86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes.The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of  Feedback inhibition of strategically positioned enzymatic steps is a commonly used strategy in the shikimate pathway but it is implemented in different ways for various organisms Escherichia coli produces three isoforms of DAHP synthase, each of them being sensitive to Tyr, Phe, or Trp E. coliIn prokaryotes, fungi, algae, and plants, the aromatic amino acids synthase . AnotherMycobacterium tuberculosis possesses just one DAHP synthase (MtDS) and one intracellular, monofunctional chorismate mutase (MtCM). Whereas MtDS alone is strongly inhibited by the simultaneous binding of the three aromatic amino acids α fold, as well as from the eukaryotic (AroQβ) and the secreted (AroQγ) CMs, and were thus grouped as the AroQδ subclass δ representative that was investigated in structural detail Actinomycetales, which includes corynebacteria and mycobacteria, generally belong to the AroQδ subclass and are involved in a similar enzyme complex with the corresponding DAHP synthases In contrast, Without MtDS, MtCM activity is reduced by two orders of magnitude. From the crystal structures of free MtCM and the MtCM-MtDS complex it is clear that MtDS residues do not directly participate in the acceleration of the chorismate to prephenate rearrangement We chose the method of directed evolution E. coli; PDB: 1ECM) (a) they are proximal to MtDS in the MtCM-MtDS complex and are also in contact with active site residues. The six last residues are within 6 Å of MtDS  They show structural and chemical dissimilarities to the prototypical AroQα protein, EcCM, which does not rely on activation by a complex partner. EcCM has two residues in the C-terminal portion of helix 3, Ser84 and Gln88, that are crucial for its activity. Upon Gln88Ala mutation, EcCM activity drops by a factor of 2×104, and Ser84 is considered important for orientation of the substrate molecule in the active site (c) They show good conservation in the AroQδ alignment  lignment . In factike EcCM .E. coli strain KA12 was adapted. KA12 carries a chromosomal deletion of the genes pheA and tyrA encoding the two bifunctional enzymes CM-prephenate dehydratase and CM-prephenate dehydrogenase, respectively. It can grow on minimal medium devoid of Phe and Tyr (M9c), if provided with the helper plasmid pKIMP-UAUC carrying the genes pheC and *tyrA for monofunctional versions of prephenate dehydratase and prephenate dehydrogenase, respectively tet promoter upstream of the CM gene i.e., M9c +F; To select for MtCM variants that are still capable of productive interactions with MtDS, a previously established selection system aroG encoding MtDS, and the library plasmid pKT-CM, which encodes partially randomized MtCM variants. Since pKT-CM has an otherwise identical structure to pKTNTET, expression of the aroQδ mutant genes is also controlled from Ptet.E. coli DAHP synthases might affect MtCM activity. However, this is highly unlikely, since MtCM is a member of the distinct AroQδ subclass not present in E. coli, and the endogenous DAHP synthase isoenzymes, which belong to a different subtype than MtDS, are not part of a feedback regulatory circuit for controlling CM activity A possible concern is that endogenous i.e., the presence of both pKIMP-ACG and pKTNTET). These results showed that KA12/pKIMP-ACG can be used as a new selection system to explore the interactions between the two enzymes.The new KA12/pKIMP-ACG selection system was calibrated using wild-type MtCM (on pKTNTET) and appropriate negative controls. It was demonstrated  that at 4 to 1.8×106 clones per library. Sequencing of 65 clones from non-selective plates confirmed that the chemical oligonucleotide synthesis and the procedures used in the construction of the libraries did not inappropriately bias the composition of the libraries at the nucleotide level, which would have skewed the ensemble of amino acid sequences available for selection . This coincides with the conservation of a small residue  in the alignment of natural AroQequences . The cryequences  providesequences , as wellproteins , and thu surface  [10]. Itith MtDS  [10].Residue Arg87 is only moderately conserved phylogenetically  and alsoδ proteins in the multiple sequence alignment  is compatible with pKT-CM (oripUC), carries a chloramphenicol resistance marker and the tetracycline repressor, which controls expression of an adjacent Ptet regulated T7 RNA polymerase gene in response to the concentration of the inducer Tet. A problem often encountered with T7 RNA polymerase-driven gene expression is an undesirable high basal activity (“leakiness”) prior to addition of inducer lac promoter-controlled T7 RNA polymerase gene tetPT7 promoter system (The new plasmid pT7POLTS (r system .kcat/Km) within a factor of 2 of the wild-type MtCM . In contrast, retaining a positively charged amino acid in place of Arg87  correlates with high activation factors, suggestive of a functionally preferred electrostatic interaction in the complex invitro, whereas a hydrophobic  or uncharged polar residue  at this position weakens MtDS activation. The regulatory behavior of the purified MtCM variants was also assessed in response to the presence of the metabolic end products of the chorismate mutase branch. Interestingly, a strong response to inhibition by Phe and Tyr generally coincides with a high degree of activation by MtDS  were performed . AlthougIn summary, the selection experiments with C-terminally randomized MtCM libraries resulted in a set of distinct regulatory variants with roughly similar intrinsic basal CM activities but varying widely in their potential to become activated by MtDS. Simultaneously, the activation potential correlates roughly with the degree of the physical interaction with MtDS observed by native PAGE and the sensitivity to feedback inhibition of the complex, as illustrated in i.e., randomizing mutagenesis, selection invivo, and analysis of surviving library members– to quickly survey essential protein-protein interactions in the MtCM-MtDS enzyme complex. The pattern of sequence conservation for MtCM’s seven C-terminal residues that we observed in our laboratory evolution experiments essentially coincides with the pattern that emerged during natural evolution of homologous CMs. From two complementary selection experiments, carried out in the absence and in the presence of MtDS, we were able to discriminate between residues needed mainly for the basic integrity of AroQδ CMs and those required for catalytically productive complex formation, respectively.We have applied the strategies of directed evolution –M. tuberculosis cell is regulated by rapid dynamic control of the ratio between the poorly active free MtCM and the over 100 fold more active MtCM when in complex with MtDS Kd, app of the hetero-octameric complex Because the biosynthesis of the aromatic amino acids Phe and Tyr is energetically very costly for organisms Actinomycetales-specific CM-DS complexes Finally, high-resolution structures of some of the strongly deregulated variants selected here, such as clones 1–6 or 2–17, could inform the rational design of molecules that interfere with protein-protein interactions E. coli strain KA12 invivo assays, protein production and standard cloning. Details on plasmids pKTCM-HC, pKTDS-H, and pKTDS-HN invivo was achieved by reengineering the selection system towards a tetracycline-inducible promoter system. pKTCM-HCDXhoI was constructed from pKTCM-HC 5′-TGGAAATCGTGGAGTCCCAACCTGT) and 204-MTCM2N . The obtained PCR fragment was subsequently used as a mega-primer together with oligonucleotide 206-MTCM2S (CATATGCGTCCAGAACCCCCACATCA5′-ACCGATGT) on pKTCM-HC as a template. The NdeI/XhoI-digested fragment (318 bp) was ligated to the 4561 bp NdeI-XhoI fragment from pKTCM-HC, yielding the 4879 bp pKTCM-HCDXhoI plasmid. This plasmid was used to construct pMG242, containing a shortened but functional version of the aroQδ gene, which encodes an MtCM variant starting at Met5. Ligation of the NdeI/XhoI-digested product (261 bp) of the PCR with oligonucleotides 297-SHO-S (CATATGCTGGAGTCCCAACCT5′-CAA) and 204-MTCM2N on template pKTCM-HCDXhoI to the 4561 bp fragment of the NdeI/XhoI-digested pKTCM-HC plasmid yielded the 4822 bp pMG242. Plasmid pKSS-TM4 was used as the template for library construction. pKSS-TM4 contains a non-functional portion of the aroQδ gene on a pKSS backbone GAGCTCACCATCATCATCACCACTTCTTCTGGTATGCTCGAGTCCCAACCTGT5′-CTCAC) and 342-MCM-KpnI-N (GGTACCTTAGCGGCCCAAGCGCAAAAGCAGGATGGCCAGA5′-TACTT) on template pMG242. The 278 bp KpnI-SacI fragment was ligated into the correspondingly cut pKSS fragment of 2855 bp, yielding pKSS-TM4 (3133 bp). Plasmid pKTCMtet2-HC (3058 bp) contains the aroQδ gene driven by a tandem PtetPT7 promoter system, as well as the tetR gene for controlled repression of the tet operator within Ptet. It was obtained by ligating the 261 bp NdeI-XhoI fragment of pMG242 to the 2797 bp NdeI-XhoI fragment of plasmid pKTH-400-5 (3100 bp) aroQδ-negative control and as acceptor vector for the libraries, plasmid pKTCTET-0 (4096 bp) was constructed by ligating the 2797 bp NdeI-XhoI fragment of pKTCMtet2-HC with the 1299 bp NdeI-XhoI stuffer fragment from pMG242-0. Plasmid pMG242-0 (5860 bp) is derived from pMG242 by inserting a Tet resistance stuffer fragment. It was constructed in two steps, first by introducing a silent AscI site into the MtCM gene of pMG242, yielding pMG242-sil, and subsequently, by cloning an AscI/XhoI-digested Tet resistance determinant fragment into the AscI/XhoI-digested pMG242-sil. For the construction of pMG242-sil, a 193 bp fragment generated with primers 299-Ascsil-S (GGCGCGCCGAGGTTTCCAAGGCCAT-3′5′-TTAGTCAAGC) and 204-MTCM2N on template pMG242 was used as a megaprimer for a second PCR on the same template together with 297-SHO-S, giving a 278 bp fragment. This fragment was cut with NdeI and XhoI (261 bp) and inserted into the NdeI/XhoI-digested pKTCM-HC vector backbone (4561 bp) to give pMG242-sil (4822 bp). The fragment carrying the Tet resistance determinant was amplified from a DNA sequence of pBR322 CTCGAGCCGTGTATGAAATCTAA-3′5′-CAAA) and 162-STS (GGCGCGCCCATTCAGGTCGAGGT-3′5′-CAAA). The resulting 1222 bp PCR fragment was digested with XhoI and AscI to yield a 1207 bp stuffer fragment (encoding the Tet resistance determinant) that was then ligated with the 4653 bp XhoI/AscI-digested pMG242-sil to give plasmid pMG242-0 (5860 bp). Plasmid pKTNTET was constructed by restriction digestion of plasmids pMG244 and pKTCTET-0 with NdeI and SpeI. The respective 296 bp and 2765 bp fragments were ligated and yielded plasmid pKTNTET (3061 bp). pKTNTET encodes the His6-tagged MtCM sequence defined in this work as wild-type (wt) MtCM  and 203-MTCM2CN (5′-CGATACACTAGTTATTAGTGACCGAGGCGGCCACGGCCCAAT) on template pKTCM-HC. pMG243 (4879 bp) was constructed from the 4561 bp NdeI/XhoI fragment of pKTCM-HC and a 318 bp NdeI/XhoI-digested PCR fragment obtained from amplification of pKTCM-HCDXhoI using oligonucleotides 294-RTOK (5′-GCAAGGCCAAAATGGCGTCCGGT) and 204-MTCM2N, where the 155 bp PCR product was used as primer together with oligonucleotide 206-MTCM2S, also on template pKTCM-HCDXhoI .Tight control of MtCM gene expression invivo selection experiments for MtCM variants able to interact with MtDS, the gene for the DAHP synthase was provided on the separate, compatible plasmid called pKIMP-ACG. pKIMP-ACG is based on pKIMP-UAUC salPT7 controlled aroG from M. tuberculosis encoding MtDS, in addition to the helper functions tyrA* and pheC, and also a copy of nahR encoding the transcriptional activator of the sal promoter SpeI and subsequently treated with calf intestinal phosphatase. The product was completely digested with NarI. pKTDS-H SpeI and partially digested with SphI. Since the resulting desired 2592 bp and undesired 2515 bp fragments were not separable by agarose gel electrophoresis, they were used as a mixture for subsequent ligation (in equimolar ratio) with both the 4993 bp SpeI-NarI fragment of pKIMP-UAUC and the self-annealing oligonucleotide pair (334-trpAfw 5′-CAGCTTAGCCCGCCTAATGAGCGGGCTTTTTTTGG and 335-trpArv 5′-CGCCAAAAAAAGCCCGCTCATTAGGCGGGCTAAGCTGCATG), which forms a trpA transcriptional terminator (with NarI and SphI-compatible ends) and which was included to circumvent transcriptional coupling of the pheC and nahR genes. The ligation product was used to transform competent KA12 cells and a clone  containing the correct (2592 bp) fragment from pKTDS-H was identified by sequencing.To carry out CATATGCACCATCATCATCACCACTCTT5′- CCTTGTT) combined with either 351-CT3-N (5′-TCGACTACTAGTTATTAGTGACCGAGNNNNNNNNNACCAAGACGCAAAAGCAGGATGGCCAGAT for CT3), 428-CT-GRGrv (5′-GGTTAAAGCTTCCGCAGCCACTAGTTATTAGTGACCGAGTCGNNNNNNNNNAAGACGCAAAAGCAGGATGGCCAGAT for GRG), 350-CT4-N (5′-TCGACTACTAGTTATTANNNNNNCAGNNNGCCNNNACCAAGACGCAAAAGCAGGATGGCCAGAT for CT4), 349-CT7-N (5′-TCGACTACTAGTTATTANNNNNNNNNNNNNNNNNNNNNAAGACGCAAAAGCAGGATGGCCAGAT for CT7), 367-CT-LGHrv (5′-TCGACTACTAGTTATTANNNNNNNNNTCGACCACGACCAAGACGCAAAAGCAGGATGGCCAGAT for LGH), 365-CT-Lrv (5′-TCGACTACTAGTTATTAGTGACCNNNTCGACCACGACCAAGACGCAAAAGCAGGATGGCCAGAT for CT-L), 477-CT-GRGRrv (5′-GGTTAAAGCTTCCGCAGCCACTAGTTATTAGTGACCGAGNNNNNNNNNNNNAAGACGCAAAAGCAGGATGGCCAGAT for GRGR), or 424-CT-RLGHrv (5′-GGTTAAAGCTTCCGCAGCCACTAGTTATTANNNNNNNNNNNNACCACGACCAAGACGCAAAAGCAGGATGGCCAGAT for RLGH). The 315 bp  or 328 bp  fragments were restriction digested with NdeI and SpeI and the resulting 296 bp library fragment  was ligated in equimolar concentration  to the 2765 bp fragment of the correspondingly cut pKTCTET-0 acceptor vector. The ligated library plasmids, denoted as “pKT-CM”, were desalted and transformed into electrocompetent KA12/pKIMP-ACG or KA12/pKIMP-UAUC cells.The eight partially randomized gene libraries CT3, CT4, CT7, GRG, GRGR, LGH, CT-L, and RLGH were constructed by PCR amplification using pKSS-TM4 as a template and the sense primer 352-CTLib-S  d-(+)-glucose, 1 mM MgSO4, 0.1 mM CaCl2, 5 µg/mL thiamine-HCl, 5 µg/mL 4-hydroxybenzoic acid, 5 µg/mL 4-aminobenzoic acid, 1.6 µg/mL 2,3-dihydroxybenzoic acid, 20 µg/mL l-Trp, 100 µg/mL sodium ampicillin, 20 µg/mL chloramphenicol, and 1.5% agar) or M9c +F +Tet500 ng/mL  for selection. For determination of the library size, cells were also plated onto supplemented minimal medium M9c +FY plates, which in addition to the M9c medium ingredients also contain 20 µg/mL of each l-Tyr and l-Phe. Analysis of plates occurred after two (M9c +FY) or three days (M9c or M9c +F +Tet500 ng/mL) incubation at 30°C. Single colonies grown on selective plates were purified on LB +Amp +Cam plates . Isolated pKT-CM plasmid DNA was sequenced using oligonucleotide 131-TERM (5′-CCCTCAAGACCCGTTTAGA), except for KA12/pKIMP-UAUC libraries, where plasmid isolation and sequencing using oligonucleotide T7 (5′- TAATACGACTCACTATAGG) was performed by Microsynth. The complementation ability of a number of pKT-CM plasmids selected in the presence of MtDS was confirmed by retransformation into CaCl2-competent KA12/pKIMP-ACG cells and reassessing their growth phenotypes at 30°C on M9c, M9c +Tet80 ng/mL, M9c +F (20 µg/mL l-Phe) and M9c +FY minimal plates. pKTNTET in KA12/pKIMP-ACG was used as positive control; pKTNTET in KA12/pKIMP-UAUC and pKTCTET-0 in KA12/pKIMP-ACG served as partially and fully negative controls, respectively.The transformed cell suspension (with either KA12/pKIMP-ACG or KA12/pKIMP-UAUC transformants) was washed three times with 1×M9 salts  was used as a new and convenient plasmid-based T7 RNA polymerase gene expression system. It contains a PE. coli strains, which do not carry an endogenous Tet resistance, gene expression from Ptet was shown to be homogeneous in each cell, and it responded over 2 to 3 orders of magnitude with little cooperativity (in the case of the moderate-copy number pT7POLTS) to increasing Tet concentrations up to 100 ng/mL, before the antibiotic became toxic E. coli strains, such as strain KA12 used in this work harboring the tetR-tetA regulatory system from Tn10, the dose-response profile is fully linear and shifted to higher Tet concentrations up to 5 µg/mL + gradient across the membrane.In tetR repressor gene responsible for tight control of T7 RNA polymerase transcription. The assembly of pT7POLTS  is described in detail elsewhere Additionally, pT7POLTS carries a p15A origin of replication, a chloramphenicol resistance gene, and the 6-tagged MtCM variants  from KA13/pKTDS-HN as described earlier MtDS protein needed for kinetic assays and native gels was produced in the His-tagged format  using BSA as a standard or a calibrated Bradford assay with BSA values corrected for MtCM and MtDS-specific absorption Protein structural integrity was assessed by SDS PAGE using the PhastSystem  and by electrospray ionization mass spectrometry (ESI-MS) as detailed previously v0) of chorismate disappearance at 310 nm (ε310 = 370 M−1 cm−1). The substrate (–)-chorismate was prepared from KA12 as previously described v0 = kcat·[CM]·[S]/(Km+[S]) by using KaleidaGraph (Synergy Software) yielding kcat and Km values. The catalytic rate constant kcat was calculated per protomer of the dimeric CM.Individual Michaelis-Menten steady-state kinetic parameters were obtained for free MtCM using a continuous assay on a PerkinElmer Lambda 20 spectrophotometer by monitoring initial rates  to the values of the homogenous dataset of typically less than 15%. For optimal comparability within the MtCM dataset, we chose to list in kcat/Km values of the homogenous set only, and add a 15% standard deviation to give a conservative estimate of the variation.The kcat/Km estimates for CM activity of the MtCM variants in the presence of an excess of MtDS to ensure complex formation were performed as described previously v0 (MtDS+MtCM)) of a mixture of 30 nM MtCM variant and 300 nM MtDS were obtained by determining the background-corrected decrease of the chorismate absorbance at 274 nm (ε274 = 2630 M−1 cm−1). All CM assays with MtDS were carried out in 50 mM BTP methylamino]propane), pH 7.5, 0.5 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride), 0.2 mM phosphoenolpyruvate, and 0.1 mM MnCl2, at a chorismate concentration (range 20–30 µM) below the Km of the MtCM-MtDS complex at 30°C. Apparent activation factors were estimated from the ratio of v0 (MtDS+MtCM), normalized by MtCM-variant and the spectrometrically determined actual chorismate concentrations in the cuvette, over kcat/Km for the free MtCM variant. Remaining activity of MtCM-MtDS complexes after addition of the feedback inhibitors were determined by dividing v0 (MtDS+MtCM) measured in the presence of 100 µM l-Phe and 100 µM l-Tyr by v0 (MtDS+MtCM) obtained in the absence of these compounds. The values of n-1) for the ratios of the individual parameters calculated by error propagation of the experimental σn-1 values.S1 FigNucleotide sequence of plasmid pKTNTET.(DOCX)Click here for additional data file.S2 FigNucleotide sequence of plasmid pT7POLTS.(DOCX)Click here for additional data file.S1 TableCompilation of amino acids observed in MtCM variants before and after selection experiments (tabular version of ).(PDF)Click here for additional data file.


Total shoulder arthroplasty (TSA) can improve function in osteoarthritic shoulders, but the ability to perform activities of daily living (ADLs) can still remain impaired. Routinely, shoulder surgeons measure range of motion (ROM) using a goniometer. Objective data are limited, however, concerning functional three-dimensional changes in ROM in ADLs after TSA in patients with degenerative glenohumeral osteoarthritis.This study included ten consecutive patients, who received TSA for primary glenohumeral osteoarthritis. The patients were examined the day before, 6 months, and 3 years after shoulder replacement as well. We compared them with a control group (n = 10) without any shoulder pathology and measured shoulder movement by 3D motion analysis using a novel 3 D model. The measurement included static maximum values, the ability to perform and the ROM of the ADLs “combing the hair”, “washing the opposite armpit”, “tying an apron”, and “taking a book from a shelf”.Six months after surgery, almost all TSA patients were able to perform the four ADLs (3 out of 40 tasks could not be performed by the 10 patients); 3 years postoperatively all patients were able to carry out all ADLs (40 out of 40 tasks possible). In performing the ADLs, comparison of the pre- with the 6-month and 3-year postoperative status of the TSA group showed that the subjects did not fully use the available maximum flexion/extension ROM in performing the four ADLs. The ROM used for flexion/extension did not change significantly (preoperatively 135°-0° -34° vs. 3 years postoperatively 131° -0° -53°). For abduction/adduction, ROM improved significantly from 33°-0° -27° preoperatively to 76° -0° -35° postoperatively. Compared to the controls (118°) the TSA group used less ROM for abduction to perform the four ADLs 3 years postoperatively.TSA improves the ability to perform ADL and the individual ROM in ADLs in patients with degenerative glenohumeral osteoarthritis over the course of 3 years. However, TSA patients do not use their maximum available abduction ROM in performing ADLs. This is not related to limitations in active ROM, but rather may be caused by pathologic motion patterns, impaired proprioception or both. Since the Neer prosthesis was developed in the 1950s, shoulder arthroplasty has advanced considerably. Today, total shoulder arthroplasty (TSA) can significantly improve function in osteoarthritic shoulders, but a pTen consecutive patients  with a mean age of 65.0 years [SD ± 4.7] and an intact rotator cuff who received TSA for primary glenohumeral osteoarthritis were included in this study. The patients were examined the day before, 6 months and 3 years after shoulder replacement as well. The results of the 6 month follow-up were published in 2010 as ‘pilot study’, there wIn all shoulders of the TSA group, a deltopectoral approach was used as described by Neer et al.. In no pThe control group included 10 subjects  who had no shoulder conditions at the time of the examination upon study entry. No surgery was performed on the controls. Matched controls had a mean age of 64 years [SD 7.3]. All controls were right-hand dominant.All tests for this study were conducted by a single examiner. In accordance with the World Medical Association Declaration, the study protocol was approved by the ethics committee of the Heidelberg medical school S-305/2007), and informed consent was obtained from all patients and controls. The present study was adhered to the STROBE guidelines. The patients were examined the day before shoulder arthroplasty, 6 months, and 3 years after surgery. The reference data set of the control group was collected once during the first follow-up time of the intervention group. A 12-camera motion analysis system  operating at 120 Hz was used to observe the motion of the patient. The spatial resolution of the system was about 1 mm. We used the HUX model as described previously by Rettig et al., and inf-13. HUX U test.For flexion/extension and abduction/adduction the corresponding angles between the body’s long axis and the humerus were accounted for . The body’s long axis is fixed to the thorax-TF; hence, compensatory movement of the thorax can be monitored and distinguished from shoulder movement. To determine the maximum values, the maximum ROM at flexion/extension, abduction/adduction, and also internal and external rotation was dynamically assessed. Angles of flexion/extension and abduction/adduction were expressed as projection angles relative to the proximal anatomical coordinate system. The maximum rotation, defined by the globe convention, was meaSix months after surgery, almost all TSA patients were able to perform the four ADLs (3 out of 40 tasks could not be performed by the 10 patients); 3 years postoperatively all patients were able to carry out all ADLs (40 out of 40 tasks possible) (Table In performing the ADLs, comparison of the pre- with the 3-year postoperative status of the TSA group showed that the subjects did not fully use the available maximum flexion, abduction and adduction ROM in performing the four ADLs Figure . In exteFigure The purpose of this study was to examine whether TSA is able to restore normal ROM in ADLs in patients with degenerative osteoarthritis of the glenohumeral joint over the course of 3 years. We analyzed shoulder motion pre- and postoperatively by three-dimensional video motion analysis using the HUX model as described previously and showMotion analysis of the upper extremity is challenging because of the great glenohumeral ROM. As the gold standard, maximum ROM is measured with a goniometer which, however, gives inaccuracies of about 5° to 10° degrees and fails to distinguish glenohumeral joint motion from trunk, spine, or scapulothoracical motion,18-27. PThe results show that TSA improves the ROM in ADLs but when performing ADLs, TSA patients do not use the maximum available ROM in flexion, abduction and adduction. How can that be explained?In flexion and adduction the controls also didn’t use more ROM in performing the four ADLs. Therefore we conclud, that it is not necessary to use more flexion and adduction ROM to perfrom the four ADLs. In abduction, however, controls needed 118° of their maximum available 113° to perform the ADLs, while the patients only use 76° of their maximum available 101°. Maybe they used compensatory motion patterns? In the literature, a study of Magermans et al. measuredPostoperatively, TSA usually allows each patient to perform all required ADLs painlessly. Considering the maximum ROM in the TSA group, TSA patients were not using their maximum available abduction ROM to perform the ADLs 3 years postoperatively. This is not related to limitations in active abduction ROM, but rather may be caused by impaired proprioception. As the study group showed in a previous investigation, proprioTSA improves the ROM in performing ADLs in patients with degenerative glenohumeral osteoarthritis. TSA patients do not use their maximum available abduction ROM in performing ADLs. This is not related to limitations in active ROM, but rather may be caused by pathologic motion patterns, impaired proprioception or both.TSA: Total shoulder arthroplasty; ADL: Activity of daily living; ROM: Range of motion; HUX: Heidelberg Upper Extremity Model; Cmb: Combing the hair; Wsh: Washing the opposite armpit; Aprn: Tying an apron; Shlf: Taking a book from a shelf; SD: Standard deviation.The authors declare that they have no competing interests. All authors, their immediate family, and any research foundation with which they are affiliated did not receive any financial payments or other benefits from any commercial entity related to the subject of this article.MM made the concept and design, and performed the acquisition of data, analysis and interpretation of data and wrote the manuscript. MN participated in data acquisition, interpretation of data, statistics and helped prepare the manuscript. TD made substantial contributions to data acquisition and interpretation from the institutional motion analysis laboratory. FZ did the surgery, participated in the interpretation of data and OR was the technical developer of the Heidelberg Upper Extremity model and participated in the interpretation of data. MK participated in data acquisition from the institutional motion analysis laboratory and statistics. SW was involved in the development of the motion analysis model, data acquisition, data interpretation and manuscript approval. PK did the design of the study, data acquisition, interpretation of data, manuscript preparation and gave final approval of the version to be published. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/15/244/prepub