Clostridium (Ruminiclostridium) thermocellum secretes a multi-enzyme system called the cellulosome to solubilize plant cell wall polysaccharides. During the saccharolytic process, the enzymatic composition of the cellulosome is modulated according to the type of polysaccharide(s) present in the environment. C. thermocellum has a set of eight alternative RNA polymerase sigma (σ) factors that are activated in response to extracellular polysaccharides and share sequence similarity to the Bacillus subtilis σI factor. The aim of the present work was to demonstrate whether individual C. thermocellum σI-like factors regulate specific cellulosomal genes, focusing on C. thermocellum σI6 and σI3 factors. To search for putative σI6- and σI3-dependent promoters, bioinformatic analysis of the upstream regions of the cellulosomal genes was performed. Because of the limited genetic tools available for C. thermocellum, the functionality of the predicted σI6- and σI3-dependent promoters was studied in B. subtilis as a heterologous host. This system enabled observation of the activation of 10 predicted σI6-dependent promoters associated with the C. thermocellum genes: sigI6 , xyn11B (Clo1313_0522), xyn10D (Clo1313_0177), xyn10Z (Clo1313_2635), xyn10Y (Clo1313_1305), cel9V (Clo1313_0349), cseP (Clo1313_2188), sigI1 (Clo1313_2174), cipA (Clo1313_0627), and rsgI5 (Clo1313_0985). Additionally, we observed the activation of 4 predicted σI3-dependent promoters associated with the C. thermocellum genes: sigI3 , pl11 (Clo1313_1983), ce12 (Clo1313_0693) and cipA. Our results suggest possible regulons of σI6 and σI3 in C. thermocellum, as well as the σI6 and σI3 promoter consensus sequences. The proposed -35 and -10 promoter consensus elements of σI6 are CNNAAA and CGAA, respectively. Additionally, a less conserved CGA sequence next to the C in the -35 element and a highly conserved AT sequence three bases downstream of the -10 element were also identified as important nucleotides for promoter recognition. Regarding σI3, the proposed -35 and -10 promoter consensus elements are CCCYYAAA and CGWA, respectively. The present study provides new clues for understanding these recently discovered alternative σI factors.The Gram-positive, anaerobic, cellulolytic, thermophile  Clostridium (Ruminiclostridium) thermocellum is a Gram-positive, anaerobic, cellulolytic thermophile that produces one of the most efficient enzymatic systems to digest cellulose [C. thermocellum have been the subject of study for many years [C. thermocellum secretes a multi-enzyme complex termed the cellulosome that is anchored to the cell surface [ellulose . The celny years , and theny years . To solu surface ,5. Altho surface ,6,7.C. thermocellum cellulosome consists of a nonhydrolytic scaffoldin subunit CipA that integrates various catalytic subunits into the complex [C. thermocellum strain, the scaffoldin can attach 8 or 9 catalytic subunits; e.g., the CipA scaffoldin of strain DSM 1313 attaches 8 catalytic subunits, whereas that of ATCC 27405 attaches 9 catalytic subunits [C. thermocellum can express over 80 different cellulosomal components encoded in its genome, which include an arsenal of different saccharolytic enzymes, such as, cellulases, hemicellulases, pectin-degrading enzymes and a chitinase [C. thermocellum to unwrap its preferred substrate, cellulose, that is covered with different types of polysaccharides in the plant cell wall [C. thermocellum should possess biomass-sensing mechanisms that allow the cells to detect which polysaccharide(s) is(are) present in the environment and regulate the relevant genes accordingly the enzymatic requirements. At present, however, the regulation of cellulosomal genes is poorly understood.The  complex ,9. Depensubunits . Additiosubunits . C. therhitinase ,13. Thisell wall –16. HencC. thermocellum, our research group discovered a collection of eight alternative σ factors and their cognate membrane-associated anti-σ factors that may play a role in regulating genes encoding cellulosomal enzymes and other proteins [C. thermocellum genome, these alternative σ factor genes are positioned adjacent to their anti-σ factor genes in an operon-like organization [C. thermocellum σI1 to σI8) are related to the B. subtilis σI [I1 to σI6) was shown to be influenced by the presence of polysaccharides  in the growth medium [C. thermocellum sigI3-rsgI3, sigI4-rsgI4 and sigI7-rsgI7 operons are up-regulated when the bacterium was grown in dilute acid-pretreated yellow poplar. Additionally, in vitro experiments showed that σI1 directed the transcription from sigI1 promoter and from the promoter of the gene cel48S [During the course of our efforts to gain knowledge about the biomass-sensing mechanisms in proteins . In the nization . This setilis σI ,18, and h medium . Furtherh medium  showed te cel48S  that ence cel48S ,20.C. thermocellum anti-σI factors of σI1 to σI6 (RsgI1 to RsgI6) embody three domains: (I) a C-terminal carbohydrate-binding module (CBM) localized on the outer cell surface, (II) an internal transmembrane/wall-spanning segment, and (III) an N-terminal cytoplasmic portion (RsgI-N) which would bind the cognate σI factor [C. thermocellum RsgI proteins resemble B. subtilis RsgI, a negative regulator of its cognate σI factor [I factors was demonstrated in vitro [The I factor ,18. The I factor ,18. Morein vitro .The C-terminal domains of the RsgIs showed binding capacities to different polysaccharides, including cellulose , xylan (RsgI6), and pectin (RsgI3) ,21,22. AC. thermocellum alternative σI factors resemble to some extent the ECF (extracytoplasmic function) σ factors [I-like factors and ECF σ factors is related to their "architectures" . While the ECF σ factors are formed with only two of the four domains of the σ70 family of proteins (σ2 and σ4) [I-like factors have only one predictable functional domain associated with the amino-terminal sequence, σ2, and the sigma domain σ4 is absent [4, the σI factors contain a novel 100-residue conserved C-terminal domain termed σI-C [I promoters.The multiple  factors –26, sinc factors ,25. We as absent . In lieumed σI-C , that miC. thermocellum σI-like factors is a recent report of Sand and co-workers [xyn10Z (or Clo1313_2635 according to the DSM 1313 genome annotation), xyn11B (Clo1313_0522) and xyn10D (Clo1313_0177) were under the control of σI6. Previously, Nataf and co-workers [celS (cel48S) was likely under the control of σI1. Therefore, the present work was devoted to demonstrating whether individual C. thermocellum σI factors regulate specific cellulosomal target genes. Taking advantage of the fact that the transcription start sites of C. thermocellum sigI6, xyn10Z and xyn11B were mapped previously in our research group [I-dependent promoters in the genome of C. thermocellum DSM 1313. This analysis allowed us to identify 40 possible σI-dependent promoters upstream of the sigI-like genes and certain cellulosomal genes of C. thermocellum. To corroborate the functionality of the 40 predicted promoters, we fused their DNA sequences to a promoterless lacZ reporter gene. To overcome the lack of genetic tools in C. thermocellum, we used a B. subtilis Δ(sigI-rsgI) strain as a heterologous host and studied the activation of the 40 predicted promoters by C. thermocellum σI6 and σI3. This strategy allowed us to show that C. thermocellum σI6 could recognize the predicted promoters associated with sigI6, sigI1, rsgI5, xyn11B, xyn10D, xyn10Z, xyn10Y, cel9V, cseP and the major scaffoldin cipA. Additionally, C. thermocellum σI3 could recognize the predicted promoters detected upstream of sigI3, pl11 (Clo1313_1983 encodes a family 11 polysaccharide lyase (PL11) containing a CBM35 and a dockerin), ce12 (Clo1313_0693 encodes for a protein that contains two family 12 carbohydrate esterase (CE12), a CBM35 and a dockerin) and cipA. The combination of these methodologies revealed a putative C. thermocellum σI6 and σI3 promoter consensus. Our results show that C. thermocellum σI6 and σI3 factors expressed in B. subtilis can recognize its potential promoters, supporting our hypothesis that the multiple C. thermocellum σI–like factors might regulate cellulosomal genes.Until now, the knowledge we have regarding the regulation of cellulosomal genes by -workers  which sh-workers  showed tch group ,27, we pC. thermocellum strain DSM 1313 (LQ8) was obtained from the DSMZ . The B. subtilis strains constructed in this work are isogenic derivatives of the B. subtilis strain PY79 (laboratory stock) [B. subtilis PY79 that were constructed in this work is shown in B. subtilis BKE13460 was obtained from the BGSC . Escherichia coli DH5α  was used for plasmid propagation during plasmid construction.y stock) . AdditioE. coli and B. subtilis were grown routinely at 37°C in liquid (at 250 rpm) or on solid LB-agar Broth . During β-galactosidase activity assays, B. subtilis was grown in Spizizen´s minimal medium (SMM) employing 5 g/L fructose as carbon source and supplemented with trace elements. The SMM contained (per liter) 2 g (NH4)2SO4, 14 g K2HPO4, 6 g KH2PO4, 1 g Na3Citrate·2H2O, and 0.2 g MgSO4·7H2O. The trace elements used were (per liter) 125 mg MgCl2·6H2O, 5.5 mg CaCl2, 13.5 mg FeCl2·6H2O, 1 mg MnCl2·4H2O, 1.7 mg ZnCl2, 0.43 mg CuCl2·2H2O, 0.6 mg CoCl2·6H2O, and 0.6 mg Na2MoO4·2H2O. When appropriate, antibiotics were included at the following final concentrations: 100 μg/mL ampicillin (Amp), 50 μg/mL kanamycin (Kan), 100 μg/mL spectinomycin (Spt), 5 μg/mL chloramphenicol (Cam) or 3 μg/mL erythromycin (Erm). The induction of genes under the PxylA promoter was carried out with D-xylose . All chemicals were purchased from Sigma-Aldrich .C. thermocellum DNA sequences were PCR-amplified using C. thermocellum DSM 1313 genomic DNA as template. The upstream and downstream regions of the B. subtilis sigI-rsgI operon were PCR-amplified using B. subtilis PY79 genomic DNA as template. The lox71-erm-lox66 cassette was PCR-amplified using B. subtilis BKE13460 genomic DNA as template. Amplification of DNA for cloning was performed using TaKaRa Ex Taq . Colony PCR was performed using Hy-Taq Ready Mix . PCR primers were purchased from hy·labs (Hy Laboratories Ltd). Restriction enzymes, alkaline phosphatase, and ligase were purchased from Fermentas . PCR and agarose-gel products were isolated and purified using the hy·labs Gel/PCR Extraction Kit (Hy Laboratories Ltd). Purification of plasmids was carried out using the Presto™ Mini Plasmid Kit . All clones were verified by PCR and sequencing in the Instrumentation and Service Center of the Life Sciences Faculty at Tel Aviv University.The oligonucleotide primers used in the present study are shown in B. subtilis sigI-rsgI operon including its promoter using resistance to Erm as a selective marker. The upstream (464 bp) and downstream (505 bp) regions of the B. subtilis sigI-rsgI operon were PCR-amplified using primer pairs P1-P2 and P3-P4, respectively. The lox71-erm-lox66 cassette was amplified using primer pair P5-P6. Subsequently, the three PCR products were cloned simultaneously with the In-Fusion HD Cloning Kit into a linearized pUC19 vector (generated by PCR and provided with the kit) following the kit protocol, obtaining the pLOXErysigIrsgIBs plasmid  glycerol. The strains constructed in the present work are listed in e method . ChromosB. subtilis PY79 devoid of its sigI-rsgI operon, B. subtilis PY79 was first transformed with the pLOXErysigIrsgIBs plasmid, and the cells were selected with Erm, obtaining the B. subtilis CO01 strain. Subsequently, B. subtilis CO01 was transformed with the pDR244 plasmid (obtained from the BGSC) that encodes the sequence of the Cre recombinase and has a thermosensitive origin of replication. The cells were plated on LB-agar containing Spt and were incubated at 30°C. Several individual colonies were then streaked on a plain LB-agar plate and incubated overnight at 42°C to cure pDR244. The resulting colonies were screening for plasmid curing (Spt sensitivity) and the loss of the lox71-erm-lox66 cassette (Erm sensitivity). Finally, a single colony was streaked on plain LB-agar plate and grown at 37°C. The loss of the lox71-erm-lox66 cassette was confirmed by PCR with the primer pair P118-P119 (B. subtilis CO02 strain (To construct a 118-P119  thus obt2 strain .600 between 0.1–0.2 and grown at 37°C (250 rpm). When the cells reached mid-log growth phase (approx. 0.4–0.5 OD600), the culture was separated into two tubes, and one tube was supplemented with xylose  whereas the other was used as a blank. Then, the cells were allowed to grow for another hour at 37°C (250 rpm). Finally, the cells were recovered by centrifuging at 16,000 g for 5 min, washed twice with Z-buffer  and recovered in 0.5 mL of Z-buffer.To measure the β-galactosidase activity, strain samples were taken from the -80°C glycerol stock and inoculated in 5 mL of SMM with Cam. Subsequently, the cells were grown overnight at 37°C with shaking (250 rpm). The next day, the cells were inoculated in 2.5 mL of SMM to an OD2CO3. To calculate the β-galactosidase activity, a standard curve with 4-MU was prepared. One unit of enzyme activity was defined as the amount of β-galactosidase that releases 1 μmol of 4-MU per minute. All the β-galactosidase activities were normalized with cell density (OD600).Enzymatic activity was measured with the fluorogenic substrate (4 mg/mL) 4-methylumbelliferyl β-D-galactopyranoside  in a microplate reader . The cells, recovered in Z-buffer (150 μL), were placed in a 96-well plate and 2 mL of MUG (4 mg/mL) were added to initiate the enzymatic reaction. The release of the fluorescent compound 4-methylumbelliferone (4-MU) was measured (using the excitation filter 360/40 and the emission filter 460/40) every 10 min at 30°C with medium agitation for one hour. The reaction was stopped by adding 100 μL of 1M NaB. subtilis 168 and C. thermocellum DSM 1313 sigI genes and their promoter sequences  were used as BLAST [http://www.ncbi.nlm.nih.gov/). In order to prevent a possible loss of promoter candidates during BLAST mining, we used both discontiguous megablast ("more dissimilar sequences") as well as blastn ("somewhat similar sequences") as implemented at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Pairwise and multiple sequence alignments were performed with the CLUSTALW program [http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html), or the ClustalW2 at the EMBL-EBI . WebLogos [http://weblogo.berkeley.edu/).Primary DNA sequence analyses and DNA motif searches were performed using the Clone Manager 9 Professional Edition software . The as BLAST  queries  program  using eiWebLogos  were genC. thermocellum sigI-like gene promoters, we performed multiple sequence alignments of the experimentally detected σI-dependent promoter sequences. The initial analysis was performed using the sigI promoter sequences experimentally identified in B. subtilis, Bacillus licheniformis ATCC 14580, Bacillus thuringiensis serovar israelensis ATCC 35646 and Bacillus sp. strain NRRL B-14911 [I-dependent genes bcrC and mreBH, which are involved in cell envelope integrity and homeostasis during heat stress in B. subtilis [sigI-like genes in various species of the order Bacillales. The alignment is shown in Bacillales sigI-rsgI operons can be observed. These basic putative promoter motifs can also be observed in Bacillales σI-dependent promoters shown in Bacillales σI promoter consensus sequence is ACCCCC for the -35 element and CGAA for the -10 element  has a very high similarity to that of C. thermocellum (96.2% similar based on 16S rDNA) [C. thermocellum sigI2, sigI3, sigI4, sigI7, sigI8, and those of C. straminisolvens sigI1, sigI2, sigI3, sigI4, sigI6, sigI7 and sigI8 were predicted. As shown in The deduced consensus sequences of the lgorithm . The ana-workers . Additio6S rDNA) ,38. The I-dependent promoter sequence for both C. thermocellum and C. straminisolvens sigI5 genes, owing to the low similarity of their upstream intergenic regions. Interestingly, whereas most of the sequences of the rsgI genes overlap with sequences of their cognate sigI genes, sigI5 and rsgI5 genes are separated by an intergenic region of 97 nucleotides, which contains a predicted σI-dependent promoter . Our analysis shows that the cellulosome-producing bacteria which use multiple sigI-like genes probably maintain different levels of similarity in promoter sequences to fine-tune the regulation of individual sigI-like genes, as well as cellulosomal target genes. With the predicted promoter sequences of the multiple sigI-like genes of C. thermocellum, C. straminisolvens, C. clariflavum, A. cellulolyticus and P. cellulosolvens we suggest that AAA of the "extended -35" and CGWA of the -10 elements represent the general motifs for σI-dependent promoters of cellulosome-producing bacteria.Comparison between the predicted promoters of the larities . For exa similar . This obI factors autoregulate their own expression, and hence the genes that are under their control should have similar promoter sequences, we performed a search of putative promoter sequences of the cellulosomal genes of C. thermocellum. The search was performed by exploiting the conserved sequences in the "extended -35" (AAA) and -10 (CGWA) elements of the general motifs in the sigI-like gene promoters of cellulosome-producing bacteria. The analysis was facilitated with the recent identification of the transcriptional start sites of C. thermocellum xyn10Z and xyn11B by Sand and co-workers [Based on the assumption that σ-workers . In TablC. thermocellum, we used B. subtilis as a heterologous host to test the ability of C. thermocellum σI6 and σI3 factors to recognize the C. thermocellum predicted promoters. A similar strategy has been successfully used by several research groups to analyze regulatory proteins from different Firmicutes species as Clostridium difficile, Enterococcus faecalis and Oceanobacillus iheyensis [B. subtilis and C. thermocellum , sigI1, rsgI5, xyn10D and cel9V . The LacZ activities of these 10 predicted σI6-dependent promoters are shown in the To avoid the interference of the native I operon . The pre-workers  and that-workers . The 7 pke genes  and the romoters  were fuster gene  and inteyE locus . Subsequactivity . Our anaC. thermocellum σI6 in B. subtilis allowed the recognition of its own promoter. This result is in agreement with the recent report of Sand and co-workers [C. thermocellum σI6 is autoregulated. Interestingly, five of the 10 activated promoters correspond to genes encoding the cellulosomal associated GH9, GH10 and GH11 glycoside hydrolases . Furthermore, the highest β-galactosidase (LacZ) activities were obtained with the predicted promoters of the two xylanase genes xyn10Z and xyn11B  and C. straminisolvens, showed identical predicted σI6 promoter upstream of a single xyn11A gene of strains ATCC 27405 and JW20  by the C. thermocellum σI6 factor suggests a more complex regulon for this kind of alternative σ factors. As already mentioned, sigI1 and rsgI5 are also proposed to be involved in the regulation of cellulosomal genes [sigI1 and rsgI5 promoters by the C. thermocellum σI6 factor suggests the possibility of crosstalk between different C. thermocellum σI factors and an overlap of their respective regulons.As expected, the heterologous expression of the -workers , which sd xyn11B . Moreoveand JW20 . Bioinfoand JW20 . This sual genes . Hence, I6 recognition, we performed an alignment using the experimentally validated promoter sequences recognized by C. thermocellum σI6. To improve the analysis, the 10 experimentally validated σI6-dependent promoter sequences of C. thermocellum DSM 1313 were compared with orthologous promoter sequences of C. straminisolvens JCM 21531. The result is shown in I6-dependent promoters share two highly conserved sequences. The suggested C. thermocellum σI6-promoter consensus motifs are CNNAAA for the -35 element and CGAA for the -10 element (where N represents any base). The spacing between the suggested -35 and -10 elements is between 13 and 14 nucleotides  at the 5' of the -35 element reduced dramatically the LacZ activity. Interestingly, the mutation from A to T in the CGA sequence (xyn10Z mut3) increased the LacZ activity by 33%. This result shows that although the nucleotides C and G in the CGA sequence of the -35 element is less conserved, they play an important role in recognition by C. thermocellum σI6.In the -35 element, next to the highly conserved C, a less conserved CGA sequence can be observed in the WebLogo generated with the 10 σisolvens . Interesd xyn11B . In ordexyn10Z mut4) and in the AAA sequence  of the -35 element abolished or reduced dramatically the LacZ activity. The least "sensitive" nucleotide was the middle A (xyn10Z mut6) in the AAA triplet of the -35 element. Regarding the -10 element, the most conserved bases were also highly sensitive to mutations. Individual changes in the highly conserved CGAA sequence showed that the first 3 nucleotides CGA  are more sensitive to changes than the last A at the 3' of the CGAA sequence (xyn10Z mut14). Finally, changes of the highly conserved AT sequence at the 3' of the -10 element showed a dramatic reduction of LacZ activity when the sequence was changed to TA (xyn10Z mut12). However, when the AT sequence was changed to CC, LacZ activity was not detected (xyn10Z mut13). All these results confirm the importance of the highly conserved sequences of the -35 and -10 elements, as well as the less conserved nucleotides C and G of the CGA sequence at the 5' of the -35 element.As expected, individual mutations in the highly conserved nucleotide C . Furthermore, during the quantification of σI3-dependent promoter activities, the promoters of pl11 and ce12 showed the highest activities  suggesting a possible overlap between the regulons of C. thermocellum σI3 and σI6 . The spacing between the -35 and -10 elements is between 13 and 14 nucleotides, resembling the organization of the σI6 promoter . This observation is in accordance with previous experiments performed by Nataf and co-workers [C. thermocellum was grown on cellulose, the expression of the sigI6 gene was up-regulated 2.5-fold; and when the cells were grown on cellulose and xylan, sigI6 was up-regulated at least 10-fold. Moreover, the anti-σI6 factor, RsgI6, bears an extracytoplasmic C-terminal sensing module that belongs to the glycoside hydrolase family 10 (GH10). Interestingly, the RsgI6 GH10-family module is highly similar to Xyn10D . Bahari and co-workers [xyn10Y and xyn10D were up-regulated when C. thermocellum was grown in dilute acid-pretreated yellow poplar, containing 65% cellulose, 4% xylan and 31% lignin.Four of the 10 promoters that were activated by -workers  which sh-workers  showed t-workers , showed C. thermocellum σI3, two of the four promoters that were activated by C. thermocellum σI3 belong to genes implicated in the solubilization of pectin (pl11/Clo1313_1983 and ce12/Clo1313_0693). Interestingly, the anti-σI3 factor RsgI3, has an extracytoplasmic sensing module that is composed of two tandem PA14 superfamily motifs that were shown to bind pectin by Kahel-Raifer and co-workers [I6 likely plays a role in the regulation of xylan-degrading enzymes, σI3 likely plays a role in the regulation of pectin-degrading enzymes.Regarding -workers . Taken tC. thermocellum sigI1 and rsgI5 genes are part of the proposed genes involved in the regulation of cellulosomal genes in response to environmental polysaccharides [I-dependent promoters for sigI1 and rsgI5 were recognized by C. thermocellum σI6  one can introduce additional, synthetic cellulosomal genes in C. thermocellum and use their products for improvement of either saccharolytic activity or, alternatively, ethanol production. Continued analysis and harnessing of the various σ and anti-σ factors in C. thermocellum will allow us to control and enhance the capacity of this ecologically prominent and industrially relevant bacterium for deconstruction of plant-derived polysaccharides en route to the production of biofuels.Our results and observations reveal several promising options to improve the performance of the industrially prominent bacterium S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.


Once a replaced prosthetic graft is infected, it is usually necessary to re-replace the thoracic aorta to achieve complete resolution of the infection. It is, however, an exceedingly invasive approach to perform such a repeat surgery on patients in a poor condition. We have managed both re-replacement of an infected prosthetic graft and conservative therapy with vacuum-assisted wound closure (VAC) without re-replacement. These two treatment modalities were retrospectively assessed.n = 14) and no-replacement group .Retrospective clinical chart review was undertaken on 21 patients with prosthetic graft infection after thoracic aortic replacement between December 1999 and December 2012. Surgical outcomes were evaluated between the two groups: re-replacement group . In terms of long-term outcome, one patient in group R and one patient in group NR died of rupture of a residual aortic aneurysm, and one patient in group NR died of renal disease during follow-up .Re-replacement of an infected prosthetic graft after a thoracic aortic operation still carries a significant risk for mortality. VAC therapy may provide an acceptable option for such a subgroup of patients with this serious condition. Infection of a prosthetic graft after thoracic aortic surgical procedure has been a significant life-threatening risk for decades . For sucn = 14) and VAC therapy .Between December 1999 and December 2012, 21 patients who were diagnosed with prosthetic graft infection after thoracic aortic replacement underwent two modalities of treatment. Cases of aorto-esophageal fistulae were excluded from the analysis because of their distinctive clinical features and unique staged treatment procedures. The diagnosis of prosthetic graft infection was made according to the overall findings such as a high fever, upregulation of inflammatory biochemical indicators, aberrant CT findings, and positive blood culture. Cases of shallow subcutaneous wound infection or osteomyelitis of the sternum without evidence of graft infection were also excluded from the analysis. The treatment strategies included re-replacement therapy of an infected prosthetic graft  should be used in each case depended upon multiple factors including a patient’s general condition. When a pseudoaneurysm was present, the VAC system installation was not adequate. An apparent intra-graft development of vegetations also precluded VAC therapy, and re-replacement of the graft was performed instead. Lateral thoracotomy infection was not a good indication of VAC therapy either. In both groups (R and NR), broad-spectrum antibiotics were initially administered and adjusted according to the culture results.In group R, the chest was opened for drainage and irrigated with saline followed by immediate re-replacement of the infected graft in six patients or the mediastinum was irrigated (or packed with povidone-iodine bond gauze in early years) once a day for 2–31 days  to reduce the amount of bacteria prior to a re-replacement surgical procedure in eight patients. As described above, an aortic homograft was the choice of the first-line substitute when available; otherwise, a rifampicin-bond prosthetic graft was used for graft replacement.® apparatus became available in Japan, and this pressure is maximal for this device. Exchange of the whole system, including the polyurethane sponges and routine irrigation, was performed once a day at least for the subsequent several days. Closure of the chest was undertaken after confirming negative bacterial culture in the wound.In group NR, the VAC system was installed as described elsewhere . In briet test. Categorical variables, expressed as percentages, were analyzed using either the Chi squared or Fisher’s exact tests. Survivals in the two groups were compared using the Kaplan–Meier method. A p value less than 0.05 was assumed to indicate statistical significance.All calculations were performed using the SPSS software . Continuous variables were expressed as mean ± standard deviation and compared using the The age was 56.4 ± 21.1 years (range 7–78 years) in group R and 70.9 ± 10.0 years (range 52–78 years) in group NR. Pre-operative patients’ demographics in each group are shown in Table Perioperative factors and surgical outcomes are summarized in Table As a substitute for an infected prosthetic graft, an aortic homograft was used for re-replacement in nine cases (64.3 %) and another prosthetic graft in the remaining five cases (35.7 %) including when it was used as a supplemental material during the Ross procedure. Technical modifications in group R included a pre-operative use of the VAC system for case No. 5 in an attempt to reduce the amount of bacteria prior to a re-replacement operation and a postoperative installation of the VAC system immediately after a re-replacement procedure in case No. 12 until the chest was eventually closed.Staphylococcus aureus (MRSA) 10 months after the re-replacement surgery. His mediastinum was then re-explored for VAC therapy. As a result, three patients died in the hospital because of systemic infection in spite of thorough replacement of the graft.There were five in-hospital deaths in group R, and one of the hospital survivors died of a ruptured dissecting descending thoracic aortic aneurysm during follow-up. The mean duration of follow-up was 52.8 ± 41.5 months. In particular, patient No. 1 died of a stroke on postoperative day (POD) 19 after re-root and arch replacement, and patient No. 5 developed low cardiac output syndrome with uncontrolled systemic infection and died on POD 8 after the re-replacement of an ascending aortic graft. Patient No. 7 died of an intraoperative rupture of the distal aortic arch during the procedure with a homograft. Patient No. 9 died of sepsis on POD 7 after re-descending aortic replacement, and patient No. 10 also died of sepsis on POD 20 after re-thoracoabdominal aortic replacement. At follow-up, the wound in case No. 8 became erosive and tested positive for methicillin-resistant In group NR, patient No. 5 died of sepsis on the 27th day after installation of the VAC system. With regard to the long-term outcome, patient No. 1 died of renal disease during the follow-up of 67 months, and patient No. 3 died of a residual-aneurysm rupture during the follow-up of 11 months. The mean follow-up period in group NR was 43.2 ± 28.5 months. Infection of the same site was recurrent only in case No. 7 of group NR. This patient had undergone a replacement of the ascending aorta for acute aortic dissection elsewhere. He developed the graft infection several months later and was treated with VAC therapy at another hospital. He developed recurrent mediastinitis and was thereafter transferred to our institution. His mediastinum was re-explored, and subsequently the VAC system was re-installed. The sternum had been almost debrided at the previous hospital; therefore, the skin was re-approximated after resolution of the mediastinitis. Unfortunately, he developed third-time mediastinitis associated with the graft infection 4 months later. We re-installed the VAC system and continued this therapy for 29 days, and his chest was closed using the rectus abdominis flap technique. After the final chest closure, there has been no sign of recurrent infection during follow-up.The median interval between a previous procedure and re-replacement or VAC installation for graft infection was 12.0 months in group R and 8.0 months in group NR. The median duration of VAC therapy was 15.0 days.The in-hospital survival rates of group R and group NR were 64.3 and 85.7 %, and 5-year survival rates according to the Kaplan–Meier analysis were 48.2 and 68.6 %, respectively.It has long been assumed that a fundamental treatment strategy should include re-replacement of the infected prosthetic graft with an in situ substitute or an extra-anatomical bypass graft –10. ConvAn adjuvant therapy was employed at the time of the chest closure in all the cases in group NR. In other words, the coverage of the prosthetic grafts and obliteration of a mediastinal dead space using an omentum or muscle flaps may be crucial for long-term prevention of recurrent infections . The sucAlthough the VAC system was proven as safe and effective, this method cannot be used in all cases. When we encounter a pseudoaneurysm or intra-graft vegetation after re-exploration, adequate surgical replacement is unavoidable. For a lateral thoracotomy incision site, installation of the VAC system for the infected prosthetic graft is difficult because of the depth of the lesion, width of the pleural space, friability of lung tissue, and the difficulty in maintaining an adequate physical position throughout postural changes. Hence, each case has to be individually considered whether it can be treated conservatively with the VAC system, or what kind of drainage or surgical procedure and to what extent the graft re-replacement should be performed. Even nowadays, adequate selection of strategy according to a patient’s individual status is necessary when little evidence is available to guide surgeons in treating these patients .There were different numbers of cases in the groups and a relatively small total sample size. In addition, the VAC system could be applied only to selected cases; consequently, the backgrounds of the patients in the two groups are different.The prognosis of a prosthetic graft infection after a thoracic aortic procedure is not yet satisfactory, but compared with surgical re-replacement, VAC therapy is an acceptable and less invasive method for resolving this challenging situation. Although selection of cases and appropriate conditions for installation of the VAC modality are necessary, it can be considered a reasonable option to achieve complete resolution of a life-threatening infection.


International Journal of Molecular Sciences have established the Best Paper Award to recognize the most outstanding articles published in the areas of molecular biology, molecular physics and chemistry that have been published in the International Journal of Molecular Sciences. The prizes have been awarded annually since 2012 [The Editors of the nce 2012 ,4.International Journal of Molecular Sciences Best Paper Award” for 2016. Nominations were selected by the Editorial Board from all papers published in 2015. The awards are issued to reviews and research articles separately. Following extensive review by the Editorial Board, five top-voted research articles and the three top-voted reviews have been awarded the “International Journal of Molecular Sciences Best Paper Award” for 2016. The papers are listed as follows, and in no particular order:Original Research Article Award:A Variant in the Osteoprotegerin Gene Is Associated with Coronary Atherosclerosis in Patients with Rheumatoid Arthritis: Results from a Candidate Gene StudyCecilia P. Chung, Joseph F. Solus, Annette Oeser, Chun Li, Paolo Raggi, Jeffrey R. Smith and C. Michael SteinInt. J. Mol. Sci.2015, 16(2), 3885–3894; doi:10.3390/ijms16023885http://www.mdpi.com/1422-0067/16/2/3885Available online: Awarding Committee Comments“This article addresses the relevant issue of increased atherosclerosis (and cardiovascular risk) in patients with rheumatoid arthritis. Identification of a polymorphism in gene encoding osteoprotegerin (involved in bone metabolism and associated with atherosclerosis) and, above all, its association with atherosclerosis in these patients, highlight novel mechanistic findings in this inflammatory disease.”By Dr. Marcello Iriti“A variant in OPG gene is associated with coronary atherosclerosis…This paper is a good example of the use of applied statistics to identify risk factors in a relevant patient population, and should serve as a benchmark for studies employing similar approaches.”By Dr. Joseph V. Moxonin Vivo and in Vitro Models of Rat OsteoarthritisAmeliorative Effects of PACAP against Cartilage Degeneration. Morphological, Immunohistochemical and Biochemical Evidence from Salvatore Giunta, Alessandro Castorina, Rubina Marzagalli, Marta Anna Szychlinska, Karin Pichler, Ali Mobasheri and Giuseppe MusumeciInt. J. Mol. Sci.2015, 16(3), 5922–5944; doi:10.3390/ijms16035922http://www.mdpi.com/1422-0067/16/3/5922Available online: Awarding Committee Comments:“Authors describe an anti-inflammatory and antiapoptotic chondroprotective factor in an elegant in vivo/in vitro model of osteoarthritis. This suggests a novel therapeutic approach in joint inflammation, as well as an activator of cartilage regeneration in degenerative diseases.”By Dr. Marcello Iriti“A nice study with both in vivo and in vitro data to demonstrate the association and protective effect of the neuropeptide PACAP in osteoarthritis.”By Dr. Vera Sau-Fong Chanin Vivo Osseointegration of Sandblasted/Acid Etched/Anodized Dental ImplantsCell Adhesion and Mu-Hyon Kim, Kyeongsoon Park, Kyung-Hee Choi, Soo-Hong Kim, Se Eun Kim, Chang-Mo Jeong and Jung-Bo HuhInt. J. Mol. Sci.2015, 16(5), 10324–10336; doi:10.3390/ijms160510324http://www.mdpi.com/1422-0067/16/5/10324Available online: Awarding Committee Comments:“A new type of surface modified titanium dental implant improved osseointegration in an in vitro/in vivo model, i.e., increased cell adhesion and bone-to-implant contact. This paper will certainly be of interest in the field of implant dentistry and dental sciences.”By Dr. Marcello Iriti“This paper presents findings to show the enhanced biocompatibility of application of the potential action of a new type of surface-modified titanium material for dental implant, revealing its potential translational impact.”By Dr. Vera Sau-Fong ChanDimethyl Fumarate Protects Neural Stem/Progenitor Cells and Neurons from Oxidative Damage through Nrf2-ERK1/2 MAPK PathwayQin Wang, Sergei Chuikov, Sophina Taitano, Qi Wu, Arjun Rastogi, Samuel J. Tuck, Joseph M. Corey, Steven K. Lundy and Yang Mao-DraayerInt. J. Mol. Sci.2015, 16(6), 13885–13907; doi:10.3390/ijms160613885http://www.mdpi.com/1422-0067/16/6/13885Available online: Awarding Committee Comments:“A quite sound paper about the role of dimethyl fumarate-induced Nrf2 pathway in neuronal progenitor cells maintenance and protection against oxidative stress. Such results may certainly contribute to a better targeting of Multiple Sclerosis-associated signaling pathways.”By Dr. Anthony Lemarié“A very interesting mechanistic study focusing on dimethyl fumarate effects in multiple sclerosis. This drug reduces oxidative stress in neural stem/progenitor cells, increasing the expression of neuroprotective transcription factors at both levels of RNA and protein, and upregulating anti-oxidative stress genes. This paper suggests further insights into new targets for treatment of this chronic neurological disease affecting young adults.”By Dr. Marcello Iriti“The authors showed through a well constructed set of experiments that dimethyl fumarate conferred protection to neural stem/progenitor cells from oxidative damage. The findings of this study will form the basis of new experiments that will enhance our understanding of multiple sclerosis and assist in development treatments that will be of benefit to people who suffer from this condition.”By Dr. Terrence PivaIntegrated Bioinformatics, Environmental Epidemiologic and Genomic Approaches to Identify Environmental and Molecular Links between Endometriosis and Breast CancerDeodutta Roy, Marisa Morgan, Changwon Yoo, Alok Deoraj, Sandhya Roy, Vijay Kumar Yadav, Mohannad Garoub, Hamza Assaggaf and Mayur DokeInt. J. Mol. Sci.2015, 16(10), 25285–25322; doi:10.3390/ijms161025285http://www.mdpi.com/1422-0067/16/10/25285Available online: Awarding Committee Comments:“The authors integrate, in a unique way, information from epidemiology, genomics and bioinformatics, thereby significantly advancing our understanding on the links between human disease and exposure to endocrine disrupting chemicals.”By Dr. Helmut Segner“Integrated bioinformatics, environmental epidemiologic and genomic approaches...An innovative approach allowing researchers to mine previously published data, consolidate existing knowledge, and synthesise new hypotheses.”By Dr. Joseph V. Moxon“A really comprehensive integrated bioinformatics  approach identified common molecular traits in breast cancer and endometriosis caused by exposure to endocrine disruptors, i.e., altered environmentally and estrogen-responsive genes in both conditions. A great example of gene–environment interaction in cancer development.”By Dr. Marcello Iriti“Through the use of environmental epidemiologic, genomic, and bioinformatics the authors showed that exposure to polychlorinated biphenyls (PCBs) increase an individuals risk factor of breast cancer or endometriosis, and they share some common environmental and molecular risk factors. This was a well performed study and has yielded some interesting results which will help us further understand how PCBs exert their detrimental effects on individuals, which will assist in the development of treatments which can confer protection to their actions.”By Dr. Terrence Piva“An interesting analysis using public databases and bioinformatics tools to reveal common environmental and molecular risk factors between two estrogen-sensitive conditions.”By Dr. Vera Sau-Fong ChanReview Paper Award:Starting to Gel: How Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell WallsCătălin Voiniciuc, Bo Yang, Maximilian Heinrich-Wilhelm Schmidt, Markus Günl and Björn UsadelInt. J. Mol. Sci.2015, 16(2), 3452–3473; doi:10.3390/ijms16023452http://www.mdpi.com/1422-0067/16/2/3452Available online: Awarding Committee Comments:“As Editor of this article, I previously appreciated it, a piece of cell wall research. The seed coat epidermis provides an alternative system to investigate the production of cell wall polysaccharides, particularly the synthesis and modification of pectin, thus improving our understanding of plant cell walls.”By Dr. Marcello Iriti“A structured review on a fascinating topic.”By Dr. Vera Sau-Fong ChanSequencing Overview of Ewing Sarcoma: A Journey across Genomic, Epigenomic and Transcriptomic LandscapesLaurens G. L. Sand, Karoly Szuhai and Pancras C. W. HogendoornInt. J. Mol. Sci.2015, 16(7), 16176–16215; doi:10.3390/ijms160716176http://www.mdpi.com/1422-0067/16/7/16176Available online: Awarding Committee Comments:“A really OMICS approach! By sequencing Ewing sarcoma at the genome, epigenome and transcriptome level, novel predictive markers and candidates for immuno- and targeted-therapy in young patients can be identified, as summarized in this review.”By Dr. Marcello Iriti“A very comprehensive and informative review with a nice touch on implications in targeted therapy—a good read to gain understanding in Ewing Sarcoma genomics.”By Dr. Vera Sau-Fong ChanLong Non-Coding RNAs in Endometrial CarcinomaMaria A. Smolle, Marc D. Bullock, Hui Ling, Martin Pichler and Johannes HaybaeckInt. J. Mol. Sci.2015, 16(11), 26463–26472; doi:10.3390/ijms161125962http://www.mdpi.com/1422-0067/16/11/25962Available online: Awarding Committee Comments:“A very novel topic: lncRNAs (long non-coding RNAs)! Specific and altered expression patterns of lncRNAs in endometrial carcinoma compared to normal endometrial tissues are reviewed, as well as the potential of deregulated lncRNAs as biomarkers. In fact, a number of lncRNAs are differentially expressed in normal, hyperplastic and dysplastic endometrium which can be targeted in order to achieve anticancer activity.”By Dr. Marcello Iriti“The authors reviewed the role different long non-coding RNAs (lncRNAs) play in the development of different types of endometrial carcinomas (EC). The review was well written and discussed the two major forms of EC and as such will form a useful reference for those studying the role of lncRNAs in the development of different endometrial carcinomas.”By Dr. Terrence Piva“Understanding lncRNAs in cancer biology is one of the hottest research areas—this article provides a timely review on recent development in endometrial carcinoma.”By Dr. Vera Sau-Fong ChanWe are pleased to announce the winners of the “International Journal of Molecular Sciences and their respective scientific research fields. On behalf of the Editorial Board of the International Journal of Molecular Sciences, we would like to congratulate the winning teams for their excellent work. In recognition of their accomplishments, they will receive the privilege of publishing an additional research article or review paper free of charge in open access format in the International Journal of Molecular Sciences, after the usual peer-review procedure.These eight exceptional papers represent valuable contributions to the International Journal of Molecular Sciences, and thank the Editorial Board of the International Journal of Molecular Sciences for assessing and helping with the selection of this “Best Paper Award”.We would like to take this opportunity to thank all the nominated research groups of the above exceptional papers for their contributions to the International Journal of Molecular Sciences are committed to meeting the needs of the molecular research community by providing extensive and timely reviews of all submitted manuscripts, and by providing an open access forum for the publication of your results. Please consider submitting your work to the International Journal of Molecular Sciences, and we look forward to announcing your manuscript as a winner of the International Journal of Molecular Sciences Best Paper award in the future.The Editorial Board and Editorial Staff at the


B cell epitopes corresponding to interaction zones between proteins and immune system are a mean to explore intrinsic features of proteins linked to allergenicity. The project PREDEXPITOPE was funded by the French National Research Agency to develop a bioinformatics strategy for prediction of B cell epitopes of allergens. The objective was to distinguish epitopic from non epitopic areas on the basis of frequently used criteria of surface exposition. In silico prediction was confronted with experimental results on wheat allergens.Epitope sequences and description were exported from Immune Epitope DataBase (IEDB). Epitopes were mapped to 61 3D structures, extracted from PDB site. A pipeline program computed relative surface exposure of amino acids for each PDB file. Data were loaded into R environment for statistical comparison between epitopic and non epitopic amino acids. B epitopes were searched on wheat allergens for which 3D structure could be known. Sera were obtained from patients with food allergy to wheat or baker's asthma.On the basis of the 61 antigen structures no clear-cut classification of epitopic and non-epitopic values could be statistically achieved. The amino acids within epitopes are not distributed above any threshold for both relative surface accessibility and protrusion index. The wheat serpin was a good candidate to validate our bioinformatics conclusion. A 3D structural model was constructed and areas of strong surface accessibility could be selected. These areas did not correspond to those bound by patient's IgE.Our work did not deny the requisite for B-cell epitopes to be exposed on surface. However, it indicates that the level of surface exposure has no discriminatory ability. The difficulty in establishing a system of prediction lies in the heterogeneity of data on B-cell epitopes, which varies from a few amino acids actually in contact with the antibodies to shorter or longer peptides, able to interact with these antibodies. The integration of experimental context or epidemiological aspects in epitope data bases may improve such a prediction.


Here, Fe3O4 nanoparticles and silver nanoparticles were synthesized by using starch under alkaline condition. Hydrodynamic diameter of starch and starch coated silver nanoparticles were determined under heat treatment and different pH. This data also display absorbance peak of silver nanoparticles synthesized by starch under different pH conditions . Iodometric titration confirmed that both components of starch (amylose and amylopectin) can adsorb on the surface of Fe3O4 nanoparticles.Green synthesis of nanoparticles by using starch has recently attracted considerable attention due to their biodegradability, non-toxicity, and cost effectiveness. The data presented in this article are related to the article entitled “Evaluation of antibacterial activity of Ag/Fe Specifications TableValue of the data•Data regarding the heat and alkaline treatment on starch structure will be useful for size-controlled synthesis of nanoparticles by starch.•3O4 nanoparticles will be useful to exploit the mechanism of the nanoparticle formation.Investigation of affinity of starch components (amylose and amylopectin) to adsorb on Fe•The importance of ammonia concentration in silver nanoparticles synthesized by starch. It can change pH and the number of electron to reduce silver ions.13O4 NPs, supernatant and starch with iodine are demonstrated.The data display the characterization of starch and silver nanoparticles synthesized by starch under different condition. Also, UV–vis spectra of Fe22.13, FeCl3.6H2O, FeSO4.7H2O, NH3.H2O, potassium iodide, and iodine were purchased from Sigma Aldrich.Soluble starch from potato, AgNO2.2Starch molecules were analyzed under heat treatment (at 95 °C for 20 min) with or without subsequent alkaline treatment by DLS. During the heating process, the starch granules first swell and then burst, the semi-crystalline structure is lost and the smaller amylose molecules start leaching out of the granules. 2.33O4 nanoparticles were synthesized according to the modification procedure previously reported 3·6H2O (1.49 g) and FeSO4·7H2O (0.765 g) were added to 200 ml of the starch solution , which was then heated at 60 °C under nitrogen atmosphere, then NH3·H2O (8 mol l−1) was added drop wise to reach pH 10. After 3 h, the synthesized nanoparticles were separated from the supernatant by an external magnetic field and were washed several times by deionized water. To assess which component of potato starch adsorbed on the Fe3O4 nanoparticles, the supernatant, Fe3O4 nanoparticles and starch were titrated with 0.1 N iodine solution (I2/KI).Fe3O4 and iodine solution has broad peak at 530 nm. It can be attributed to the overlap between absorbance of starch (amylose and amylopectin)–iodine and iron–iodine complexes.As shown in 2.43 (0.3 g) was added to 100 ml of the starch solution  then the pH was adjusted  by ammonia, it was heated for one hour at 80 °C. As shown in In order to synthesis of silver nanoparticles, AgNO


Morning stiffness is assessed daily in the diagnostic process of arthralgia and arthritis, but large-scale studies on the discriminative ability are absent. This study explored the diagnostic value of morning stiffness in 5,202 arthralgia and arthritis patients and the prognostic value in early rheumatoid arthritis (RA).Recognition Clinics (EARC) of Leiden (n = 807) and Groningen (n = 481) or included in the Rotterdam Early Arthritis Cohort (REACH) study (n = 353), the associations  between morning stiffness and presence of arthritis at physical examination were studied. In early arthritis patients, included in the Leiden Early Arthritis Clinic (EAC)  and Evaluation et Suivi de POlyarthrites Indifférenciées Récentes (ESPOIR) (n = 813), associations with fulfilling the 2010-RA criteria after one year were assessed. In 2010-RA patients included in the EAC  and ESPOIR (n = 677), association with the long-term outcomes of disease-modifying antirheumatic drug (DMARD)-free sustained remission and radiological progression were determined. Morning stiffness was defined as a duration ≥60 minutes; sensitivity analyses were performed for other definitions.In arthralgia patients referred to the Early Arthritis RC odds ratio (OR) 1.49 (95% CI 1.001 to 2.20), Groningen EARC OR 2.21 (1.33 to 3.69) and REACH OR 1.55 (0.97 to 2.47) but the areas under the receiver operating characteristic curve (AUCs) were low . In early arthritis, morning stiffness was associated with 2010-RA independent of other predictors , ESPOIR OR 1.68 ). Duration of ≥30 minutes provided optimal discrimination for RA in early arthritis. Morning stiffness was not associated with radiological progression or DMARD-free sustained remission.In arthralgia, morning stiffness (≥60 minutes) associated with the presence of arthritis; Leiden EAMorning stiffness in arthralgia and early arthritis is associated with arthritis and RA respectively. This supports the incorporation of morning stiffness in the diagnostic process.The online version of this article (doi:10.1186/s13075-015-0616-3) contains supplementary material, which is available to authorized users. Morning stiffness is common in patients with rheumatoid arthritis (RA); it affects the ability to function in the morning , the quaThe scientific data on the diagnostic value of this symptom are surprisingly scanty. In the literature, it has been mentioned that morning stiffness is a poor discriminator between RA and other rheumatologic disorders ,5. HowevAlthough it is known that morning stiffness is associated with the disease activity , functioAll datasets and cohorts used are described in more detail elsewhere -16.Recognition Clinics (EARC) from Leiden and Groningen (the Netherlands) or included in the Rotterdam Early Arthritis Cohort (REACH) (the Netherlands). The EARCs were initiated to reduce referral delay by GPs. It was observed that the GP delay contributed to two-thirds of the total delay between symptom onset and first visit to a rheumatologist in the Netherlands and that GPs frequently applied a ‘wait-and-see’ approach if they were unsure of the presence of arthritis. Therefore, GPs were instructed to refer to the EARC if they were undecided. The EARCs are early access clinics in which experienced rheumatologists screen patients on the presence of arthritis by physical examination; no laboratory investigations are done [RC between September 2010 and August 2013 and the Groningen EARC between October 2010 and January 2014. The EARC is primarily part of our care. Patients visiting the EARCs were not subjected to procedures that were done for scientific purposes, such as blood taking for biobanking. Therefore, in line with the Dutch law ‘Wet medisch-wetenschappelijk onderzoek met mensen’ , patients were not asked to sign an informed consent form.In short, the arthralgia patients were referred to the Early Arthritis are done ,17. The The REACH study is an inception cohort that was initiated in the Rotterdam area, the Netherlands, in 2004. Inclusion required either pain or loss of movement in ≥2 joints or >1 swollen joint and ≥2 of the following items: unable to clench a fist in the morning, pain when shaking someone’s hand, pins and needles in the fingers, difficulties wearing rings or shoes, a family history of RA, morning stiffness >1 hour, unexplained fatigue, all <1 year . The patThe early arthritis patients were included in the Leiden Early Arthritis Clinic (EAC) and the Evaluation et Suivi de POlyarthrites Indifférenciées Récentes (ESPOIR) cohort. The Leiden EAC is a population-based inception cohort that started in 1993. Inclusion required the presence of arthritis of ≥1 joint at physical examination and symptom duration <2 years . The patRCs, patients answered questionnaires on the presence and duration of morning stiffness  had no evident arthritis but were also not classified as having ‘no arthritis’ because the rheumatologists suspected these patients of RA development; these patients were excluded from analyses.In arthralgia patients, the outcome was the presence of arthritis ascertained at physical examination by experienced rheumatologists . In both EAIn early arthritis, we aimed to assess the diagnostic value of morning stiffness and here the outcome was the presence of RA after one year. RA was defined as fulfilling the 2010 ACR/EULAR criteria for RA during the first year. An advantage of the 2010-RA criteria is that morning stiffness is not included, preventing circle reasoning. Since during the first weeks the diagnoses may not yet be definitive, the classification after year one was evaluated. These first two parts evaluated the diagnostic value.Third, within 2010-RA patients, the prognostic value was assessed by studying two long-term outcomes. Structural damage was assessed using serial hands and feet radiographs that were scored according to the Sharp/van der Heijde (SHS) method with known time order and blinded to clinical data. In the EAC, radiographs were scored of patients included between 1993 and 2006. The follow-up was seven years in the EAC and three years in ESPOIR. The within-reader intraclass correlation coefficients (ICCs) were 0.91 and 0.87 for two readers in the EAC and 0.97 in ESPOIR. DMARD-free sustained remission, the opposite of disease persistence, was defined as the sustained absence of arthritis after discontinuation of DMARD therapy, including biologics and glucocorticoids (systemic and intra-articular), for the entire period of follow-up, which was at least one year . In the t tests, Mann–Whitney tests or chi-square tests when appropriate. Associations of morning stiffness in arthralgia and early arthritis were done using logistic regression analyses. All analyses were adjusted for age and gender . In early arthritis further adjustments were made for anti-citrullinated protein antibody (ACPA), rheumatoid factor (RF), swollen joint count (SJC), erythrocyte sedimentation rate (ESR) and symptom duration at baseline. The test characteristics , positive/negative predictive values (PPV/NPV) and area under the receiver operating characteristic curve (AUC) were calculated. This curve was used to derive the morning stiffness duration with the optimal discriminative ability (Youden’s index). Associations between morning stiffness at baseline and radiographic progression were studied using multivariate normal regression analysis with log-transformed radiographic data as response variable as described elsewhere [Characteristics were compared using Student lsewhere ,20. Anallsewhere . SPSS veRC, 807 arthralgia patients were seen, in the Groningen EARC, 481 arthralgia patients and in the REACH, 353 patients were included. Arthritis was observed in 372 (46%), 267 (56%) and 181 (51%) patients respectively. Table RC, Groningen EARC and REACH respectively. The specificities ranged between 73 to 85% but the sensitivities were low (21 to 38%). The low sensitivity indicated that the majority of all patients with arthritis did not have morning stiffness for ≥60 minutes. The PPVs were 54%, 69% and 60% respectively ; therefore, no duration with optimal discriminative ability was ascertained. When morning stiffness was defined as ≥90 minutes , the specificity increased to 85 to 93%  and 60 (15 to 120) minutes. Other baseline characteristics are presented in Table S1 in Additional file P <0.001 in EAC and Spearman rho 0.54, P <0.001 in ESPOIR) Early arthritis patients with moderate severity (VAS 34 to 67 mm) had an OR on RA of 1.87 (95% CI 1.48 to 2.36) and 2.04 (1.32 to 3.17) and the patients in the most severe category (VAS ≥68 mm) had ORs of 2.38 (95% CI 1.89 to 3.00) and 2.46 (95% CI 1.53 to 3.96) respectively. Using the VAS, the AUCs were 0.62 (95% CI 0.60 to 0.65) in EAC and 0.62 (95% CI 0.57 to 0.68) in ESPOIR. The AUCs of morning stiffness duration and morning stiffness severity were not statistically significantly different.Both cohorts also recorded the severity of morning stiffness. In both cohorts morning stiffness duration and severity were correlated .RA patients reporting morning stiffness (≥60 minutes) at baseline did not have more severe radiographic progression  included in several European cohorts. The arthralgia datasets had different inclusion criteria. The studied EAs short.  AlthoughThe early arthritis cohorts also had different inclusion criteria. ESPOIR included patients that were considered susceptible to have or develop RA, whereas the EAC included all types of early arthritis patients. Consequently, the percentage of patients fulfilling the 2010 criteria at year one and the PPV were higher in ESPOIR than in the EAC and the difference in morning stiffness between RA and non-RA patients was larger in the EAC. Nonetheless, the ORs, AUCs and test characteristics were almost similar between the cohorts, supporting the consistency of the findings.et al. proposed that the suspicion of RA is increased in the presence of ≥3 swollen joints, a positive squeeze test or morning stiffness ≥30 minutes. Data supporting the latter recommendation were not published before [Emery The outcome in early arthritis was fulfilling the 2010-RA criteria after one year; generally diagnoses are definite then . We did Our findings that morning stiffness is associated with 2010-RA, independent of other characteristics , are in contrast with the absence of morning stiffness in the 2010 criteria . This isOur study in early arthritis assessed morning stiffness in two ways: the questions related to the duration and the severity (on a VAS). It has been reported that such descriptions poorly define the experiences of patients, which encompass temporal patterns, intensities and functional disability in the early morning ,25. To oA limitation of our data on the severity of morning stiffness is that VAS data were missing in 19% of EAC RA patients at baseline and in almost 50% of the RA patients who achieved DMARD remission at the time of remission. A previous study on 143 patients reported that morning stiffness severity is more responsive than morning stiffness duration . We did Another limitation of this study is that no longitudinal evaluations on the arthralgia patients were done. Recently, a prediction rule for arthritis development in ACPA-positive arthralgia patients was derived and morning stiffness ≥1 hour was part of this rule . This suAlthough the circadian rhythm is associated with nightly activation of the inflammatory processes and elevation of pro-inflammatory cytokines (for example TNF-α and IL-6) -30, the The additional relevance of present data is that it provides evidence, for the first time on a large study, that incorporating morning stiffness in the diagnostic process of joint symptoms in daily practice is valuable. A duration of morning stiffness of 30 minutes had the best combination of sensitivity and specificity (74 to 77% and 48 to 52% respectively). However, in case one prefers to be specific (specificity ≥80%) a cutoff duration of 60 minutes is preferable.


STARCH BRANCHING ENZYME II (SBEII) using overexpression in potato tubers.Starch is biosynthesised by a complex of enzymes including various starch synthases and starch branching and debranching enzymes, amongst others. The role of all these enzymes has been investigated using gene silencing or genetic knockouts, but there are few examples of overexpression due to the problems of either cloning large genomic fragments or the toxicity of functional cDNAs to bacteria during cloning. The aim of this study was to investigate the function of potato SBEII intragene consisting of potato cDNA containing a fragment of potato genomic DNA that included a single intron was used in order to prevent bacterial translation during cloning. A population of 20 transgenic potato plants exhibiting SBEII overexpression was generated. Compared with wild-type, starch from these tubers possessed an increased degree of amylopectin branching, with more short chains of degree of polymerisation (DP) 6–12 and particularly of DP6. Transgenic lines expressing a GRANULE-BOUND STARCH SYNTHASE (GBSS) RNAi construct were also generated for comparison and exhibited post-transcriptional gene silencing of GBSS and reduced amylose content in the starch. Both transgenic modifications did not affect granule morphology but reduced starch peak viscosity. In starch from SBEII-overexpressing lines, the increased ratio of short to long amylopectin branches facilitated gelatinisation, which occurred at a reduced temperature (by up to 3°C) or lower urea concentration. In contrast, silencing of GBSS increased the gelatinisation temperature by 4°C, and starch required a higher urea concentration for gelatinisation. In lines with a range of SBEII overexpression, the magnitude of the increase in SBEII activity, reduction in onset of gelatinisation temperature and increase in starch swollen pellet volume were highly correlated, consistent with reports that starch swelling is greatly dependent upon the amylopectin branching pattern.A hybrid SBEII has been achieved in a non-cereal plant. The data show that overexpression of SBEII using a simple single-intron hybrid intragene is an effective way to modify potato starch physicochemical properties, and indicate that an increased ratio of short to long amylopectin branches produces commercially beneficial changes in starch properties such as reduced gelatinisation temperature, reduced viscosity and increased swelling volume.This work reports the first time that overexpression of The online version of this article (doi:10.1186/s12896-015-0143-y) contains supplementary material, which is available to authorized users. Starch serves as the most important carbohydrate storage reserve for plants, and provides the foundation for human and animal nutrition as well as a raw material for numerous food and industrial applications . Starch waxy mutant of maize (Zea mays L.)  had a lower gelatinisation temperature, whereas that from wheat (Triticum aestivum L.) had an unaltered gelatinisation temperature but altered pasting properties [Solanum tuberosum L.) where GBSS had been down-regulated produced gels with improved stability and clarity [Amylose and amylopectin are synthesised in the plastids, where they assemble into a semicrystalline granule. Amylopectin is the major component, making up 70–80% of starch in most species. Amylose is synthesised by granule-bound starch synthase (GBSS), whereas a large complex of enzymes is required to synthesise amylopectin. This complex consists of four soluble starch synthases  and two types of starch branching enzyme , with various debranching enzymes, kinases and other enzymes also involved -4. The aoperties . Starch  clarity .amylose extender mutant of maize that lacks SBEIIb, amylopectin had fewer branches and a higher proportion of longer chains [Oryza sativa L.), down-regulation of SBEIIb decreased the proportion of short branches and increased the proportion of intermediate and long branches [Hordeum vulgare L.), only when both SBEIIa and SBEIIb were down-regulated was a significant reduction in the frequency of amylopectin branches observed [In vitro experiments with rice enzymes found that SBEIIb transferred chains of DP6 and DP7, SBEIIa transferred chains of DP6–15, and SBEIa produced a range of short and intermediate chains of DP ≤ 40 [Differences in amylopectin branching affect granule crystallinity, which together with differences between species in granule size and shape result in altered thermal, pasting and biophysical properties ,8. Amylor chains  that arebranches . In barlobserved . Indeed,observed . In vitr DP ≤ 40 .amylose extender mutant [SBEI and SBEII produced potato starch that was >70% apparent amylose and had a very high phosphorus content [In potato only two isoforms of SBE are involved, SBEI  and SBEII  ,14. SBEIr mutant , resulte content .SBEIIb gene, and expressing it in a rice SBEIIb-deficient background [GBSS promoter. An additional way to increase the proportion of branched amylopectin in the granule is to down-regulate GBSS and reduce amylose content, so knockdown GBSS lines were also generated for comparison.Overexpression of SBE activity to modify starch structure is a much more difficult proposition than knockdown, due to the enzyme’s deleterious effects on bacteria during cloning of the constructs . Overexpckground . We aimeckground , whose sckground  due to iSBEII was unable to be propagated in Escherichia coli [SBEII intragene containing a single intron to prevent bacterial translation was assembled from cDNA and genomic DNA fragments , moderate overexpression  to high overexpression . Line 1047–19 exhibited approximately wild-type mRNA abundance of SBEII but with an additional anomalous larger SBEII transcript presumably derived from an aberrant insertion event. To confirm that mRNA accumulation of the structurally unrelated starch synthase enzymes SSII and SSIII was not affected by manipulation of SBEII expression, the same gel blot was stripped and re-hybridised. Sequential re-hybridisation with labelled probes for SSII and SSIII showed that the mRNA abundances of these two genes were essentially indistinguishable from wild-type and empty vector controls , followed by an in-gel activity reaction and staining for SBE reaction products . The greatest enhancement was in lines 1047–15 and 17, which possessed SBEII activity almost 3-fold that of wild-type  method was used to examine the effects of altered s Figure . Polyglus Figure A, C. The2 Figure B. The gr2 Figure A. Howeve2 Figure B, which GBSS , the chain length distribution of polyglucans was very similar to wild-type  than amylose. An increased birefringence in the GBSS knockdown line was also observed using differential interference contrast microscopy  combined with confocal laser scanning microscopy Figure F. APTS lGBSS expression and overexpression of SBEII markedly altered the onset of gelatinisation temperature (To), but in opposite ways , To was increased by ~4°C, to 59.3 to 60.0°C. In the transgenic line 1041–4, with unaltered GBSS expression, To was not increased relative to controls. In contrast, overexpression of SBEII in various 1047 lines reduced To by up to 3°C (Table To (WT > 1047–5 = 1047–23 > 1047–12 ≥ 1047–15 ≥ 1047–17) correlated strongly (r = −0.931) with the trend of increasing SBEII activity , temperature of gelatinisation conclusion (Tc) and gelatinisation enthalpy (ΔH) showed similar trends to To in all of the lines.The different contents of amylose, short-branched amylopectin and phosphorus in the starch would be expected to result in altered thermal and gelatinisation properties. Both knockdown of ys Table . In wild 1041–1,  and 16, To of starch from line 1047–17 and the increased To of starch from line 1041–3. Other than the onset temperature, pasting profiles were similar in shape, but with a reduction in peak viscosity of ~10% for line 1047–17 and ~24% for line 1041–3. Final viscosities were also reduced in both transgenic lines.An examination of starch pasting properties using a rapid visco analyser (RVA) detected differences between the lines Figure , includiThe greatest difference in physicochemical properties was seen in the gelatinisation of starch granules in urea. In 4 M urea, starch granules from line 1041–3 showed very little gelatinisation, with a swollen pellet volume only one-third that of wild-type starch granules Figure A. StarchSBEII overexpression had a clear effect on the swollen volume of the starch granules in 4 M urea . In contrast, the increase in swollen pellet volume was strongly but negatively correlated (r = −0.976) to the reduction of To , is due to disruption of the molecular order in the crystalline regions and results in irreversible granular swelling and starch solubilisation. The temperature required for starch gelatinisation, and the swelling volume achieved, are affected by granule size and shape, the amylose:amylopectin ratio, amylopectin branching, phosphorus content and other factors. Overexpression of re Table  and at are Table . Granule amylose ,35. Thuson Table . In SBEISBEII-overexpressing line 1047–17, the swelling volume in 4 M urea was increased to approximately 65% of the maximal volume achieved (in 9 M urea), compared with only 20% for wild-type  mutant of maize [Studies on cereal starch have suggested that swelling power is exclusively due to the properties of the amylopectin, with amylose and lipids inhibiting swelling . In SBEI in rice ,29, or kn potato ,38. The SBEII expression, there was a correlation between SBEII activity, the degree of reduction in To and the degree of increase in the swelling volume in 4 M urea. In other studies, comparisons between transgenic potato lines found that swelling power was decreased when either amylopectin branching was reduced (knockdown of SBEI/SBEII), or phosphorus content was reduced ) [GWD that reduced starch C6-linked phosphorus content by 85% had little effect on To orTp, although swelling power was considerably reduced [SBEII overexpressing lines, despite a 25% reduction in phosphorus content. Thus, increased amylopectin branching rather than small changes in amylose or phosphorus content most likely produced both the lowered To and increased swelling power. Consistent with the data presented here, increased branching in rice starch was also reported to increase swelling volume [In lines with a range of kinase)) . A knock reduced ,40. We og volume , althougg volume .SBEII increases the proportion of short chains of DP6-12, consistent with in vitro experiments that found potato SBEII transfers a high number of chains of DP6 [SBEII that caused an increase in average chain length [To found in the GBSS knockdown lines was most likely due to the reduced amylose content, since the degree of amylopectin branching and the phosphorus content were not substantially altered , the approach could be used to create entirely intragenic plants where all the transferred DNA (including T-DNA borders) is derived from the potato genome [Additionally, although modifying the amylopectin branching pattern  enzymes -21. Sinco genome . The useo genome .SBEII in Escherichia coli, so an alternative strategy was employed. A suitable short intron to prevent translation of a functional SBEII protein in bacteria was identified at amino acid 439, and primers  gDNA using primers FW3 and RW10 produced a 374 bp fragment that contained an intron of 231 bp.It did not prove possible to propagate a complete cDNA of potato SBEII cDNA was PCR-amplified using primers SBE-A and RW11 (incorporating a AflII site) to give cDNA1, and the downstream portion using primers F4 (incorporating a SacI site) and SBE-B to give cDNA2. All PCR products were verified by DNA sequencing. cDNA1 and the genomic fragment containing the intron were ligated together using the AflII site, then the cDNA1:gDNA fragment (blunt-SacI) and the cDNA2 fragment (SacI-blunt) were ligated in a three-way ligation into expression vector GBSSPTpUC57 (a gift from Dr A. J. Conner), between the GBSS promoter and the GBSS terminator. This produced a 4.46 kb construct pDB1047 consisting of GBSSpro:cDNA1:gDNA:cDNA2:GBSSterm  of potting mix and grown to maturity in a containment glasshouse in Palmerston North  during the southern summer and autumn months (January to May). Glasshouse conditions were approximately 15 h daylight per day, with supplementary cooling and heating providing a maximum daytime temperature of 27°C and a minimum night-time temperature of 16°C. The plant aerial portions were allowed to senesce naturally. Three tubers per plant were peeled, chopped into small cubes and frozen in liquid nitrogen before being stored at −80°C for RNA or enzyme assay. RNA was prepared using the hot borate method , and 10  Gilbert  buffer a2, 50 mM 2-mercaptoethanol, 12.5% (v/v) glycerol), incubated on ice for 5 min then centrifuged twice at 16,000 × g for 15 min [et al. [2/KI as described by Nishi et al. [Tuber tissue was powdered in liquid nitrogen, and 100 mg was extracted with 100 μL of extraction buffer , dry starch powder was lightly sprinkled onto carbon adhesive tabs attached to aluminium stubs and was viewed uncoated in a FEI Quanta 250 SEM  using variable pressure mode to reduce charging .Aqueous, unstained suspensions of starch grains were observed by brightfield, differential interference contrast (DIC) and simple polarising microscopy (crossed polars but no phase plate) using an Olympus Vanox AHT3 microscope . Brightfield microscopy was also used to view starch stained with iodine using a stain solution of 0.2% Iet al. [The reducing ends of starch molecules were fluorescently labelled within the starch granules using APTS as described by Blennow et al. . Opticalet al. [6 Da, and eluted with 10 mM NaOH at 50°C. Carbohydrate content in the effluent was recorded on-line by measuring refractive index ). A large peak at the end of the elution profiles (due to NaOH) was omitted.Aliquots of starch were suspended in 2 M NaOH, gelatinised at 100°C then dissolved in water as described by Blennow et al. . The solThe percentage of amylose present in the starch was measured using the Megazyme K_AMYL Amylose kit . Starch branching patterns were assessed using a micro-scale FACE procedure to measure chain length size distribution . The deg−1 starch added to give a suspension containing 70% water. The pans were hermetically sealed and left at room temperature (20°C) for at least 2 h. DSC conditions were: equilibration at 10°C for 5 min, then heating at 5°C min−1 to 100°C. The heat flow curves were analysed using TA Universal Analysis 2000 software version 4.5A build 4.5.0.5 using the “integrate peak sigmoid horizontal” option over the range 50–80°C. Variables calculated were peak onset temperature (To), peak maximum temperature (Tp), peak conclusion temperature (Tc) and peak area (ΔH), which provides an estimate of enthalpy in J g−1.Thermal analysis of gelatinisation properties was performed using a Q2000 differential scanning calorimeter (DSC) . Starch (~5 mg) was accurately weighed into Tzero aluminium hermetic pans (TA Instruments), and 2.33 μL water mg−1, maintained at 95°C for 5 min, then cooled to 50°C at 6°C min−1, with constant stirring at 160 rpm.Starch pasting properties were evaluated using a rapid visco analyser (RVA) . Approximately 1 g of starch was accurately weighed and dried at 130°C to constant weight. A starch suspension was prepared at 7% (w/w) on a dry weight basis in distilled water, with a total weight of 28 g. The suspension was equilibrated at 50°C for 1 min, heated to 95°C at 6°C ming for 20 min, then allowed to stand at room temperature for 1 h. The volumes of the swollen pellet were determined by subtracting the volume of the supernatant from the original 1 mL volume.The swelling volume of starch was determined using a urea procedure . Freeze-P = 0.05). Since for SBEII activity the data was relative and was scaled so that wild-type was 1, the wild-type data was not included in the ANOVA; pooled standard errors from the ANOVA were used in one-sample t-tests to test whether each mean was significantly different from 1. Correlation coefficients were calculated between means. Analysis was carried out using Genstat .Means were compared using one-way ANOVA. Least significant differences (LSDs) were used to establish which means differed significantly (


To develop a systematic review by assessing and comparing the different complications that occurs in bone graft surgery using the mandibular body, ramus and symphysis as donor sites.In order to respond to the following question, a systematic review was developed: does the use of intraoral mandibular body and ramus as donor sites in bone graft surgery, produce fewer and less severe complications in comparison to the use of the mandibular symphysis in patients that present bone resorption that needs augmentation using autologous grafts? The review was carried out between January 1990 and 2015, during which only clinical essays with a minimum follow-up period of six months were included.The initial search yielded a total of 2912 articles, of which 6 were finally selected. In total, 259 graft surgeries were performed; 118 using the mandibular body and ramus as donor sites, and 141, the symphysis. The most frequent complications that arose when using the mandibular symphysis were temporary sensory alterations in the anterior teeth (33.87%), followed by sensory alterations of the skin and mucosa (18.57%). As for the mandibular body and ramus donor sites, the most frequent complications relate to temporary sensory alterations of the mucosa (8.19%) and to minor postoperative bleeding (6.55%).The analyzed results show a higher prevalence and severity of complications when using mandibular symphysis bone grafts, producing more discomfort for the patient. Therefore, it would be advisable to perform further clinical essays due to the lack of studies found.Key words:Alveolar ridge augmentation, autogenous bone, mandibular bone grafts, chin, mandibular symphysis, mandibular ramus. The implant management of the partial or completely edentulous patient is nowadays a daily and routine treatment with proven success rates in the long term . To achiAmong the described bone volume restoration procedures , the useAutologous grafts can be classified as extraoral or intraoral . IntraorThere are multiple donor sites described in the literature that can be used as bone grafts. Among them are: the mandibular body and ramus , the symAlthough there are several articles regarding the complications that follow graft surgery, few compare in the same study the complications that arise from the use of one type of graft to the other. This fact will not help the professional in making the decision of what type of graft is the most suitable, bearing in mind that it is a common surgical procedure. Therefore, the purpose of this study is to conduct a systematic review by evaluating and comparing the various complications that occur in the mandibular body, ramus and symphysis grafts as donor sites.A protocol was developed prior to the review covering all aspects regarding the systematic review methodology as per Prisma guidelines , includiQuestion at issue: ¿Does the use of mandibular ramus intraoral bone grafts produce fewer and less severe complications in patients that present bone resorption that needs augmentation using autologous grafts, compared to mandibular symphysis bone grafts?Population to be studied: The population of interest in this review corresponds to patients presenting bone resorption in need of restoration of bone volume using intraoral autologous bone grafts.Type of intervention and comparison: The type of intervention to be studied in the review was bone graft surgery using intraoral autologous grafts extracted from the mandibular body or ramus, being compared to bone graft surgery where the graft is also of intraoral autologous origin, but obtained from the symphysis.Selection of studies: The selected studies included clinical trials on humans during a minimum follow-up period of six months. The area from where the graft was obtained was clearly identified as well as the complications that occurred in the donor site following the grafting, specifying how the complication was measured .All clinical trials that included patients with the following conditions were automatically excluded from the review: uncontrolled metabolic illnesses, head radiotherapy in the past 24 months, intraoral or intravenous bisphosphonate treatment during a period that exceeded 3 years, patients with mental disorders, heavy drinkers and/or smokers (more than 10 cigarettes per day), or drug addicts.Results: The resulting complications in bone graft surgery using the mandibular body and ramus and symphysis as donor sites in the form of block or particulate grafts were the variables to be analyzed, paying more attention to the following complications: pain, postoperative hemorrhage, sensory alterations  in skin, mucosa or teeth, infection, pulp necrosis and mandibular fractures.Search Strategy: The search and following selection of articles was performed by using 4 electronic databases . Only the articles which complied with the inclusion criteria and written between January 1990 and 2015 were considered.The search was completed using the following terms: “Population AND Intervention AND Outcomes” .Only the articles written in Spanish or English were included. Revision of the cross-references of the initial list of selected references was carried out.Manual search in the following journals was developed: Journal of Oral and Maxillofacial Surgery, International Journal of Oral and Maxillofacial Surgery, Clinical Oral Implants Research, Clinical Implant Dentistry and Related Research, Journal of Oral Implantology and International journal of Oral and Maxillofacial Implants.Review Methodology: Following the initial search, the review of the resulting titles and abstracts was performed by two independent reviewers (D.R. and C.C.). Only the studies which complied with the requested inclusion criteria were included; articles whose title and/or abstract did not clearly match with the inclusion criteria were subsequently read in full text. Thus, a complete list of full text articles was obtained for the reviewers to read and select. In cases of disagreement between reviewers, a third reviewer was consulted (J.LQ.). To avoid selection bias, the reviewers concealed the name of the Journal, the institution of origin and the names of the authors of the articles during the review. Special attention was paid to duplicate publications.Quality assessmentFollowing the Cochrane recommendations “assessing risk of bias in included studies” , the quaThe initial search yielded a total of 2912 articles  were not considered, given the impossibility of distinguishing complications specific to graft surgery from those related to third molar extractions). Of the 252 intervened patients, 259 underwent bone graft surgeries, 118 of which were mandibular body and ramus grafts, and 141, symphysis grafts. In regards to the ramus grafts, in 67 cases, burs were used to undertake the osteotomy procedure ; in 51 cases the osteotomy was performed with a saw and round bur or fissure bur generally used for the lower osteotomy procedure. In 118 cases, the osteotomy was performed only in the cortical bone. In 43 cases collagen sponges were left in the site, and in 5 cases compression bandages were used, leaving the remaining patients with no treatment. Regarding the chin area grafts, in 46 cases the graft was extracted with fissure burs, in 50 cases trephines with diameters between 7 and 11 mm were used, and a saw was used in 45 cases.A total of 252 patients were treated   included sensory alterations of the skin and mucosa, followed by postoperative bleeding with 6.55% of TC, 4.37% of TC came as a result of temporary teeth sensory disturbances and permanent sensory disturbances with 1.09% of TC. As for the complications occurred in the symphysis donor site, the most frequent were those associated to temporary sensory disturbances of the teeth reaching 33.87% of TC, followed by the temporary sensory disturbances of the skin and mucosa with 18.57% of TC, permanent sensory disturbances with 12.02% TC, bruising with 7.1% of TC, blemishes with 5.46% of TC, wound dehiscence with 1.63% of TC, and finally, pulp necrosis with 1.09% of TC. Despite the different existing alternatives, the autologous bone graft still remains as the chosen graft for bone volume restoration procedures  being quHowever, one of the major disadvantages of this type of graft is the donor site morbidity. This phenomenon, on the contrary, does not occur with other different types of bone substitutes, being this their main advantage. These bone substitutes have excellent clinical results in small flaws, but in larger defects their predictability is reduced, reason why the autologous bone graft is considered as the first treatment option .The most commonly used intraoral autologous grafts are those that originate from the mandibular body and ramus and symphysis areas, which, according to the analyzed studies, have the following different types and complications that can be divided into 4 groups:1.- Pain, discomfort and aesthetic resultset al. . Tooth sensory alterations using the mandibular body and ramus graft correspond to a total of 8 cases, being all temporary and described only by Cordaro et al.  This stuet al. (et al. (Cordaro et al. (Cordaro et al. (et al. (By using the chin area as the donor site, in 64 cases there were sensory disturbances of the lower anterior teeth; of these, 2 corresponded to pulp necrosis and 62 to temporary sensory disturbances. Cordaro et al.  are the  (et al.  considero et al.  presenteo et al.  with 12  (et al.  with 2 a (et al.  also wit4. Other complicationset al. (et al . (The reviewed studies also describe other types of complications, which present a low prevalence. Cordaro et al.  indicate(et al . , which o(et al .  describeIn conclusion, it can be said that despite the small number of investigations which comply with the selection criteria included in the review, it appears that the majority of complications occur when using the chin area as the donor site. This area, as well as presenting a higher prevalence and severity of complications, generates greater discomfort for the patient, showing high levels of sensory disturbances in teeth, skin and mucosa, both temporary and permanent, in addition to aesthetic changes, despite being the latter rare.Thus, it is necessary to carry out a greater number of randomized clinical trials in the future, assessing a larger number of patients, along with the possible complications. It is recommended that clinical trials detail sensory recovery periods and the type of treatment used for recovery.