Arthritis is the most common disease in elderly individuals. Many medications for osteoarthritis treatment have the potential for side effects. Using a bioinformatics tool and preclinical studies, ChondroT, 5 herbal complexes, was identified from Ganghwaljetongyeum, which is a18 herbal complex, which has often used to treat osteoarthritis. The goal of this study is to evaluate short-term safety of ChondroT.This will be a multicenter, randomized, double-blind, and placebo-controlled trial. There will be a 2-week run-in period before random allocation to 3 groups, ChondroT 1.0 g, 2.0 g, and placebo groups. Total duration of the clinical trial will be 14 weeks including a 4-week washout follow-up. Participants will be followed-up every 4 weeks, and the effect and the safety will be assessed at visit 2, 3, and 4. All participants are asked to maintain the medication schedule in this protocol. The primary outcome will be measured using pain visual analog scale (VAS) after 8 week treatment and secondary outcomes will include pain VAS score after 4 week treatment, SF-36 survey score, patient's global assessment, physical function test, and the change of erythrocyte sedimentation rate, and C-reactive protein. The repeated-measure analysis will be used for the primary efficacy based on full analysis set and per-protocol.This study has restrictive inclusion, exclusion criteria, and a well-controlled intervention, and it will be the first randomized controlled trial to evaluate the efficacy and safety of ChondroT formula in osteoarthritis patients. The trial according to this protocol may provide a new intervention in the treatment of osteoarthritis. The symptoms are characterized by inflammation, pain, and stiffness in the musculoskeletal system and they range from localized self-limiting conditions to systemic autoimmune processes.–4 depending on the type of drugs or individuals involved.Various modalities to manage knee joint pain range from conservative management, such as exercise, oral medication including nonsteroidal antiinflammatory agents, and joint injection, to surgical treatment. All medications for osteoarthritis have the potential for side effects It is a complex herbal decoction composed of 18 plants. In order to improve the efficacy and convenience of pharmaceutical prescription, a previous bioinformatics study identified the 5 medicinal herbs with the greatest potential. These were Ostericum koreanum Maximowicz , Lonicera japonica Thunberg , Clematis mandshurica Ruprecht , Angelica gigas Nakai , and Phellodendron amurense Ruprecht . The 5 herbs were extracted with water and named ChondroT. A previous study confirmed that ChondroT had a chondroprotective effect and demonstrated multitarget mechanisms related to inflammation and arthritis which suggest ChondroT's therapeutic potential for the treatment of arthritis.Ganghwaljetongyeum (GHJTY), a mixture of traditional herbs, also has been widely used to improve the symptoms of osteoarthritis in China and Korea. Our previous study showed that GHJTY was effective on rheumatoid arthritis by inhibiting the production of pro-inflammatory mediators and proliferation of synoviocytes.In this study, we designed this study protocol to test if this new drug, ChondroT, is effective on symptoms of osteoarthritis patients by randomized, double-blind, placebo-controlled, multicenter clinical trials.22.1This will be a multicenter, randomized, double-blind, and placebo-controlled trial. Participants will be recruited through poster or advertisement from arthritis outpatient department in Dongshin University Korean medicine hospitals in Gwangju or Mokpo (DSGOH or DSMPOH). There will be a 2-week run-in period before random allocation to 3 groups, ChondroT 1.0 g, 2.0 g and placebo groups. Total duration of the clinical trial will be 14 weeks including a 4-week washout follow-up. Participants will be followed-up every 4 weeks, and the effect and the safety will be assessed at visit 2, 3, and 4  of this study has been approved by ethical approval from the institutional review board (IRB) in DSGOH  and DSMPOH . For those eligible patients, researchers will give participants the detailed information of this study. Participants or their family should give written informed consent, and then will be enrolled in the study.Subjects may be required to quit the study in case of serious adverse events or adverse drug reaction, and then they will be reported to institutional review board in both hospitals. In addition, patients may leave this study at any time without any disadvantage or constraint.Trials are being conducted by supervision of the clinical trial center in both hospitals and monitored by an independent contract research organization (CRO). Especially, a specific clinical research coordinator will coordinate all procedures for participants to improve participants’ adherence to intervention protocols. If there is any change in protocol, it will be approved by IRB again before implementation.The results will be published on the website of ClinicalTrials.gov in accordance with the CONSORT 2010 Statement and in special journals.2.3 a daily mean pain visual analog scale (VAS) (100 mm Pain VAS) ≥ 40, during the past 1 week (0–100); male or female aged 50 to 80 years.Inclusion criteria were as follows: diagnosed as knee osteoarthritis according to the clinical diagnosis of American College of Rheumatology clinical classification criteria;Exclusion criteria were as follows: receiving immunosuppressant, corticosteroids, cyclosporine, antipsychotics, osteoarthritis medications including painkillers  or health functional foods to improve joint or cartilage health; having renal functional disorder , liver functional disorder , connective tissue, multiple sclerosis, inflammatory disease, tumor, trauma, rheumatoid arthritis, autoimmune disease, severe genu valgum or vagus, secondary arthritis, hip disease, or other forms of inflammatory arthritis disease; having a past history such as knee surgery including arthrocentesis drainage or gastrointestinal operations (except for simple appendicitis or hernia) or abuse of drug or alcohol; being treated with traditional medical interventions, steroids, or mucosal supplements injection  in the knee joint or physical therapy; participating in other clinical trials within 2 months before the screening.2.4All participants will take 2-week run-in period before randomization, then receive a treatment of 3 groups, ChondroT 1.0 g (2 chondroT tablets + 2 placebo tablets), ChondroT 2.0 g (4 chondroT tablets) or placebo tablet  and per-protocol set (PPS) will be used for the efficacy assessment. According to the principle of intentionality treatment, all patients having the efficacy assessment record at least one time will be brought into the FAS. PPS will include patients who complete the trial and do not violate the protocol.The primary goal of this study is to test whether ChondroT, a herbal complex, is effective on osteoarthritis patients. Repeated-measures analyses will be used to assess between-group differences in the modeled change in scores from baseline to 12 weeks. Independent variables for fixed effects will be treatment , visit , interaction between treatment and visit, baseline measures, sex, age, and so on.For safety analysis, Chi-square test, Fisher exact test, or Wilcoxon rank test will be conducted for assessing the differences of incidence of adverse events  between ChondroT and placebo groups.All analyses will be performed using the software of Statistics Analysis System, blinded to treatment allocation.2.8Considering the phase of this study (2a) to evaluate short-term safety of a drug, we have not calculated the sample size based on the previous studies. The sample size is 72 in total and 24 for each group. Assuming a dropout of about 20% and sufficient statistical power of 80% at a significant level of 0.05 in 2 tailed test, about 57 patients may be expected to complete the trials finally.3Arthritis is the most common inflammatory disease in elderly individuals. The symptoms are characterized by inflammation, pain, and stiffness mainly in the musculoskeletal system.–4 Therefore, demands for the development of safer medication have been increased. Our previous study showed that GHJTY, a 18 herbal complex, was effective on rheumatoid arthritis by inhibiting the production of pro-inflammatory mediators and proliferation of synoviocytes. Other previous studies identified that the 5 medicinal herbs had the greatest potential, named ChondroT, from GHJTY to improve the efficacy and convenience of pharmaceutical prescription using bioinformatic method and confirmed that ChondroT had a chondroprotective effect and demonstrated multitarget mechanisms related to inflammation and arthritis which suggest ChondroT's therapeutic potential for the treatment of arthritis.Many medications for osteoarthritis have the potential for side effects.The phase of this study is trial 2a to evaluate short-term safety of the drug. It will be a study with restrictive inclusion, exclusion criteria, and a well-controlled intervention, and will be the first randomized controlled trial to evaluate the efficacy and safety of ChondroT formula in osteoarthritis patients. However, this protocol may have limitations of the small sample size which may lead to bias. The trial according to this protocol may provide a new intervention in the treatment of osteoarthritis.4Conceptualization: S. Lee.Writing – original draft: S. Lee.Writing – review & editing: S-J. Kim.


Grapevine rupestris vein feathering virus and Grapevine yellow speckle viroid 1 were recorded in the Czech Republic for the first time.Comprehensive next generation sequencing virus detection was used to detect the whole spectrum of viruses and viroids in selected grapevines from the Czech Republic. The novel NGS approach was based on sequencing libraries of small RNA isolated from grapevine vascular tissues. Eight previously partially-characterized grapevines of diverse varieties were selected and subjected to analysis: Chardonnay, Laurot, Guzal Kara, and rootstock Kober 125AA from the Moravia wine-producing region; plus Müller-Thurgau and Pinot Noir from the Bohemia wine-producing region, both in the Czech Republic. Using next generation sequencing of small RNA, the presence of 8 viruses and 2 viroids were detected in a set of eight grapevines; therefore, confirming the high effectiveness of the technique in plant virology and producing results supporting previous data on multiple infected grapevines in Czech vineyards. Among the pathogens detected, the  The infection of plant tissues by viral pathogens can cause significant economic losses to agriculture crops . Many diThe availability of such a broad-spectrum detection technique provides powerful support to certification processes, which are supposed to guarantee the clean health status of propagation materials .In addition, the use of the metagenomic approach in vegetatively propagated species would help in preventing the spread of viruses  as well https://www.ncbi.nlm.nih.gov/guide/all/). Genomic resources specific to viruses can be accessed at https://www.ncbi.nlm.nih.gov/genome/viruses/.The NGS method generates a very large amount of sequencing data from each sample, and thus the high demand for proper data processing and analysis arises. UNIX-based open-source platforms for the entire process exist, as well as commercial ones such as CLC Genomic Workbench, Geneious, or Lasergene. The need for reference sequences is primarily provided by the NCBI, with its large and freely available databases  present in plant tissues , 8. TherTechniques for the molecular detection of grapevine viruses are based on the analysis of RNA, isolated either from vascular tissues or from softer tissues such as leaves and petioles. NGS techniques more frequently use RNA isolated from leaves and petioles as the starting materials , 9.The present work was aimed at demonstrating the NGS technique's utility for the detection of grapevine viruses using phloematic tissues as the starting material, and in obtaining a picture of the virome of different Czech grapevine cultivars.All plants analyzed in the work belong to the public collection of the Crop Research Institute, Prague, Czech Republic. They are officially recognized as genetic resources under “The National Program on the conservation and utilization of genetic resources of microorganisms important for food and agriculture”. Their free use for research purpose is guaranteed by the Czech national legislation, namely Act no. 148/2003 Coll., on the conservation and utilization of genetic resources of plants and microorganisms important for food and agriculture.Grapevine cuttings were collected in November 2013 from grapevines maintained under controlled conditions in a screenhouse of the Crop Research Institute, Prague, Czech Republic, ensuring freedom from infection of some additional pathogens.All the grapevines had already been partially characterized, and the present work was focused on those plants known to contain larger numbers of viruses, as well as those showing symptoms of an unclear etiology. Further criteria for plant selection were their having been different cultivars and having different origins from divergent parts of the Czech Republic. A descriptive list of the samples is provided in Vitis berlandieri × Vitis riparia), originating from Modřice, South Moravia, coordinates 49.1252N, 16.6031E. It was tested for the first time in 2005 during the ELISA (enzyme-linked immunosorbnet assay)-based grapevine virus survey [Grapevine leafroll-associated virus-1 (GLRaV-1), Grapevine virus A (GVA), Grapevine virus B (GVB), and Grapevine fleck virus (GFkV). Testing by RT-PCR also revealed infection by the Grapevine rupestris stem pitting-associated virus (GRSPaV) [Plant G1 is grapevine rootstock Kober 125 AA (origin s survey . During (GRSPaV) .Cuttings from the plant were taken in 2005, from which a new plant was grown and cultivated at the Crop Research Institute Prague (CRI) in a screenhouse, where it was labeled as TI23. The plant did not show any symptoms of virus infection.Plant G2, labeled as TI21 in the screenhouse of CRI , of grapevine variety Pinot Noir, had recently been bought in a market. It was selected as being representative of certified propagated material. The origin of the plant was Mělník, Central Bohemia, Czech Republic. The plant showed mild mosaic symptoms, and was positive in RT-PCR, using generic primers for tymoviruses .Grapevine Pinot gris virus (GPGV) [e.g., stunting, mottling and leaf deformations) [Plant G3, labeled as TI25 in the CRI screenhouse, is a interspecific grapevine hybrid of variety Laurot, having originated from a vineyard of genetic resources held in the Mendel University in Brno, Faculty of Horticulture, Lednice, South Moravia. The cultivar is a product of crossing Merlan (Merlot × Seibel 13 666) × Fratava (Frankovka × Svatovavřinecké); while the Seibel 13666 is a complex interspecific variety, used as a donor of resistance against fungal diseases. The G3 plant is the first grapevine in the Czech Republic found to be infected by the newly emerged mations) .Plant G4, labeled as TI15 in the screenhouse of the CRI, is grapevine variety Müller-Thurgau, having originated in the Karlštejn Research Station of Viticulture, Central Bohemia, coordinates 49.9350611N, 14.1652414E. This Research Station is a unit of the Crop Research Institute, Prague, Czech Republic. The plant was previously found to be infected with GLRaV-1, GVA, and an unspecified tymovirus. The plant was showing symptoms of a mild mosaic, and downrolling of the leaf blades.Plant G5, labeled as TI30 in the screenhouse of the CRI, grapevine variety Guzal Kara, having originated from Modřice, South Moravia, coordinates 49.1252N, 16.6043E, was showing symptoms of irregular and dichotomous growth, typical for nepovirus infection, although no nepovirus had thus far been detected. In 2006, the plant was also replanted into the screenhouse of the Crop Research Institute, Prague.Plants G6-G8 were originally selected from production vineyards in Perná, South Moravia for containing different viruses based on previous commercial tests. These plants showed symptoms such as leaf mottling, mosaic, and leaf deformations; but not symptoms typical for GPGV . The graTotal RNAs were extracted from 1 g of scraped phloem according to . The lowhttp://www.ncbi.nlm.nih.gov/) with an e-value threshold of 10−6 in both. Contigs mapping to the Vitis vinifera L. genome, as well as those mapping to bacterial and fungal genomic fragments or their viruses were omitted from further work. A list of the potential viruses and viroids present in the analyzed samples was created, and reference sequences were selected for mapping of NGS reads. As the first choice, sequences from the RefSeq database (NCBI) were used, because they contain full-length sequences of particular viruses and viroids. For viruses with a lower homology with RefSeq-originated data, sequences with a higher homology were used for mapping, despite the fact they were not of full length. This was especially the case for GRSPaV, GVA, and GVB viruses. A list of reference sequences is presented in The sequence quality was checked by use of fastQC-0.10.1 , and a FAfter mapping the unique reads on the reference sequences, the presence of the studied viruses in grapevine vascular tissues were then determined. The data carried with BLAST and mapping analyses adjudged that viruses whose genome coverage were at least 20%, and with a sequencing depth greater than 5, were expected to be present in the sample. Further confirmation of the presence of a virus or viroid was done by RT-PCR as a second method, and a positive reaction in RT-PCR was the definitive criterium for estimating the presence of a virus or viroid.A genome coverage threshold of 20% was used according to the authors' experience, based especially on the analysis of the presence of real viruses in examined grapevines. A sequencing depth of 5 was used according to published works –23.™ Plant Total RNA Kit . Reverse transcription was performed with a Transcriptor First Strand cDNA Synthesis Kit , using random hexamers for reaction priming according to the manufacturer's suggestions. FastStart Taq DNA Polymerase  was used for PCR. The conditions and primers used for the detection of individual viruses and viroids are listed in The isolation of RNA from vascular tissues was performed using a SpectrumIn the case of unclear results, and a first record of the pathogens, the products of RT-PCR, were cloned into pGEM-T Easy vector and sequenced. At least five clones from every sample were commercially sequenced. The resulting sequences were compared, the genetic divergence was estimated, and a phylogenetic tree was constructed using the MEGA7 program  to ensur6, G2 = 1.5×106, G3 = 2.7×106, G4 = 2.1×106, G5 = 1.9×106, G6 = 1.3×106, G7 = 8.9×105, G8 = 8.2×105 unique reads, respectively , Closteroviridae (Grapevine leafroll-associated virus 1), and Betaflexiviridae ; whereas, the two viroids (Hop stunt viroid and Grapevine yellow speckle viroid 1) belong to the family Pospiviroidae.Plants of various varieties and geographical origins were included in the analyzed set; primarily from Moravia , but also from wine regions in Bohemia . Libraries representative of the sRNA populations extracted from grapevines G1-G8, and sequenced by Illumina technology, contained G1 = 1.6×10ectively . Furtherapevines . The eigThe sequences obtained were submitted to GenBank NCBI under the following accession numbers: KT000353-KT000360 (HSVd), KP693444-KP693448 (GPGV), KT000346-KT000352 (GYSVd-1), KT000361-KT000362 (GFkV), KT000363-KT000365 (GLRaV-1), and KT000366-KT000371 (GRSPaV). Sequences of GVA, GVB, GRVFV, and GSyV-1 were not completed because of the low coverage of the full-length reference sequences.The results of sRNA sequencing were checked by RT-PCR detection; thus meaning that all detected pathogens (viruses and viroids) had their presence proven in the tested materials by at least two methods see . All mapTo compare the read mapping efficiency of two frequently used programs , belonging to the genus Foveavirus, family Betaflexiviridae, was confirmed to be present in all eight plants. A high coverage of mapped reads occurred in the majority of vines (6 out of 8) ranging from 97% to 100%; with low coverage in plants G1 and G4, reaching only 46% and 25%, respectively. The greatest amount, corresponding to sRNA GRSPaV, was detected in the G3  and G5 plants , with 100% coverage of the genome. Regarding the high level of GRSPaV genome variability, we used as a reference sequence KX274277.1 for mapping on sample G4 with 25% resulting genome coverage; the mapping on reference NC_001948.1 provided genome coverage that was too weak (only 16%); however, the other results suggested the presence of GRSPaV.Grapevine Pinot gris virus (GPGV), genus Trichovirus, family Betaflexiviridae, is a newly-emerged virus, recently identified in Italy [in Italy , and subin Italy , 34, 35,in Italy , 36. GPGGrapevine virus A (GVA), genus Vitivirus, family Betaflexiviridae, was detected in plants G1 and G4. GVA is a common grapevine virus in the Czech Republic [Republic . It had Grapevine virus B (GVB), genus Vitivirus, family Betaflexiviridae, was detected in plant G1. The virus is very rare in the Czech Republic [Republic . In factGrapevine leafroll associated virus 1 (GLRaV-1), genus Ampelovirus, family Closteroviridae, was detected in plants G1, G4, and G6. The virus had the greatest number of reads among all of the viruses. This is caused by the fact that it has the largest genome (more than 18 kb) compared to e.g., the 6–8 kb in fovea-, tricho-, and tymoviruses. GLRaV-1 occurrence is common in the Czech Republic [Republic .Grapevine fleck virus GFkV, genus Maculavirus, family Tymoviridae, was detected in plants G2 and G7. GFkV is a common grapevine virus in the Czech Republic according to the previous surveys [ surveys .Grapevine rupestris vein feathering virus (GRVFV), genus Marafivirus, family Tymoviridae, was detected in plants G1, G4, G7, and G8. RT-PCR with primers designed on the contigs and sequencing of the cloned PCR product confirmed the presence and proper taxonomic identity of the virus in all of the plants. The sequence of the PCR product was submitted into GenBank under reference number KX465108.This is the first confirmation of the presence of GRVFV in the Czech Republic.Grapevine Syrah virus 1 (GSyV-1), genus Marafivirus, family Tymoviridae, was detected in plants G1 and G3. The presence of this virus on the examined plants was also confirmed by [irmed by .Grapevine yellow speckle viroid 1 (GYSVd-1), genus Apscaviroid, family Pospiviroidae, was found to be present in 7 out of the 8 plants. To our knowledge, this is the first described occurrence of Grapevine yellow speckle viroid 1 in the Czech Republic. The viroid is reported to occur worldwide; e.g., in Italy [in Italy , USA [37in Italy , and Newin Italy .Hop stunt viroid (HSVd), genus Hostuviroid, family Pospiviroidae, was detected in all eight grapevine plants. HSVd was earlier detected in Czech grapevines by [The next viroid, vines by . HoweverVitis sp.Plant G1, which is grapevine rootstock Kober 125AA, does not show symptoms of virus disease, although it is infected with at least 7 viruses and viroids. As this genotype is an indicator for rugose wood complex viral disease, it should show some symptoms, as the plant is infected with several viruses responsible for expression of the disease mentioned. However, after grafting inoculum from this plant into the sensitive LN33 indicator, symptoms will appear . It is tPlant G2, showing symptoms of a mild mosaic, was found to be infected with several pathogens including GPGV . Mosaic Plant G3, showing symptoms of mosaic and vein banding, was known to contain GPGV. The presence of GPGV was confirmed together with other viruses and viroids. The typical symptoms of GPGV were not observed.Plant G4, showing symptoms of downrolling of its leaf blades, was found to contain GLRaV-1, GVA, GRSPaV, and viroids. GLRaV-1 is the cause of the downrolling of the leaf blades.Plant G5, showing dichotomous growth, was expected to contain some nepovirus. However, no nepovirus was detected by NGS, only GRSPaV and viroids. The symptoms are probably due to another endogenous factor causing a phytohormone imbalance .Plants G6 to G8 were showing symptoms of leaf mottling and deformations, but not those typical for GPGV. All three had a confirmed infection by NGS of GPGV. Since all three come from the cultivar Chardonnay, we can conclude that GPGV can cause symptoms of leaf mottling and deformations in this cultivar under Czech conditions.Based on the results of sRNA NGS detection, RT-PCR with specific primers to the corresponding pathogens were applied to RNA isolated from vascular tissues to confirm virus and viroid presence in the analyzed plants. The results are given in Using the RT-PCR method, GYSVd-1 in plant G6 was not detected, while the mapping of reads covered 57% of the reference sequence. Additionally, not one contig was identified to be of GYSVd-1 origin. The other seven plants were positive for the viroid in RT-PCR, with coverage ranging from 91–100%. We can conclude that the viroid is not present in plant G6.GPGV was detected by RT-PCR, even in plant G4; although the coverage was only 18%, which is under the 20% threshold. The numbers of mapped reads are shown in Tymoviridae, Grapevine rupestris vein feathering virus (GRVFV) was detected in plant G1. Its presence was confirmed by RT-PCR and sequencing of the PCR product. As mentioned in the introduction about plants tested in the present work, the G1 plant was originally positive in ELISA for GFkV. Since no GFKV is present in plant G1 (but GRVFV is), the antibodies were also probably accidentally raised against another virus other than GFkV. GRVFV was also detected in plants G4, G7, and G8.GFkV was detected by NGS in plants G2 and G7, and also confirmed by RT-PCR. In its first occurrence in the Czech Republic, another virus from the family For all of the reasons delineated, and due to the end-point nature of RT-PCR, as well as the massively parallel nature of sRNA NGS, sensitivity comparisons of the two methods can be very difficult. This subject should continue to be studied diligently. Further, it needs to be ascertained what kind of coverage and sequencing depth is sufficient to determine a positive sample. A comprehensive database of virus sequences should be used for NGS contig identification, and the one with the best matching score should then be used for genome mapping. For many grapevine viruses, full-length genome sequences from the Central European region are still missing.The diagnostics of RNA pathogens (viruses and viroids) was carried out using sRNA sequencing. This method seems very promising, using the metagenomic approach –4, consiThe plants used in this study were all tested and previously found positive for the presence of some RNA viruses. However, the spectrum range of RNA pathogens detected was still unexpected. In every one from the evaluated set of 8 plants, at least 3 RNA pathogens were detected. This study also provides an unique comparison of various varieties, grown in different wine regions of the Czech Republic. The relatively high occurrence of GLRaV-1 and GFkV in the Czech Republic is a well-known fact , 41. HowDistribution of foveavirus GRSPaV in the Czech Republic has not yet been studied; however, thus far, our experience with grapevine virus detection shows that its occurrence in Czech grapevines will be very high , 42.This study is also the first work to prepare libraries for sRNA sequencing from grapevine vascular tissues.  used wooThe virus detection percentage rates obtained in this study among the set of 8 plants from 5 localities are: GRSPaV—100%, GPGV—75%, GRVFV—50%, GLRaV-1–38%, GFkV—25%, GVA—25%, GSyV-1–25%, and GVB—13%. Viroid HSVd was detected in 100% of the plants, GYSVd-1 in 88% of the plants. This result is especially interesting due to the different geographical origins of the plants tested. According to the requirements of Czech legislation , plant propagation materials have to be tested for GFLV, ArMV, GLRaV-1, and GLRaV-3 (as well as for GFkV in the case of rootstock mother plants), which are all widespread in the Czech Republic [Use of the coverage value set at 20% proved to be a good threshold for estimating virus presence, taking into consideration use of suitable reference sequences for mapping, mostly the one giving the best identity during the BLAST search.The fact that all of the tested plants turned out to be positive for the presence of viroids is another interesting observation of this study.As a general conclusion, the results presented in this study demonstrate the great importance of correct and proper processing of NGS data, as well as the need for localized reference data on viruses and viroids.S1 File(PPTX)Click here for additional data file.S2 File(PPTX)Click here for additional data file.S1 Table(PPTX)Click here for additional data file.


Attempts to discover genes that are involved in the pathogenesis of major psychiatric disorders have been frustrating and often fruitless. Concern is building about the need to understand the complex ways in which nature and nurture interact to produce mental illness. We analyze the epigenome in several brain regions from schizophrenic patients with severe cognitive impairment using high-resolution (450K) DNA methylation array. We identified 139 differentially methylated CpG sites included in known and novel candidate genes sequences as well as in and intergenic sequences which functions remain unknown. We found that altered DNA methylation is not restricted to a particular region, but includes others such as CpG shelves and gene bodies, indicating the presence of different DNA methylation signatures depending on the brain area analyzed. Our findings suggest that epimutations are not relatables between different tissues or even between tissues' regions, highlighting the need to adequately study brain samples to obtain reliable data concerning the epigenetics of schizophrenia. Schizophrenia is one of the most complex and enigmatic of all psychiatric disorders, being characterized by the heterogeneous presence of positive, negative, and cognitive symptoms that affect all aspects of mental activity. Twin, family, and adoption studies support the idea that schizophrenia has a strong genetic component, with an estimated heritability as high as ~80%  have been published that analyze the DNA methylation profile of schizophrenic patients. Four of these were performed in peripheral blood samples and one used the frontal cortex of schizophrenic patients , hippocampus and ACC of healthy controls and of patients with a diagnosis of schizophrenia with severe cognitive deficits in the executive function, memory and with patterns of positive and negative symptoms, respectively, by using Illumina's 450K DNA methylation microarray (Infinium HumanMethylation450 BeadChip). This microarray encompass 485,764 CpGs located along the human genome, and exceeds the number of CpGs covered by Illumina's previous EWAS, in which the analysis was limited to the CpG sites in CpG islands in the gene promoter region. Although, we previously have described the methylation status of the major neurotransmitter systems associated with the pathophysiology of schizophrenia . The FAB is a short cognitive and behavioral six subtests devised for screening of a global executive dysfunction (scores from 0 to 10 represent high cognitive impairment and from 10 to 15 represent mild cognitive impairment. Mean age ± SD = 10.3 ± 4.7). The MMSE is a short, standardized form devised for screening the cognitive mental state (scores from 10 to 20 represent mild cognitive impairment and <10 represent high cognitive impairment. Mean age ± SD = 25.4 ± 3.4). Finally, PANSS was conceived as an operationalized, drug-sensitive instrument that provides balanced representation of positive and negative symptoms and gauges their relationship to one another and to global psychopathology.We administered a comprehensive battery of neurocognitive tests to all patients in random order. The battery included the Buschke Memory Impairment Screen  and an exhaustive neuropsychological evaluation.All subjects are high-rate smokers and polymedicated patients and, therefore, it is not possible to analyze the influence of specific drug on the DNA methylation patterns.DNA isolation was performed following the protocol established by Alelu-Paz et al.  for humaAfter bisulfite conversion of 500 ng of each sample, we used 4 μl of bisulfite-converted DNA to hybridize on the Infinium HumanMethylation450 Beadchip, following the Illumina Infinium HD Methylation protocol. Detailed information about the contents of this array is available in the Infinium HumanMethylation450 Beadchip user guide and data sheet. Two previous papers described the technical schemes, the accuracy and the high reproducibility of this array  in the β-value unless specifically indicated otherwise.To calculate the DNA methylation status of the CpG sites, we selected the β-value, which represents the signal ratio from a methylated probe relative to the sum of the methylated and unmethylated probes, and takes any value between 0 (unmethylated) and 1 (completely methylated). To avoid possible sources of technical bias that might influence the results, we excluded probes located on the X chromosome and every β value that had a threshold detection value of Database for Annotation, Visualization, and Integrated Discovery program , which is able to extract biological features and meaning associated with gene lists, and which has most commonly been employed in genome-wide or near-genome-wide studies .To identify functions associated with the genes analyzed, we performed gene ontology (GO) analysis using the Z-test; p < 0.01). More interestingly, in the intergroup analysis we found significantly more methylated CpGs in schizophrenic DLPFC and hippocampus than in the corresponding healthy brain structures, while more unmethylated probes were found in the schizophrenic ACC than in the healthy ACC  shows that differentially methylated CpG positions distinguished schizophrenic from healthy samples  were discarded and 485,476 probes (99.94%) were used for further analysis. First, we compared the total number of methylated (β ≥ 0.75) and unmethylated (β ≤ 0.20) CpG sites in schizophrenic and healthy samples. In general terms, intragroup analysis showed that, in all brain structures, there were more methylated than unmethylated CpGs in the schizophrenic and normal brain , hippocampus (R2 = 0.994), and ACC . Bonferroni correction for multiple testing was applied at the <10−7 level. When we compared schizophrenic and healthy DLPFCs, we found 66 differentially methylated probes corresponding to 37 genes  in the three brain areas analyzed between schizophrenic and healthy samples. From the CpG content and neighborhood context of the 66 differentially methylated CpGs, 24.05% were in CpG islands, 21.5% in CpG shores, 6.3% in CpG shelves, and 48.1% were outside coding genes or known transcription regulatory elements  and cell surface receptor-linked signal transduction . The network analysis showed differently directed first-order protein interactions . In the known and predicted protein–protein interactions we draw attention to the associations of HLA-DRB5 with HLA-A and HLA-B -6 cytokine family and has recently been associated with persecutory delusion in the Korean population  and signal transduction . STRING analysis highlighted first-order protein interactions among IL12RB1 and IL12B, IL1B, IL6, and IL12A, all of which have previously been associated with schizophrenia  have been shown to have crucial involvement in gene activation and de-activation , which is hypomethylated in the hippocampus of samples from schizophrenic patients, could have an important role in the cognitive impairment associated with schizophrenic disabilities, since it induces neuronal cell differentiation and participates in the deterioration of working memory function -induced neurite outgrowth and neuron morphological change. CNTNAP2 may play a major role in the formation of functionally distinct domains critical for saltatory conduction of nerve impulses in myelinated nerve fibers and, therefore, its epigenetic alteration is consistent with the hypothesis that schizophrenia results from poor or miswired anatomical or functional connections  were present in at least two of the three areas included, corresponding to intergenic regions (six probes) and three corresponding to genes . No probe was repeated across all three brain areas analyzed, so we can conclude that the schizophrenic and normal human brain have different anatomical DNA methylation signatures, which are brain-region dependent.Even more, these signatures differ between brain regions. In this sense, in our study, only nine differentially methylated probes (Δβ > 0.2; Therefore, we wished to highlight the need to study brain samples to obtain reliable data in regard to the epigenetic study of this severe mental illness. The more we understand the dynamic nature of epigenetic the more we need to take care of the wise tissue selection and its adequate anatomical identification. The conjunction of these factors will allow us to deepen our knowledge in schizophrenia, avoiding data that add “noise” instead truly information, and leading to a more exact diagnose, treatment, and patients' life-quality improvement.RA, FC, JVS, AG, NA, MO, AE, AM, CG, AC, JS, ME, SR: acquired, analyzed, and interpreted data for the work. This also included, drafting the work or revising it critically for important intellectual content. RA, FC, JVS, AG, NA, MO, AE, AM, CG, AC, JS, ME, SR: Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. RA, FC, JVS, AG, NA, MO, AE, AM, CG, AC, JS, ME, SR: Final approval of the version to be submitted.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.


AbstractJansenusburgeri Foldi, 1997  was studied cytogenetically for the first time. It was shown that the species reproduces bisexually, displays XX/X(0) sex chromosome system and 2n=6/5  including two pairs of long autosomes and a pair of shorter X-chromosomes in female. The chromosome complement, adult female morphology and the life cycle of J.burgeri are illustrated. The cytogenetic data are in fact the first ones for Kuwaniini scale insects, because Kuwaniaoligostigma De Lotto, 1959 briefly cytogenetically studied by Kuwania Cockerell, 1903 and the tribe Kuwaniini, since this species shows aberrant morphological characters, specifically the total absence of abdominal spiracles and the presence of tubular ducts. Kuwaniini MacGillivray, 1921 , which currently comprises four nominal genera . Two other genera of the tribe, Neogreenia MacGillivray, 1921 and monotypic Neosteingelia Morrison, 1927, have never been studied in terms of cytogenetics and reproductive biology. In 1997 Dr. Imre Foldi described from Thailand a peculiar new species and new monotypic genus, Jansenusburgeri Foldi, 1997, placed by him in the subfamily Xylococcinae without tribal attribution. Wu and Nan . The material was suitable both for morphological investigation  chromosome system and 2n=6/5  for Xylococcinae have been studied cytogenetically till now and all available data were published without photographs of the chromosomes. Thus, Kuwaniaoligostigma De Lotto, 1959 (tribe Kuwaniini) shows 2n=16 in adult female and bisexual reproduction  and has tubular ducts in contrast to all other Kuwaninii. In my opinion, the species may be excluded from the tribe, but its correct taxonomic placement remains obscure for me.Only a few species of oduction ; the chrMatsucoccusgallicolus Morrison, 1939  was studied by Steingeliagorodetskia Nasonov, 1908  and found that the species had the bisexual reproduction, XX-X(0) chromosome system and 2n=10 in females.Xylococcinae species studied so far, Jansenusburgeri shows the lowest chromosome number, 2n=6. Of the other scale insects, the same number is known only in few species of the subfamily Monophlebinae  and in some species of neococcids (superfamily Coccoidea) – see for review Thus, amongst the four


Acquired hemophilia A (AHA) is caused by polyclonal inhibitory immunoglobulins G (predominantly IgG1 and IgG4) against factor VIII (FVIII). They react with A2, A3, or C2 domains of the FVIII molecule, blocking its interactions with active factor IX, phospholipids, and von Willebrand factor. A disturbed proportion of CD4Here we report a rare case of a patient with AHA and multiple myeloma, where the disappearance of the hematologic malignancy induced by chemotherapy resulted in a regression of the coagulation disorder.A 67-year-old man came under our observation for plasmacytoma. Significantly, his past medical history included both high blood pressure and hip replacement. His concomitant medication was only lacidipine. In 2009 the patient underwent cholecystectomy without bleeding. In 2014, he underwent a surgical excision of basal cell carcinoma in the pectoral region without bleeding. In 2014 the patient was diagnosed, at a different hematological center, with congenital mild hemophilia A and monoclonal gammopathy of undetermined significance IgG kappa. At that time, FVIII was 29.1%. He also underwent a desmopressin test, which displayed an increase of FVIII activity  up to 90% after 60 min. Elevation of IgG (2319 mg/dL) was present, while the rest of the immunoglobulins were within normal values. IgG k-type monoclonal protein was detected in the serum and urine.In 2015, a diagnosis of plasmacytoma resulted from a biopsy of abnormal tissue performed during vertebroplasty for a pathological L3 fracture. The orthopedic intervention was performed with premedication with recombinant FVIII. He came, therefore, to our attention presenting with 28% FVIII activity, prothrombin time within normal limits, and prolonged activated partial thromboplastin time (44.1 s).Serum protein electrophoretic analysis showed a monoclonal peak within γ-globulin, increased IgG, and suppression of all other components. Serum protein electrophoresis showed a monoclonal free k-light chain.Bone marrow biopsy, performed after infusion therapy of recombinant FVIII, showed plasma cells at 50%. Therefore, a diagnosis of multiple myeloma IgG kappa, stage IIA Durie-Salmon, I ISS, was made. He began treatment with intravenous bortezomib and oral melphalan and prednisone, associated with monthly infusions of bisphosphonates, for 6 cycles in total. Revaluation of the disease documented stringent complete response (sCR), but we also witnessed the normalization of blood coagulation parameters, while FVIII activity was 98%. The patient was then mobilized with high-dose cyclophosphamide and autotransplanted, retaining the sCR to this day.Diagnosis of AHA can be difficult. It is often unrecognized or misdiagnosed as other acquired hemorrhagic disorders, such as disseminated intravascular coagulation, acquired inhibitors against von Willebrand factor, and acquired factor XIII deficiency .The association of acquired hemophilia and multiple myeloma is extremely rare; there are only four such reports in the literature ,6,7,8. HAs said above, both solid tumors and hematologic malignancies, especially lymphoproliferative diseases, are able to predispose or be associated with this antibody’s growth .A small number of works investigated the genetic basis of AHA, due to immune tolerance breakdown. Pavlova et al.  pointed In conclusion, it is clear that in our case the reestablishment of immunocompetence, complete remission of multiple myeloma, and disappearance of AHA are closely related events. In fact, previous works have demonstrated that the treatment of the underlying cancer led to the disappearance of the inhibitor in 22% of patients . Finally


Oenococcus oeni (O. oeni), as the one of most important microorganisms in wine industry and the most resistant lactic acid bacteria (LAB) species to ethanol, has not been investigated regarding the selection of stable reference genes for RT-qPCR normalization under ethanol stress conditions. In this study, nine candidate reference genes  were analyzed to determine the most stable reference genes for RT-qPCR in O. oeni SD-2a under different ethanol stress conditions  ethanol). The transcript stabilities of these genes were evaluated using the algorithms geNorm, NormFinder, and BestKeeper. The results showed that dnaG and dpoIII were selected as the best reference genes across all experimental ethanol conditions. Considering single stress experimental modes, dpoIII and dnaG would be suitable to normalize expression level for 8% ethanol shock treatment, while the combination of gyrA, gyrB, and rrs would be suitable for 12% ethanol shock treatment. proC and gyrB revealed the most stable expression in 16% ethanol shock treatment. This study selected and validated for the first time the reference genes for RT-qPCR normalization in O. oeni SD-2a under ethanol stress conditions.The powerful Quantitative real-time PCR (RT-qPCR) was widely used to assess gene expression levels, which requires the optimal reference genes used for normalization.  Oenococcus oeni (O. oeni) is well known as the main starter of malolactic fermentation (MLF). Through the MLF process, O. oeni reduces the acidity naturally and improves the quality and stability of wine , one of the most common technologies for quantifying gene expression level, was often used to analyze the stress response to abiotic and biotic stresses in hsp18, citE, ctsR, clpP, ftsH, have been studied using RT-qPCR analysis in O. oeni under different stress conditions, however only one common reference gene, ldhD, was used for normalization  were used to calculate the expression stability of the candidate genes and obtain the most stable reference genes. In addition, the optimal reference genes were tested to normalize the expression of one target gene (hsp18) under ethanol stress conditions. This work is hoping to provide a basis for future study on gene expression under ethanol conditions in O. oeni SD-2a and other lactic acid bacteria (LAB) species.In this study, nine genes, Oenococcus oeni SD-2a was obtained from our own collection . This strain was previously isolated from Shandong province in China and was properly identified  in China.ied Liu, . The strO. oeni SD-2a was cultured at 28°C and pH 4.8 in three flasks with 100 mL FMATB broth, which contains glucose 5 g/L, D, L-malate 5 g/L, yeast extract 5 g/L, peptone 10 g/L, MgSO4 •7H2O 0.2 g/L, MnSO4 •4H2O 0.05 g/L, Cysteine/HCl 0.5 g/L, and tomato juice 250 mL , they were completely mixed and split into three sterile flasks. Then the flasks were performed the shock treatment (ST) by adding with 8, 12 and 16% (v/v) ethanol, respectively. Bacterial samples were collected at time zero just before the addition of ethanol and then at one, 3 h after ethanol was added , which were then frozen in liquid nitrogen and kept at −80°C until RNA extraction  as described by the manufacturer. At the end of the reaction, each sample was diluted 1:100 with nuclease-free water prior to the RT-qPCR analysis.O. oeni SD-2a (results not shown), which was also used to check exon-intron borders by matching the primers to their location. The primer sequences are listed in Table Primers of candidate reference genes and one target gene for RT-qPCR were designed using Primer Premier (version 5.0) and each has a length of about 20–25 bases, a G/C content of over 50% and a Tm of about 60°C . Each reaction was performed in triplicate with a total reaction mixture of 20 μl final volume containing 2 μl diluted cDNA, 0.4 μl of each primer, 10 μl of ChamQ SYBR qPCR Master Mix, and 7.2 μl of RNase-free water. PCR conditions were indicated as follows: 95°C for 30 s, 40 cycles of 95°C for 10 s, 60°C for 30 s. To confirm product specificity, a melting curve analysis was performed after each amplification is a statistical algorithm, which is based on the principle that the expression ratio of two reference genes should be constant in all samples, regardless of the experimental condition, or sampling time  was also calculated by stepwise inclusion of a less stable gene until the (n+1)th gene has no significant contribution to the newly calculated normalization factor NFNormFinder is a Visual Basic application tool for Microsoft Excel used to calculate the stability values of each candidate reference genes by combining intra- and inter-group variations of gene expression  and the coefficient of variation (CV). The candidate reference genes can be ranked from the most stable one with the lowest variation, to the least stable one with the highest variation. Any candidate reference gene with the SD higher than 1 will be considered as inconsistent.Pfaffl et al.  have estIdealRef is the algorithm recently developed by Palombella, based on the principle published by Vandesompele et al. . The rawhsp18) was carried out with the same cDNA samples used for the selection of reference genes .In order to validate the selection of reference genes, the expression profile analysis of one target gene  the optimal multiple reference genes from all samples, (2) the optimal multiple reference genes from each treatment, (3) the least stable reference gene from each treatment.rrs showed the most abundant transcript level with the lowest Ct value 8.57, while proC was the least abundant with the highest Ct value 24.12. dnaG revealed the least gene expression variation , while rrs and ldhD with CV of 6.61 and 6.34% were regarded as the most variable  across all samples pooled together. When all 21 samples were analyzed together, dpoIII, dnaG, and gyrA were defined as the most stable genes with the M-value of 0.431, 0.448, and 0.482, respectively. While rpoA and ddlA were defined as the least stable genes with the M-value of 0.917 and 0.999, respectively. In 8% ethanol, we also found that dpoIII, dnaG, and gyrA  were the most stable genes, whereas proC and ddlA  were the least stable genes . ldhD and rpoA  showed the greatest variation, which were ranked eighth and ninth, respectively  were identified as the most stable genes, while rpoA and ddlA  as the least stable genes  identified by IdealRef software were consistent with those selected by the overall ranking across all samples  between two sequential normalization factors (NFn and NFn+1). As shown in Figure 2/3 values were respectively 0.099 and 0.117, lower than the cut-off value of 0.15, indicating that two genes would be sufficient for normalization under these conditions. Therefore, we could consider dpoIII and dnaG as the optimal multiple reference genes in 8% ethanol, while proC and gyrB in 16% ethanol. However, in 12% ethanol, three reference genes were required for normalization with an acceptable V3/4 value of 0.137. Hence, gyrA, gyrB, and rrs would be used as the optimal multiple reference genes for normalization in 12% ethanol.In order to determine the optimal number of reference gene used for accurate normalization, geNorm performed a stepwise calculation of the pairwise variation (Vhsp18) was carried out to validate the selection of candidate reference genes. According to the reference gene strategies mentioned above, besides the optimal multiple reference genes selected from each treatment, we also used the least stable gene in each treatment: proC in 8% ethanol, rpoA in 12% ethanol and ldhD in 16% ethanol, and the optimal multiple reference genes selected from all samples: dpoIII and gyrA in 8% ethanol, dnaG, dpoIII, and gyrA in 12% ethanol and dnaG and dpoIII in 16% ethanol, to normalizer the expression level of hsp18.An expression profile analysis of the target gene (hsp18 increased continuously at 1 and 3 h in 8% ethanol, using dpoIII and dnaG as the optimal reference genes. Similar expression pattern was also revealed in 12% ethanol when normalization was carried out using dnaG, dpoIII, and gyrA as the optimal reference genes. In contrast, when normalizing using proC as reference gene, which was the least stable reference gene in 8% ethanol, the expression exhibited also continuously but sharply increasing. Meanwhile the relative expression was obviously overestimated, much higher than when normalizing using the optimal reference genes, dpoIII and dnaG, at all time points. The same expression trend was observed in 12% ethanol when normalizing using the least stable reference gene, rpoA. Normalizations with the optimal reference genes selected from all samples were also respectively performed in 8 and 12% ethanol. The expression levels were consistent with those obtained when normalization with the optimal reference genes from each treatment. However, in 16% ethanol, when normalization was performed using proC and gyrB as the optimal reference genes, the relative expression increased sharply at 1 h and unchanged at 3 h. The relative expression showed a different trend compared to when normalized with the least stable gene, ldhD, or with the optimal reference genes from all samples, dnaG and dpoIII.As shown in Figure O. oeni has evolved different mechanisms to cope with the ethanol stress condition in wine (Guzzo et al., O. oeni, but the accuracy of its result was directly affected by the expression stability of reference genes (Vandesompele et al., As the most resistant LAB species to ethanol, dnaG, dpoIII, and gyrA were the most stable reference genes when all samples pooled were analyzed together, however different experimental conditions emerged their own optimal reference genes. This is more evident that the validation of the reference genes for specific experimental condition is required prior to use in RT-qPCR normalization. In 8% ethanol, dpoIII performed as the most stable gene, followed by dnaG and gyrA. In 12% ethanol, gyrA and gyrB were identified as the most stable gene, while rrs ranked third position. In 16% ethanol, the top-ranked gene was proC, which was not evaluated in the previous studies of O. oeni, whereas gyrB and dnaG were ranked second and third respectively. In our study, dpoIII, dnaG, gyrB, and gyrA showed better expression stability, while in some studies, they were used together as reference genes for RT-qPCR normalization in O. oeni, but not evaluated particularly (Costantini et al., O. oeni, such as ddlA and ldhD varied greatly in our experimental conditions. ddlA were ranked fourth, seventh and seventh in the overall rangkings of 8, 12, and 16% ethanol, respectively. ldhD, together with rpoA, was considered as the least stable genes in all experimental conditions, so in this work, it was not chosen as reference gene for the normalization. This result is consistent with that described by Cafaro et al. (rrs, encoding 16S rRNA, has been reported unstable in some studies with the disadvantage of high transcript level (Desroche et al., rrs than other genes, however in 12% ethanol, the expression stability of rrs was stable, which was ranked in top position by geNorm analysis and third in the overall ranking, was good (Figure rrs would be used for normalization to validate the selection result in 12% ethanol. Furthermore, in order to neutralize the impact of the high abundance of rrs, the templates for rrs should be diluted more times than the samples for the other genes in RT-qPCR analysis, however the different dilution ratios can lead to more human errors (Sun et al., In our study, three popular statistical algorithms, geNorm, NormFinder, and BestKeeper, were used for the evaluation of the expression stability of nine candidate reference genes in three different ethanol conditions. geNorm confirmed the most stable gene with a low pairwise variation among reference genes (Vandesompele et al., o et al. . rrs, end Figure . Thus, ihsp18. In this study, the results showed that the expression patterns were influenced by the reference gene strategy obviously. When being normalized with the least stable gene, the relative expression levels of hsp18 were overestimated in all experimental conditions (Figure hsp18 gene acts in the early response to stress conditions (Beltramo et al., proC and gyrB as reference genes was different from using dnaG and dpoIII (Figure hsp18 gene was proposed as molecular marker to select good MLF starters by Coucheney et al. (hsp18, which confirmed hsp18 as a useful tool to evaluate the ability of O. oeni strains to survive in wine and to perform MLF. Moreover, Betteridge et al. (hsp18 as an indicator to determine the high ethanol tolerance phenotype of O. oeni. Therefore, according to the expression results, O. oeni SD-2a seems to be a high ethanol tolerance strain and a good starter for MLF process.In order to validate the selection of reference genes, in this study, we employed three reference gene strategies to normalize the relative expression of one stress response gene, s Figure . The expI Figure . These ry et al. . Capozziy et al.  reportede et al.  considerO. oeni could be used as alternative sources to identify novel candidate reference genes, however the validation of these novel genes using RT-qPCR or literature-based searches was required (Alexander et al., O. oeni SD-2a using RNA-seq has been reported by Liu et al. (O. oeni SD-2a under ethanol stress condition more accurate and robust. In this study, we selected nine candidate genes, commonly used for normalization in O. oeni, based on literature review. These genes are still very popular with the researchers and still used for RT-qPCR data normalization in many studies.Concluding, microarray and RNA-seq datasets from u et al. . HoweverO. oeni. The results will benefit future gene expression studies in O. oeni SD-2a and facility the selection of reference genes of other LAB strains under ethanol condition.This study is the first to systematically analyze reference genes for RT-qPCR under ethanol stress conditions in HL and SP conceived the idea of the study. SP, LL, and HZ designed and performed the experiments. SP and HW analyzed the data and wrote the paper. HL revised the paper.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 identify the prevalence of and risk factors for knee pain and radiographic knee osteoarthritis (RKOA) and to investigate the relationship between decreased lower extremity muscle mass (DLEM) and knee pain severity.Using data from the Korea National Health and Nutrition Examination Survey, 3,278 participants who were ≥50 years old and who underwent dual x-ray absorptiometry, plain knee radiographs and completed a knee pain questionnaire were enrolled. Lower extremity muscle mass (LEM) was defined as the sum of the fat-free soft tissue mass of the legs, and lower extremity muscle mass index (LMI) was calculated as LEM/body weight (%). DLEM was defined as an LMI more than two standard deviations below the mean of a gender-matched young reference group. Categorical variables were presented as numbers (weighted %).The prevalence of knee pain and RKOA were 22% (n = 721) and 34.7% , respectively. Multivariate logistic regression analysis showed being female , older , less educated , stiffness , bed rest , RKOA  and DLEM  were associated with knee pain. Participants with simultaneous RKOA and DLEM complained of more severe pain (pain score 7.18 ± 2.48) than those with knee pain without RKOA or DLEM (5.02 ± 2.44), those with only RKOA (6.29 ± 2.50), or those with only DLEM (6.78 ± 2.18) (P<0.001). These results remained after multivariate analyses of variance (MANOVAs).The prevalence of knee pain and RKOA were 22% and 34.7%, respectively, in the general Korean population. DLEM was an independent risk factor for knee pain and it was associated with increased pain severity, regardless of RKOA. The number of elderly people in the world is increasing. With aging, chronic pain, especially knee pain, is becoming a serious social problem, causing impairments in physical function and quality of life, as well as increased morbidity. Although previous studies have revealed multifactorial risk factors for knee pain, including obesity, radiographic changes, female gender, older age, stiffness and previous knee injury, the underlying etiology for knee pain remains poorly understood –4. The pKnee osteoarthritis (OA) is the leading cause of productivity loss and long-term disability, particularly in the elderly , 7. AlthBody composition changes with aging, including reduced muscle mass or strength and increased fat proportions. Sarcopenia is defined by decreased muscle mass and impaired muscle function; it progresses with aging and is associated with frailty, falls, mortality, and pain risk . In addiThe first objective of this study was to identify the prevalence of knee pain, RKOA, and risk factors in the elderly Korean population using data from the fifth Korean National Health and Nutritional Examination Survey (KNHANES V 1–2). Second, we attempted to determine whether DLEM influenced knee pain severity in both the elderly Korean population and in participants with RKOA.The KNHANES is a nationally representative cross-sectional health survey periodically conducted by the Korean Ministry of Health and Welfare since 1998 to assess the health and nutritional status of the Korean population. In Korea, all citizens are covered by health insurance, regardless of income or region, and anyone can be selected to participate in the KNHANES survey. The KNHANES used a stratified, multistage, clustered probability sampling method to select a representative sample of the Korean population. In the case of KNHANES V 1–2, a total of 6,567 households selected from 384 survey regions responded to the questionnaire-based survey and were categorized by region, gender, age, and average household size. The details of the KNHANES V have been previously described elsewhere , 18. ThiAll questionnaires were administered by well-trained government survey staff, primarily consisting of nurses and nutritionists. Smoking status , monthly income  and educational level  were assessed via individual interviews. All participants reported whether or not they experienced bed rest due to any disease in the three months previous to their interview.2). BMI scores were categorized into three main groups: normal (BMI < 25 kg/m2), pre-obese (25-< 30kg/m2) and obese (≥ 30 kg/m2) according to World Health Organization (WHO) recommendations  Previous studies demonstrated that aging, female gender, obesity, radiographic changes, older age, stiffness and lower economic status were related to knee pain –4. ConsiSocioeconomic characteristics such as income and education play a significant role in the severity of knee pain , 3, 4. Ast Health and Nutrition Examination Survey, even though the possible mechanisms were still unknown [In the present study, current smoking was negatively associated with knee pain severity in a univariate analysis, although it did not remain significant in a multivariate analysis. Previously, only a few studies have found an association between smoking and knee OA or knee pain; however, these results were controversial and the causal pathway between smoking and knee pain was still unclear. Some studies have reported that smoking was positively associated with knee OA or pain in the presence of greater cartilage loss , while o unknown . This re2, which is typically accepted in Western countries. However, in this study, we defined obesity as BMI ≥ 25 kg/m2, because the mean BMI of our participants was 24.04 ± 0.06 kg/m2  and only 3% of patients had a BMI ≥ 30 kg/m2. It is well known that Asian people are usually less obese than Western people, therefore, many Asian countries, including Korea [2 as the cut-off value for obesity. Consistent with our study, one study demonstrated that a BMI ≥ 25 kg/m2 was not related to knee pain after controlling for age and gender [2 when they investigated the association between radiographic knee OA and knee pain [2 as a cut-off point for obesity in this study was suitable; this different criterion for obesity may explain why obesity was not related to knee pain in the current research, unlike in findings from previous studies.Obesity is another well-known risk factor for knee OA and knee pain .33. The ng Korea , Japan [ng Korea , and Taing Korea , use a Bd gender . In addinee pain . TherefoStiffness was the factor most strongly related to knee pain in this study. This result was consistent with previous studies. Schiphof et al., reporting on data from the Rotterdam population-based cohort study, showed that subjects with stiffness had a higher risk for knee pain  in . They spSeveral studies have investigated the relationship between thigh muscle mass and knee pain , 37, 38.Recently, one study investigated the relationship between lower leg muscle mass and knee pain using DXA . ConsistPrevious studies have revealed a discrepancy between radiographic changes and knee pain in patients with RKOA. Numerous factors, such as age, morning knee stiffness, high BMI, temporal patterns of knee pain, depression, and anxiety, have been proposed to explain this discordance , 34. HowThere are several hypotheses regarding the relationship between DLEM and the knee pain severity. First, muscle mass is a major determinant of muscle strength and is important for pressure distribution throughout the joint . TherefoThis study has several limitations. First, we did not evaluate longitudinal knee pain patterns or the cause-effect relationship between knee pain and risk factors, due to the cross-sectional nature of this study. Second, there was no personal medication information in the KNHANES survey questionnaire; therefore, we could not exclude the direct effects of analgesics, such as non-steroid anti-inflammatory drugs, which are highly correlated with pain levels. Third, it is well known that scores based on the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC)—rather than an NRS scale—are more appropriate for pain assessment in patients with OA. In the KNHANES V1-2 survey, however, pain was assessed with an NRS scale. Accordingly, we were unable to determine the relationship between DLEM and functional status based on the KNHANES V1-2 dataset. Forth, this study was not able to analyze the effects of potential risk factors such as depression, anxiety, knee alignment, edema, or ligament injury in elderly patients because those factors were not surveyed in the KNHANES questionnaire.In conclusion, the prevalence of knee pain and RKOA were 22% and 34.7%, respectively, in the general Korean population. Decreased lower extremity muscle mass was an independent risk factor for knee pain in both the general population and in participants with knee OA. In addition, DLEM was positively related to pain severity. Therefore, we suggest that DLEM might be an independent reason for knee pain in the general population and also in subjects with knee radiographic changes. Further studies are needed to identify the role of DLEM in knee pain.


Antibiotic resistance poses an important threat to global public health and has become a challenge to modern medicine. The occurrence of antibiotic-resistant bacteria in a broad range of foods has led to a growing concern about the impact that food may have as a reservoir of antibiotic resistance genes. Considering Minas Frescal Cheese (MFC)—a typical Brazilian white soft cheese—and its economic and cultural values, in this study, medically relevant antimicrobial-resistance genetic markers (AR genes) were screened, and the occurrence of integrons were evaluated in manufactured MFC using culture-independent approaches. Through a fingerprinting analysis, the tested MFCs were brand-clustered, indicating reproducibility along the production chain. A common core of resistance markers in all brands evaluated and related antimicrobials such as β-lactams, tetracyclines, quinolones, and sulfonamide was detected. Several other markers, including efflux pumps and aminoglycosides-resistance were distributed among brands. Class 1 and 2 integrons were observed, respectively, in 77% and 97% of the samples. The presence of AR genes is of special interest due to their clinical relevance. Taken together, the data may suggest that the production chain of MFC might contribute to the spread of putative drug-resistant bacteria, which could greatly impact human health. Furthermore, detection of class 1 and class 2 integrons in MFC has led to discussions about resistance gene spread in this traditional cheese, providing evidence of potential horizontal transfer of AR genes to human gut microbiota. As long as antimicrobial-resistant bacteria are being detected worldwide, associated to activities other than health-care and including food chain production, the phenomena will continue to pose a major threat to humans, animals, and environmental health and will become the focus of scientific investigation . For sevAntimicrobial resistance in foodborne pathogens involves direct risk for consumers’ health, impairs successful antibiotic treatment of infections, and increases the number of hospitalizations . In addiResistance-encoding genes can be located on chromosomes or on mobile genetic elements, such as plasmids, transposons, and integrons, making them available for horizontal gene transfer . IntegroStaphylococcus, Listeria monocytogenes, and coliforms as quality indicators, other drug-resistant putative bacteria (DRB) may occur, posing a health issue [Dairy products such as milk and cheese have been implicated as potential sources for transmission of antimicrobial-resistant bacteria, with a direct link between the animal microbiota and the human gastrointestinal tract . Minas Fth issue ,18,19. To date, despite MFC having already been associated with multidrug-resistant bacteria, and considering the major microbial groups related to human health, there are no data on the cheese resistome, or even the clinical resistome including the occurrence of integrons, which would potentially be associated with antimicrobial-resistance genetic marker (AR genes) transfer. Most of the studies evaluating the prevalence of AR genes in cheese samples are based on methodological approaches such as isolation and identification of the resistant bacteria, followed by a molecular analysis of their AR determinants ,21,22,23To contribute to aspects of clinical resistome and microbiological safety of manufactured MFC, this study aimed to perform a screening of clinically relevant antimicrobial-resistance genetic markers and evaluate the occurrence of integrons by culture-independent techniques based on direct analysis of total DNA from cheese samples. In addition, the application of the repetitive extragenic palindromic sequences polymerase chain reaction (rep-PCR) fingerprinting technique to compare the microbial community profile of MFC samples and evaluate its reproducibility was also evaluated.w/v) sterile sodium citrate , using a shaker at 250 rpm for 20 min. Aliquots (50 mL) were centrifuged , and the supernatant containing the fat layer was discarded. The pellet obtained was resuspended in 10 mL of Tris-EDTA (TE) ; pH 8) and then centrifuged at 14,000× g for 10 min. This step (addition of wash TE buffer and centrifugation) was repeated twice, and the obtained pellet was resuspended in 200 µL TE buffer. DNA was extracted from the cell pellet using the QIAamp DNA Stool Mini Kit  according to the protocol. DNA concentration was determined by fluorimetry using the QubitTM 2.0 fluorimeter, with the Qubit dsDNA HS Assay kit . DNA integrity was assessed by 0.8% agarose gel electrophoresis. The resulting purified DNA was stored at −70 °C until use. Samples from 35 cheeses from seven different commercially manufactured, trademarked, Minas soft cheeses  were purchased in retail stores in Juiz de Fora . To obtain Brazilian sanitary certification, cheeses are made from pasteurized bovine milk. Following acquisition, the samples were kept in an ice box and sent to the laboratory for immediate processing. Total DNA was extracted directly from 5 samples (same batch) of seven different cheese brands. Twenty-five-gram cheese portions were weighted and homogenized in 225 mL of 2% (w/v) agarose gel for 2 h at a constant voltage of 100 V in 1× Tris/Acetate/EDTA buffer (TAE). A 100 bp DNA ladder  was used as a molecular-size marker. Gels were stained using ethidium bromide, and the images were visualized in ultraviolet light transilluminator . Fingerprints were analyzed using BioNumerics 5.1 . The similarities between the profiles were calculated using the Jaccard coefficient, and the dendrograms were constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA).The rep-PCR analysis was performed using the total DNA extracted directly from the cheese samples. The PCR reactions were performed according to Gaber et al. , with mow/v) in 1× Tris/Borate/EDTA (TBE) buffer. DNA bands were visualized after gel staining with ethidium bromide on ultraviolet light transilluminator. Amplicon sizes were estimated by a 100 bp DNA ladder (Ludwig Biotec). Negative controls were performed as reactions without template DNA. As a positive control, amplicons were purified using the QIAquick Gel Extraction Kit (Qiagen), and the DNA fragments obtained were sequenced in an ABI Prism 3730 DNA sequencer  and were further blasted against the nucleotide database of the National Center for Biotechnology Information (NCBI). The clustering based on the occurrence of antibiotic resistance markers was performed by a principal component analysis (PCA) using a PAST program (version 3.14).A set of 40 resistance genetic markers of human clinical relevance , including genetic markers related to efflux pumps and class 1, 2, and 3 integrons, were screened from the metagenomic DNA of the samples by conventional PCR, using specific primers and amplification conditions previously described . PCR proRepetitive extragenic palindromic sequences PCR was considered in this study as a culture-independent method, using the total DNA extracted from the cheese to compare the microbial community profile among samples from the same batch and to provide evidence of its reproducibility. The dendrogram obtained by rep-PCR clustering of cheese samples is presented in blaZ β-lactamase gene was detected in 100% of the samples and tetB, blaTEM, sul1, and sul2 genes in more than 80%. Despite the high percentage of occurrence, only three resistance markers related to β-lactams were detected, representing 21.4% of the total markers evaluated for this class (14 markers). Resistance markers to tetracyclines were the most prevalent in the analyzed samples. All markers evaluated in this study (eight markers) were detected among the samples, despite the low occurrence of the tetO gene.Considering the occurrence of antibiotic resistance markers, tetB, bla-TEM, blaZ, qnrS, sul1, and sul2), were detectable in all brands . Class 1 and 2 integrons were detected, respectively, in 77% and 97% of the samples and also characterized the common core of detectable genes in all brands , was detected in 91.4% of the samples. In general, ESBL is mainly encoded by mobile genetic elements such as integrons, transposons, and plasmids, which are easily transferable to other bacteria. Occurrence of blaTEM had already been documented in bacteria isolated from Portuguese soft and semi-soft cheeses [blaZ, was observed in all tested cheese sample. The data is impressive and may be related to the massive use of penicillin in Brazil, both in animal and human medicine. In cheese samples, blaZ has been reported in isolates of Staphylococcus aureus [β-lactams are also clinically relevant antibiotics, for which resistance genes have also been described in bacteria from several foods, including cheeses. The f cheese . Add to  cheeses . Of espes aureus ,33,34.aacA-aphD) in 60% of the cheese samples is also of concern due to its controlled use, although this gene was already associated with coagulase-negative Staphylococci isolated from cheese samples in Turkey [The presence of the aminoglycoside resistance gene (n Turkey . Aminogln Turkey .qnrB and qnrS, detected at rates of 68.6% and 71.4%, respectively, along with sulfonamide resistance genes, sul1 and sul2, detected at rates of 88.6% and 94.3%, respectively, are also of particular interest; to date, these genetic markers have not been related to cheese. According to the literature, both qnr and sul genes are thought to be integron- and plasmid-born genes and, thus, highly available to horizontal transfer and environmental persistence upon selective pressure [Quinolone resistance genes, pressure ,37,38. Bacterial multidrug efflux pumps are antibiotic resistance determinants which may be found in all microorganisms. These transporters can actively extrude a variety of substrates such as antibiotics, heavy metals, dyes, detergents, organic solvents, among others, indicating their importance in bacterial physiology under environmental selective pressure as a defense mechanism. Unspecific efflux pumps that transport several compounds can be associated with bacterial cross-resistance, i.e., simultaneous resistance to different antimicrobial drugs and other xenobiotics ,40. EfflOverall, the screening of AR genes in this study provided information on particularities of MFC processing. Although there is no available information with which to compare the results, taken together, the data may suggest that different manufacturing plants may face different contamination or even cross-contamination failures, leading to a peculiar AR profile in the final product which is available to the consumers. To date, it is not possible to address if the microbial contamination is related to the raw-material used in the dairy industry or related to human handling. However, the MFC resistome would be predictive of regional antimicrobial resistance epidemiology. In Brazil, despite the lack of statistical information about the use of antibiotics in livestock, the β-lactams and tetracyclines are drugs frequently chosen for the treatment of infections affecting dairy herds . In thisIt is well-known that genes encoding antimicrobial resistance are often linked to mobile genetic elements. In this study, class 1 and 2 integrons were detected in 77% and 97% of the cheese samples, respectively, raising issues about AR gene mobilization. According to the literature, class 1, 2, and 3 integrons are historically associated with antimicrobial-resistance gene spread . It has Overall, the high prevalence of antibiotic resistance genes in MFC samples raises issues about its clinical resistome composition and the threat of the cheese ecosystem as a potential reservoir of resistance genes. In addition, the presence of class 1 and 2 integrons may lead to the risk of AR gene mobilization to human intestinal microbiota and putative pathogens. A larger scale investigation with metagenomic studies would clarify correlations between the bacterial community of MFC and the resistance markers detected in this study.


Neglected tropical diseases (NTDs) are an important cause of death and disability in Africa. This study estimates the monetary value of human lives lost due to NTDs in the continent in 2015.The lost output or human capital approach was used to evaluate the years of life lost due to premature deaths from NTDs among 10 high/upper-middle-income (Group 1), 17 middle-income (Group 2) and 27 low-income (Group 3) countries in Africa. The future losses were discounted to their present values at a 3% discount rate. The model was re-analysed using 5% and 10% discount rates to assess the impact on the estimated total value of human lives lost.The estimated value of 67 860 human lives lost in 2015 due to NTDs was Int$ 5 112 472 607. Out of that, 14.6% was borne by Group 1, 57.7% by Group 2 and 27.7% by Group 3 countries. The mean value of human life lost per NTD death was Int$ 231 278, Int$ 109 771 and Int$ 37 489 for Group 1, Group 2 and Group 3 countries, respectively. The estimated value of human lives lost in 2015 due to NTDs was equivalent to 0.1% of the cumulative gross domestic product of the 53 continental African countries.Even though NTDs are not a major cause of death, they impact negatively on the productivity of those affected throughout their life-course. Thus, the case for investing in NTDs control should also be influenced by the value of NTD morbidity, availability of effective donated medicines, human rights arguments, and need to achieve the NTD-related target 3.3 of the United Nations Sustainable Development Goal 3  by 2030.The online version of this article (10.1186/s40249-017-0379-y) contains supplementary material, which is available to authorized users. Please see Additional file Africa has 54 countries: 1 (1.9%) high-income, 9 (16.7%) upper-middle-income, 17 31.5%) lower-middle-income and 27 (50%) low-income countries  were from communicable, maternal, perinatal and nutritional conditions; 3 985 251 (37.9%) were from non-communicable diseases; and 1 039 282 (9.9%) were from injuries  right” (p. 1).There are six main arguments for continued (and probably increased) investment to control, eliminate and eventually eradicate NTDs. First, in line with the 1948 UN Universal Declaration of Human Rights, it is the right of every person living in NTD-endemic areas to have unconstrained access to all effective preventive and treatment interventions , 99. ThuSecond, the resources required for implementing the regional strategic plan for eliminating NTDs in the African region have been estimated at US$ 2.57 billion over a six-year period, which translates to US$ 428.33 million per year , 102. ThThird, effective medicines for treating all NTDs are available and a sufficient amount of donated drugs has been pledged by their manufacturers to meet the needs in endemic countries , 103.Fourth, even though NTDs are not a major cause of death, every year they lead to a substantive loss of disability-adjusted life years (DALYs) in Africa. For example, the WHO estimated that in 2015, the African continent lost 10.3 million DALYs due to neglected parasitic diseases and intestinal nematodes .Fifth, global and continental plans and programmes for combating NTDs exist. They provide detailed guidance on the cost-effective NTD interventions that individual countries should invest in. These plans include: the WHO global strategy 2015–2020 on water, sanitation and hygiene for accelerating and sustaining progress on neglected tropical diseases ; regionaSixth, continental and global political commitment exists for ending the morbidity and mortality from NTDs. In January 2014, the Twenty-Fourth Ordinary Session of the African Union Executive Council adopted the Continental Framework on the Control and Elimination of NTDs in Africa by 2020 and committed to using it for developing and revising national NTD plans . In 2013There are seven broad limitations of the current study. First, some costs were omitted. For example, direct costs of NTD prevention programmes, and diagnosis and treatment services were not taken into account because the current study focused on years of life lost due to premature mortality. The study also excluded the indirect costs of productive labour time lost due to morbidity, including cost of time spent seeking treatment, reduced level of performance of activities/functions of daily living, and time expended by caregivers (family and friends) and those accompanying the sick to sources of care, e.g. health facilities, private pharmaceutical shops, traditional healers. The intangible/psychological costs related to stigmatisation, discrimination, pain, anxiety and bereavement were also omitted.Second, to date there is no consensus in the published literature on the discount rate that should be applied in health sector studies. In this study, we used discount rate of 3%, which has been applied frequently in past health-related studies , 113–115Third, there is no agreement in literature about whether mortality occurring at different age groups should be weighted differently. In our study, we assumed all life to be intrinsically valuable and thus a year lost in an age group was considered to be of equal value . This isFourth, various authors have underscored a few weaknesses inherent in the use of per-capita GDP as a measure of societal economic and wellbeing: (a) per-capita GDP is an average value, which is distorted by high-income earners and corporate supernormal profits, and does not reflect distribution of income, consumption and wealth . Therefostigma, pain, bereavement, anxiety and suffering [Fifth, a number of weaknesses characterise the lost output approach or HCA: (a) It assumes that the objective of health care is getting sick people back to productive employment. However, there are other objectives, such as preventing morbidity and death so that people can enjoy life (flourish), enjoy leisure and perform non-economic societal functions, etc. (b) In its pure form, the HCA would value the lives of pensioners (elderly), full-time homemakers and non-working children at zero. In this study, we value all lives using per-capita GDP prevailing in each country. (c) The HCA does not capture intangible psychological costs of NTDs, e.G. uffering , 123.Sixth, the values of life loss estimates reported in this paper are not a guide to setting priorities in the research, prevention and treatment of NTDs , 125. ThSeventh, it is common knowledge that vital registration systems in many countries in Africa are either non-existent or very weak. That is why the deaths and burden of disease estimates reported by international organisations are often projections based on second-best approaches. Thus, it is usually not possible to verify the coverage and quality of secondary mortality data among the analysed countries.Even though NTDs are not a major cause of death, they impact negatively on the productivity of those affected throughout their life-course. Thus, the case for investing in NTD control should also be influenced by the value of NTD morbidity, availability of effective donated medicines, human rights arguments and need to achieve the NTD-related target 3.3 of the UN SDG 3  by 2030.In order for the African continent to have a fair chance of ending the epidemic of NTDs by 2030, as envisioned in SDG 3, the national governments, African Union, Regional Economic Communities and all partners need to continue fighting the war against NTDs until they are all controlled, eliminated, eradicated and eventually extinct from the continent. As long as corruption and lack of accountability remain endemic due to weak leadership –129 and Additional file 1:Multilingual abstracts in the five official working languages of the United Nations. (PDF 392 kb)Additional file 2:Number of NTD deaths in Africa in 2015. (DOCX 13 kb)Additional file 3:WHO Global Health Estimates of Frontier Years of Life Lost (not age weighted or discounted). (DOCX 12 kb)Additional file 4:Non-health GDP per capita. (DOCX 13 kb)Additional file 5:Illustration of estimation of value of human life lost in a country. (DOCX 310 kb)