Moringa oleifera) seed extracts and coagulant protein for their potential application in water treatment. Pathogenic microorganisms were obtained from Ramachandra Hospital, Chennai, India. Bacterial cell aggregation and growth kinetics studies were employed for six bacterial strains with different concentrations of seed extracts and coagulant protein. Moringa seed extract and coagulant protein showed cell aggregation against six bacterial strains, whereas seed extract alone showed growth inhibition of all six bacterial strains for up to 6 h, compared to that of control. Escherichia coli and Salmonella para typhi B did not develop resistance against coagulant protein. The results imply that Moringa oleifera is likely an efficient low-molecular bioactive peptide , using liquid chromatography–mass spectrometry and HPLC, with the corresponding sequences from Napin-1A peptide posing different degrees of antibacterial activity against different pathogenic organisms.The aim of the study was to investigate the antibacterial and anticoagulant activity of Moringa ( Shigella species, Salmonella species, Klebsiella species, Escherichia coli, Enterobacter species, and parasites such as Giardia lamblia and Entamaeba histolytica [A major problem is poor water quality, and it is estimated that waterborne diseases affect about 37.7 million Indians annually; 1.5 million children under the age of five die due to diarrhea each year [tolytica . The avaMoringa (Moringa oleifera) is one of the widely cultivated species in tropical regions of Asia, Africa, and South America, and it is used in rural areas of Africa for water treatment [M. oleifera is effective against Staphylococcus aureus, Streptococcus pyrogenes, Streptococcus mitis, Streptococcus pneumoniae, Enterococcus faecalis, Escherichia coli, and Legionella pneumophila for up to 150 min [Moringa seed extract has been extensively studied to be a potent coagulant and antimicrobial agent, there are some concerns, such as i. the availability of Moringa seeds and cost; ii. difference in coagulation property of seed extracts collected from different localities; and iii. the type and quality of surface water  to be treated. A large screening study was conducted in order to find coagulant protein from plant materials as a complement to Moringa seeds for water treatment in Southern India.As an alternative, natural materials  can be used for water treatment. reatment . Previoureatment . The see 150 min . Though Moringa seed extracts and coagulant protein against thirteen different clinical pathogens isolated from patient samples in India  seeds were purchased from local shops in Southern India. Preparations of crude extracts from Moringa seeds were performed as described earlier [ earlier . Seed coEscherichia coli 0157:H7, Salmonella paratyphi B, Salmonella typhimurium, Shigella flexneri, Salmonella paratyphi A, and Kelbsiella pneumoniae. The molecular weight of coagulation and antimicrobial peptide falls under 7.5–6 kDa  as described by Chelliah et al. , in a ba.5–6 kDa b.v/v) at 4 °C for 10 min. The purity of the coagulant protein was analyzed by Tricine gel analysis according to Laemmli [The protein content of MO crude extracts and coagulant protein was estimated by Lowry’s method . The sam Laemmli . Novex Tv/v) glutaraldehyde for 25 min. The low molecular weight 7.5 KD1a band was cut into pieces of gel, using the flat end of the pestle, and extraction powder (about 1/4 to 1/3 the volume of the gel) was added to the gel at bottom of the tube. Elution buffer (0.1–0.5% SDS or acid labile surfactants) was added to the tube (20 μL/piece of gel), and protein pellets (100 μg) were re-suspended into a buffer loading 25 μL , and incubated for 5 min at 90 °C.Protein encoded bands were set in 5% (v/v), 0.1% formic acid) 98% and solvent-B  2% and 0.1% formic acid) at a flow rate of 5 µL/min. The peptides were segregated on a capillary column  (Zorbax 300SB-C18) at a transfer rate of 290 nL/min with a gradient at 2–35% solvent-B over 30 min, then from 35–90% over 10 min, followed by 90% solvent-B for 5 min, and, finally, 5% solvent-B for 15 min. The electrosprays were smeared by 2250 eV, based on a coated silica tip. The peptides were inspected using QS 3.0 software . The ranges of proteins were identified based on 300–3000 m/z values.Sequential identification of peptides by mass spectrometry–liquid chromatography–electrospray ionization–quantitative time-of-flight tandem–mass spectrometry (LC–ESI–TOF–MS–MS) were analyzed at the National Instrumentation Center for Environmental Management of Seoul National University in Korea, according to an earlier method by Chelliah et al. . AnalyseThe protein models were spawned by a structural bioinformatics web-server dedicated to homology modeling of 3D protein structures (SWISS-MODEL), licensed under the Creative Commons Attribution-Share Alike 4.0 (CC BY-SA 4.0) International License, based on the sequence generated by liquid chromatography–mass spectrometry (LC–MS). SWISS-MODEL generates theoretical models by automated homology modeling techniques developed by the Computational Structural Biology Group at the Swiss Institute of Bioinformatics (SIB) at the Biozentrum, University of Basel, Switzerland.The crude extracts and the coagulant proteins were tested for coagulation activity, using synthetic clay solution as described earlier by Sami . Clay soClinical isolates of thirteen bacterial strains: Escherichia coli, Salmonella paratyphi B, Salmonella typhimurium, Shigella flexneri, Salmonella paratyphi A, and Klebsiella pneumoniae were obtained from the Department of Microbiology, Ramachandra University, Chennai, India. Stock culture was maintained in nutrient agar media at 4 °C.Moringa CE 0.041, 0.020, 0.012, and 0.008 mg/mL; Moringa coagulant purified protein (CP) 0.006, 0.012, 0.015, and 0.03 mg/mL. CE and CP were added to the culture suspension with an initial OD of around 0.3, and incubated at 37 °C for 4 h. The cell aggregation was observed at each hour under phase-contract microscope , The Florida State University, Tallahassee, Japan), and the images were captured using a microscopic digital camera .The clinical isolates were grown in nutrient broth (NB) and incubated at 37 °C in a shaking incubator overnight, and diluted to an initial optical density (OD) of 0.1 and 0.3 at 600 nm for growth kinetics or cell aggregation test, respectively. The concentrations of the protein that was used for cell aggregation tests were: The growth kinetics studies were conducted by adding different concentrations of protein to pre-diluted overnight cultures (0.1 OD) to a final volume of 1 mL in a sterile cuvette, and incubating at 37 °C for 4–6 h, with continuous shaking. The OD at 600 nm was measured every 60 min by spectrophotometer , and values were recorded.Moringa oleifera Seed Extract (MOS)In Silico Molecular Interaction Analysis and Docking for Antimicrobial Peptides in In silico molecular docking was performed to assess the docking ability of Napin with Lipid II, 4PLB, and LipoXc. The 3D structure of napin, Lipid II, 4PLB, and LipoXc was downloaded from Research Collaboratory for Structural Bioinformatics-Protein Data Bank (RCSB-PDB). Proteins were prepared before docking, and provided to Cluspro (v2.0) . The CluBinding energy surface properties were calculated, which in turn gives the protein interface probability and the interaction site of two proteins. For surface properties calculation, Surface Racer 5.0 was used, which calculates exact accessible surface area (ASA), molecular surface area (MSA), and cavities to the inner protein inaccessible to solvent from outside. The output includes surface area of the docked proteins models for each residue, in addition to those of individual atoms .The protein concentration was found to be higher in crude extract from MO seed extract. The coagulant protein was separated from the crude seed extracts of MO by spin column chromatography (SCC). The eluted protein fraction was concentrated, and showed similar coagulation activity as that of crude extract. The molecular mass of coagulant protein was determined by SDS-PAGE . The coaMoringa coagulant protein has been reported as a complement to the chemicals in drinking water treatment process, the present study reveals that the antimicrobial property of Moringa extract provides a good substitute to treat water, thereby reducing the risk of infection caused by water contamination. The specific < 7.5 kDa peptide band from the Tricine-gel was eluted using elution buffer (0.1–0.5% SDS or acid labile surfactants) to the tube (20 μL/piece of gel); further, the eluted bands from <7.5 kDa peptides were identified in the low molecular weight peptide profile, and plant protein profiling is displayed in Moringa (MO) crude seed extracts and coagulant protein were measured using kaolin clay solution, and clay solution alone used as a control  and coagulant purified protein (CP) showed cell aggregation of six bacterial strains. The results from the aggregation experiments are shown in a. Aluko  reportedMoringa olifera < 7.5 kDa purified protein (MOCP) are attributes of its functionality as an antimicrobial agent. MOCP is a cationic protein, and contains a net positive charge that facilitates the interaction with microbial anionic lipid membranes. MOCP also possesses an amphiphilic helix–loop–helix motif that helps it to integrate into the bacterial membranes. Owing to these functional properties, MOCPs are selectively targeted, and kill several microbes, including waterborne pathogens. It has been established that membrane fusion is the most dominant mechanism of MOCP antimicrobial activity [The key structural features of activity . The floMoringa seed  seed protein showing bacterial cell aggregation and reduction in growth rate against clinical isolates.The growth kinetic experiment was performed against six different clinical pathogens with MO coagulant. The isolates were selected based on the cell aggregation and effect against CE. All the strains tested showed reduced growth rate at the concentration of 0.02 mg/mL . In comp E. coli . The rea E. coli  reportedSWISS-MODEL: Homology Modeling of Protein StructuresMolecular dynamics were considered to be an efficient tool to form a simulation model in order to find linkage between plant-based bioactive  compounds, in addition to their respective targets, which enable interactions at specific time period. The three-dimensional quantitative modeling (3D-QSAR), to understand the relationship of specific amino acids of p Progress in 3D quantitative structural activity relationship modeling (3D-QSAR), has reopened a method to compare the plant secondary metabolites and their targeted molecular function. The three-dimensional structure of napin peptide analog 5  is the shttp:/aps.unmc.edu/AP/database/mysql.php) and AMPed (https:/amped.uri.edu/index.php), is one of the most straightforward methods to verify whether a query protein has been identified as an antimicrobial peptide, based on experimental proof.According to the in silico molecular modeling docking analysis, the first loop region of napin was formed by three different amino acids, such as isoleucine (Ile), serine (Ser), and proline (Pro), between the 18th and 20th positions, whereas the secondary loop, based on glycine (Gly), glutamine (Gln), and valine , was between the 39th to 41st positions. The secondary structure of napin peptide is the electrostatic potential of the protein with various ligands. The electrostatic potential was calculated using a Poisson–Boltzmann equation-based algorithm . The bluDeinococcus radiodurans (PDB ID: 1XBP) and Staphylococcus aureus gyrase B (PDB ID 4URM). Conversely, procruciferin that binds to dihydrofolate reductase (PDB ID 3FYV) and S. aureus is found to be better. Therefore, the molecular docking analysis shows napin as an antimicrobial agent. Trimetrexate, pyrimethamine, methotrexate, and trimethoprim, well-established drug molecules, have been reported to bind with 3FRA [D. radiodurans (1XBP). Novel antibacterial compounds with substituted amino moieties derived from pleuromutilin have been reported to exert antibacterial activity by binding to 1XBP [The results show that the proteins of both napins have a high binding affinity to selective bacterial enzymes, implying that they can inhibit bacterial activity. The lowest value represents the strongest binding force, as far as global force is concerned . It can ith 3FRA . The fin to 1XBP .Some previous simulation work inside a model bacterial membrane environment on lipid-II and antimicrobials has been completed. The identification of lipid-II by vancomycin and vancomycin was modeled by Jia and colleagues . Then caMoringa seeds were investigated against six different pathogenic microorganisms. It is evident that crude extract and coagulant protein from Moringa seed protein has antibacterial activity when measured by cell aggregation and growth inhibition studies. Furthermore, based on mass spectrometry–liquid chromatography–electrospray ionization–quantitative time-of-flight tandem–mass spectrometry (LC–ESI–TOF–MS–MS), it was found that <7.5 kDa peptide of Moringa seed extracts could effectively have antibacterial activity. Furthermore, the elucidation of the mechanisms behind such aggregation and inhibition activities would widen the understanding of such mechanisms, and thus help to identify such similar coagulant molecules that can be effectively used in the water treatment process.The effects of crude extract and coagulant protein from


Toxoplasma gondii, a zoonotic food borne parasite that can infect almost all warm-blooded animals including people, and ranks 4th among 24 most significant global foodborne parasites listed by the World Health Organization/United Nations Food and Agriculture Organization . Exposure to T. gondii has been reported in wildlife and people in the Canadian North, despite low densities of feline definitive hosts. The ecology of this host-parasite system could be affected by changing climate and landscape in boreal and sub-Arctic regions, and surveillance data are critically needed. Wolverines are an economically and culturally important species in northern Canada due to their valuable fur. Fluid obtained from diaphragmatic muscle of 127 wolverines (Gulo gulo) were tested for antibodies to T. gondii using an enzyme linked immunosorbent assay (ELISA). A seroprevalence of 62% : 53–71%) was observed. This result indicates high levels of exposure, likely either through environmental contamination with T. gondii oocysts shed by infected wild felids, or consumption of carcasses/offal of other intermediate hosts containing tissue cysts with bradyzoites in tissues. We examined factors associated with seropositivity, including age, sex, harvest location, harvest location with respect to treeline, and body condition index. Adult (≥2 years) wolverines had 5.2 times higher odds of being sero-positive than juvenile (<1 years) wolverines. The highest seroprevalence was observed in wolverines from Sahtu and South Slave regions. Proportion of sero-positive wolverines harvested above and below the tree line was not significantly different (60% vs 65%). Age was the only significant predictor of T. gondii exposure in wolverines ; further studies should target larger sample sizes. This study is an example of how fluid from diaphragmatic muscle can be used for screening for T. gondii antibodies in wolverines. The diaphragm, commonly collected for screening for another food borne parasite, Trichinella, in wildlife harvested for human consumption, can be used for screening of T. gondii exposure in wildlife. Due to their predatory and scavenging lifestyle and high trophic level, wolverines could serve as a sentinel species for T. gondii. Toxoplasma gondii exposure in wolverines from Northwest Territories.Wolverine picture supplied by Emilie Bouchard (University of Saskatchewan). •Toxoplasma gondii were reported in wolverines (Gulo gulo) in the Canadian North.Using an ELISA, antibodies to •Significantly high seroprevalence was observed in adult wolverines than juveniles.•T. gondii antibodies in wolverines.ELISA can be carried out on fluid from diaphragmatic muscle for screening •Toxoplasma exposure.Wolverines could serve as sentinel species for  Felids are the only definitive host, where sexual reproduction of the parasite results in excretion of oocysts in feces. Intermediate hosts become infected by ingesting food or water contaminated with oocysts, which ultimately develop into bradyzoites in tissues. Ingestion of tissue cysts containing bradyzoites can also lead to T. gondii infection. Infection with T. gondii may occur by sexual (via semen) or transplacental routes. In humans, the infection is often asymptomatic, but clinical manifestation can range from mild to life-threatening, particularly among immunocompromised persons and fetuses , Muskoxen (Ovibos moschatus) etc.] has been reported in some studies (T. gondii in muskox from the Northwest Territories (NWT), Canada than from Greenland (5% vs. 3%)  as well as domestic cats   as well  vs. 3%)  than Nor vs. 3%)  and Alas vs. 3%)  has been animals . Toxopla hunters . An outbtic cats .Gulo gulo) are an economically important species in northern Canada because of their valuable fur. Wolverines are classified as vulnerable throughout their circumpolar range by the IUCN; however, the highest population densities in Canada occur in the mountains of the Yukon and Northwest Territories, where they are legally trapped , enzyme linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT) have been used for detection of antibodies to T. gondii in wildlife species .The first objective of this study was to determine the seroprevalence of 22.1Rangifer tarandus), moose , muskox (Ovibos moschatus), mountain goats (Oreamnos americanus), Dall sheep , beaver (Castor canadensis), wolves (Canis lupus), foxes , lynx (Lynx Canadensis), wolverines (Gulo gulo), and bears .Bordered by Nunavut to the east and the Yukon to the west, the NWT is the most populous of the three territories in Northern Canada, with an estimated population of 44,718 . Climate2.2Wolverines were collected by trappers as part of the fur harvest during 2005–2012 in NWT . Trapper2.3g for 5 min at 4 °C. The supernatant was stored at -20 °C until used for testing.Diaphragms were placed individually in double plastic bags to avoid cross-contamination between samples and left at 4 °C for 24 h to thaw. Approximately, 1 ml of accumulated tissue fluid was removed from the bag and centrifuged at 1000 ×2.4T. gondii using a commercially available multi-species ID Screen Toxoplasmosis Indirect kit as per manufacturer's instructions . Briefly, 100 μl of tissue fluid (at 1:2 in dilution buffer) were transferred to wells of an antigen coated ELISA plate and incubated for 45 min at room temperature. Wells were then washed 3 times and 100 μl 1× multispecies peroxidase (HRP) conjugate was added. The ELISA plate was incubated for 30 min at room temperature and then washed three times. Substrate solution (100 μl) was added to each well and the plate was incubated in the dark for 15 min at room temperature. After addition of 100 μl stop solution, optical density (O.D) values at 450 nm were recorded using an ELISA automated plate reader . Tests were considered valid if the mean value of the positive control OD was >0.350 and if the ratio of the mean OD values of the positive and negative controls was >3.Tissue fluid samples were tested in duplicate for the presence of antibodies against S/P percentage was calculated using controls provided by the manufacturer as per the following formula.Samples presenting an S/P ratio ≤ 40% were considered negative. If the S/P ratio was between 40% and 50%, test outcome was considered doubtful and the sample was omitted from statistical analysis. Samples with an S/P ratio ≥ 50% were considered positive.T. gondii DNA in heart tissue using magnetic capture PCR  were evaluated by binary logistic regression. The following predictors were evaluated for inclusion in a final multivariable model using univariable binary logistic regression analysis: Age , sex , harvest location , harvest location with respect to tree line (above or below tree line) and body condition (continuous factor). A relaxed level of significance (p ≤ 0.20) was used to identify variables. Stepwise forward multivariable regression analysis was performed to include risk factors and confounders in final model. Goodness of fit of the final model was evaluated by Hosmer Lemeshow test. Variables associated at a significance level of p < 0.05 were retained in the model. To estimate the degree of the association between each predictor and T. gondii sero-status, odds ratios and respective 95% confidence intervals were calculated. All statistical analyses were performed using IBM SPSS .Seroprevalence was calculated from the proportion of sero-positive results of those tested and is presented with 95% confidence intervals. One sample had a doubtful result and was not included in statistical analysis. Possible associations between outcome variable  of the analyzed wolverines. Antibodies to T. gondii were detected most frequently in adult wolverines  followed by yearlings  and juvenile wolverines . Nearly equal proportion of females  and males  were -anti T. gondii -antibody–positive. Sero-positive wolverines were found in all five regions sampled, with the highest seroprevalence from Sahtu and South Slave regions  followed by North Slave , Inuvik  and Dehcho region  A total of 127 wolverines covering a range of sex and age were trapped for this study, 112 (88%) from below tree line and 15 from above treeline. The mean age and BCI of sampled wolverines was 2.07 years  and 10.82 , respectively; male to female ratio was 3:2 . Antibodies to 3.2p = 0.02) and location (p = 0.139) appeared to be associated with exposure to T. gondii , p = 0.002) higher in adult wolverines than in juvenile wolverines.On univariable logistic regression, age  from which sera are not routinely available. We reported high seroprevalence of T. gondii in wolverines from the NWT in comparison to seroprevalence reported in other wildlife species in NWT  ] , althougrbatus)] . The 62% Nunavut  and none Nunavut . Variati Nunavut , MAT  performed well  with a commercial ELISA kit, and the use of wildlife species-specific controls whenever possible is recommended. Further studies are required on validation of serological tests for detection of T. gondii antibodies in tissue fluid/sera of wolverines.There are many reports of prevalence of antibodies to earchers ; howeverearchers . As tissearchers . This stT. gondii antibodies are not known in wolverines. Higher prevalence in adult wolverines is in agreement with surveys conducted in other wildlife species from different geographical areas, e.g. wolves (Canis lupus) from Alaska, (Chrysocyon brachyurus) from Brazil, (Lutra lutra) from England and Wales, . No significant association between age and T. gondii exposure in wolverines from Nunavut was reported  indicates widespread transmission of the parasite across NWT, and geographical differences were not significant, likely as a result of small sample size.Our finding that significantly higher proportion of seropositive adult than juvenile wolverines is not surprising due to the longevity of antibodies  and more cumulative exposure to food or water contaminated with oocysts and/or consumption of meat having tissue cysts. However, the dynamics of anti- Alaska, ; maned w Brazil, ; Eurasiad Wales, ]. On thed Wales, , in whicreported , nor in reported . A slighreported , e.g. Eureported ; grizzlyreported ; cariboureported . The bodm Mexico  as well m Mexico . ExposurLynx canadensis) are the only wild felid present in northern Canada and they are rarely seen above the treeline; thus, we hypothesized decreased opportunity for exposure to oocysts above the treeline (i.e. some parts of Inuvik and North Slave). Among 15 wolverines harvested above the tree line (13 from Inuvik and 2 from North Slave), mean straight-line distance from tree line was 22 km (range 4–99 km). Both the wolverines [IDs: W2010–086 (71 km) and NS06–92 (99 km)] from North Slave had highest straight line distance and were positive and negative for anti-T. gondii antibodies, respectively. Wolverine (ID: TU11/02) from Inuvik harvested at highest straightline distance was 33 km above the tree line and was sero-positive. Presence of sero-positive wolverines above the tree line indicates either environmental contamination with oocysts or presence of potential intermediate hosts (caribou and muskoxen). Also, as home range of wolverines varies from 100 to 800 km2, possibility of migration of sero-positive wolverines cannot be ignored. An association between the presence of antibodies to T. gondii and proximity to the treeline has been reported in wolverines from Nunavut  (Enhydra lutris), brown hare (Lepus europaeus P.), Eurasian red squirrels (Sciurus vulgaris), Pallas' cats (Otocolobus manul)  . Fatal ts manul) . FurtherT. gondii in animals indicates prior exposed to the parasites but does not always indicate if it is infected  (Sus scrofa)   and wild scrofa)  could ac5T. gondii circulates in wolverines throughout NWT. Due to their apex position in the food chain, dietary habits, and reported high seroprevalence, wolverines can serve as useful sentinels of risks of transmission to other wildlife and people from their shared environment in the Canadian north. We report the successful application of a commercial serological test to a sample substrate (diaphragmatic fluid) commonly available in harvested wildlife, which may have food safety applications. Finally, we found, as expected, that older animals were more likely to be exposed to T. gondii, while other risk factors for seropositivity should be explored with a larger sample size.Our results indicate that


Listeria monocytogenes contaminating chicken meat in Surabaya. 60 chicken meat samples were collected from supermarkets, mobile vendors, and traditional markets in Surabaya. A selective medium is used for isolation and identification of Listeria monocytogenes by chopping 25 grams of the chicken meat and to put it into the sterilized Erlenmeyer flasks. Some methods were used for the identification procedures, such as biochemical and morphological tests, antibiotic resistance test, PCR, and sequencing; also a phylogenetic analysis was conducted by a neighbor-joining analysis using Genetix Mac ver 8.0 with hlyA genes of Listeria monocytogenes recorded in GenBank, such as Lineage I (KC808543), Lineage II , Lineage III , and Lineage IV . The result shows that the prevalence of L. monocytogenes in Surabaya contaminating the chicken meat samples from the supermarkets was 10% (2/20), from the mobile vendors was 0/20 (0%), and from the traditional markets was 5% (1/20). It was seen from the band at 456 bp fragment. Furthermore, three isolates found in Surabaya were included in the new lineages which were resistant to old-generation antibiotics such as sulfamethonazole-trimetophrim (SXT) and amoxyllin sulbactam (MAS), but they were still sensitive to new-generation antibiotics such as cefotaxime (CTX) and meropenem (MEM).The objective of this study was to identify the phylogenetic analysis and antibiotic resistance of  The most popular animal protein source in Indonesia is chicken meat. It is cheap, delicious, and easy to cook; also, various dishes can be made of it. In 2018, Indonesia produced 2,144,013 tons of broiler meat; it increased then on 2016 by only 1,905,497.28 tons and the demand grows fast every year [Listeria monocytogenes [L. monocytogenes [With the increasing demand for chicken meat, people's anxiety for food-borne diseases also arises. One of the agents causing it is ytogenes . This baytogenes . Human lytogenes , 5.Listeria genus is categorized as gram-positive bacterium that contains 6 species of Listeria: Listeria monocytogenes, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria ivanovii, and Listeria grayi. One of them known as the most pathogenic to humans is L. monocytogenes which consists of 4 lineages  [According to Janzten et al. , Listeri and IV) . These bL. monocytogenes infection. Sometimes, L. monocytogenes can also be found in processed food products. L. monocytogenes contamination after food being processed is a critical point for human health. Therefore, considerable knowledge is needed so that the prevention of L. monocytogenes bacteria transmission in the environment or in food products from livestock and its dairy products can be done appropriately. Furthermore, a fast and accurate detection technique for the presence of L. monocytogenes in food is needed so that the infected individuals can be immediately treated [L. monocytogenes are poultry and beef meat stored in refrigerator; smoked and fresh meat. Human listeriosis caused by L. monocytogenes is an intermittent disease with mild-to-severe flu symptoms as well as meningitis and septicemia manifestation. The group at risk are pregnant women and immunocompromised people. In pregnant women, it can lead to abortion, premature birth, and birth defects.Fast food such as nonpasteurized meat and milk products stored for a long time at 4°C is a potential source of  treated . Rodrigu treated  also repListeria sp. infection, especially L. monocytogenes, is less frequent than from E. Coli and Salmonella bacteria. L. monocytogenes contamination in Indonesia has not been widely reported as in developed countries [L. monocytogenes are also identified [In Indonesia, food poisoning due to ountries . In Malaentified .L. monocytogenes is susceptible to a wide range of antibiotics, although some isolates have been reported resistant to many antibiotics [L. monocytogenes such as listeriolysin O (encoded by the hlyA gene) have a role in regulating virulence and pathogenicity [Typically, ibiotics . Some ofgenicity . Even ingenicity .Listeria sp. bacteria, as well as in US and Europe. The Food and Agricultural Organization (FAO) guidelines also emphasize proactive and risk-based modern food security system. Therefore, it is necessary to identify diseases affecting the population and the presence of pathogens in food and to establish risk mitigation measures [According to Harsoyo and Andini , Indonesmeasures .L. monocytogenes contaminating chicken meat in Surabaya, Indonesia, especially for Ampicillin, amoxylin sulbactam, cefotaxime, meropenem, and sulfamethonazole-trimetophrim which are commonly used by broiler farmers as antibiotic growth promoters and therapeutic antibiotics in Indonesia [The objective of this study was to identify phylogenetic analysis and the antibiotic resistance of ndonesia .A cross-sectional prospective study was carried out in Surabaya metropolitan city. 60 raw chicken meat samples were collected from supermarkets, traditional markets, and mobile vendors as the main meat suppliers for Surabaya people, which included 20 samples from each place. Simple random sampling technique was employed for all the samples that were collected aseptically from randomly selected supermarkets, traditional markets, and mobile vendors in Surabaya. The meats were placed in sterile leakproof container in cold chain box. The samples were transported to the Tropical Disease Diagnosis Center (TDDC), Institute of Tropical Disease, Airlangga University.L. monocytogenes. 25 grams of the chicken meat was put into the sterilized Erlenmeyer flasks, and 225 ml Buffered Listeria Enrichment was added and then homogenized using a vortex mixer for 2 minutes and incubated at 30°C for 24–28 hours [L. monocytogenes on green PALCAM agar medium was surrounded by black zone [β-hemolysis. Gram staining processing showed purple or violet color in the microscopic observation [Staphylococcus aureus and Rhodococcus equi [A selective medium is used for isolation and identification of 28 hours . After iack zone . The poservation . And theervation . Triple ervation . Methyl ervation . The concus equi .The prepared pure culture was taken by sterilized cotton swab and spread over the surface of Muller Hinton Agar (MHA) and left for 5 minutes. Paper disc containing antibiotics was put on MHA with pure culture separated 25–30 nm and then incubated at 35°C for 24 hours . The antL. monocytogenes was conducted with Kit Qiagen. 5 μl suspension from the extraction was used directly as template for CR amplification of hlyA gene fragments. 20 μl PCR reaction consisted of 12.5 μl master mix, 0.5 μl distilled water, 0,5 μl forward primer (F), 0.5 μl reverse primer (R), and 5 μl DNA template. The primers used were F: 5′-GCAGTTGCAAGCGCTTGGAGTGAA-3′ and R: 5′-GCA-ACGTATCCTCCAGAGTGATCG-3′ [μl PCR product was put into 2% electrophoresis gel and then used in electrophoresis medium of 100 volts for 60 minutes. After the electrophoresis, gel was then taken for observation with UV light. DNA target fragments' visualization was shown at 456 bp with UV transilluminator [DNA extraction of GATCG-3′ . PCR conuminator .μl was added with loading buffer for 1 μl. During application, the mixture was poured into every other slot. After the electrophoresis, the result was seen with UV light with 365 nm wavelength. The DNA band was then cut with a cutter ; (3) the piece of Agarose gel mixed with a DNA slice was inserted into 2 ml microtube; (4) the DNA in the gel was then purified with reagent QIAquick PCR purification kit from Qiagen by following the instructions attached in the kit.PCR product was acquired and then purified. The steps were as follows: (1) 2% agarose gel was prepared for L Agarose (Low-Melting Agarose) using ethidium bromide 1 mg/ml; (2) DNA from the PCR result about 5 The result of the purifying was then labelled through prosequencing PCR process using hlyA gene primers (forward). In this procedure, the labelling was done using labelled dideoxynucleotide triphosphate dye, BigDye Termination Kit V.1.1 from Applied Biosystems. The condition of prosequencing PCR was 1 cycle of predenaturation at 96°C for 3 minutes, followed by 25 cycles which covered denaturation phase at 96°C for 10 seconds, annealing phase at 50°C for 5 seconds, and extension phase at 60°C for 4 minutes.μl labelled DNA was put into the new microtube and then precipitated using ethanol with addition of 1/10x NaAc 3M (pH 5.2) volume for 15 μl and 2X ethanol 100% volume for 300 μl, mixed with the vortex mixer and kept at –20°C for 30 minutes; (2) centrifugation was done at 14,000 rpm for 10 minutes; then the supernatant was discarded carefully; (3) the pellet was washed with 550 μl 70% ethanol and then centrifuged at 12,000 rpm for 5 minutes and the supernatant was discarded carefully; (4) the pellet was dried with vacuum pump for 10 minutes and resuspended with 10 μl of TE pH 8 solution; and (5) the DNA was kept at –20°C and ready for sequencing procedures.DNA product from the prosequencing PCR was then precipitated with these steps: (1) 150–200 After the DNA was purified using low-melting Agarose and labelled with hlyA primer gene, there was a sequencing procedure to get the nucleotide formation. The nucleotide sequence was determined with BigDye Terminator Cycle Sequencing kit, EDTA Hi-Di formamide, and special 0.5 ml tube from Applied Biosystems and also an automatic sequencing device, ABI Prism 310 Genetic Analyzer.L. monocytogenes isolates in Surabaya was then analyzed phylogenetically using a neighbor-joining analysis using Genetix Mac Ver 8.0 with hlyA genes from L. monocytogenes recorded in GenBank: Lineage I (KC808543), Lineage II , Lineage III , and Lineage IV .Nucleotide model profile acquired from L. monocytogenes in Surabaya contaminating the chicken meat samples from the supermarkets was 10% (2/20), from the mobile vendors was 0/20 (0%), and from the traditional markets was 5% (1/20). It was seen from the band at 456 bp fragment.In In L. monocytogenes contaminating the chicken meat collected from the supermarkets or traditional markets in Surabaya, Indonesia, have been resistant to sulfamethonazole-trimetophrim (SXT) and amoxyllin sulbactam (MAS), but they were still sensitive to cefotaxime (CTX) and meropenem (MEM).From L. monocytogenes contamination found in supermarket was about 10% and traditional markets about and mobile vendors was 0% in Bandung City, Indonesia. However, it was also in line with the previous study which reported that L. monocytogenes prevalence found in chicken meat ranged between 15 and 35%, where the bacteria grew well in chicken meat which was kept at 0–8°C temperature without having been vacuumed and in 10 days the bacteria reached 108–109 cells/gram [This study was not linear with that of Sugiri et al.  that sholls/gram , 21.Ampicillin, amoxyllin sulbactam, cefotaxime, meropenem, and sulfamethonazole-trimetophrim are the most widely used antibiotic drugs in broiler farms in Indonesia as feed additive and growth promoter; they increase production and increase the efficiency of feed use . This isThe presence of antibiotics residues in chicken meat beyond maximum permissible limits is a matter of serious concern. Heat treatments can decrease the risk of some antibiotics groups but do not guarantee the complete degradation of these antibiotic residues present in meat. Some of the developed countries, including Denmark, Norway, Sweden, and the European Union, have restricted the application of antibiotics for growth-promoting purposes. Training farmers to monitor withdrawal periods, banning the use of antibiotics as preventive treatment, and adopting the veterinary feed and drugs regulation are important parameters to mitigate the emergence of antibiotic resistance in bacteria related to broiler production .Personal hygiene of chicken meat seller in mobile vendors and traditional markets in Surabaya is very poor because none of them wear masks and gloves. Rafikah et al.  explain L. monocytogenes to grow and thrive in food even at frozen temperatures. Chen et al. [L. monocytogenes was found in the food storage refrigerators, either edible food with little or no preheating process, so it can be a harmful threat for consumer. The presence of L. monocytogenes in chicken meat in traditional markets, mobile vendors, and supermarkets in Surabaya showed that during the production process, from the cutting to storage, L. monocytogenes contamination occurred; therefore, strict monitoring and preventive procedures are needed minimize it.Although meat storage in supermarkets is better and cleaner than traditional markets in Surabaya, the condition of ready-to-eat foods (RTE) storage and display in supermarkets allows n et al.  reportedL. monocytogenes isolates from poultry meat and dairy product exhibited resistance to one or more antibiotics, indicating that animal products are potential carrier for antibiotic-resistant foodborne diseases. Listeria monocytogenes is naturally susceptible to β-lactams group antibiotic such as penicillin amoxicillin, meropenem, and ampicillin, or combined with an aminoglycoside (gentamicin) as standard therapy for human listeriosis [β-lactams against L. monocytogenes inhibit the synthesis of bacterial cell wall peptidoglycan [L. monocytogenes isolated from meat and dairy farm product.Antunes et al.  and Srinteriosis , 29. β-ldoglycan . For patdoglycan ; howeverdoglycan  and Yucedoglycan  have repL. monocytogenes may transfer resistant genes to human natural microbes through mutation, plasmid mediation (self-transferable plasmids and mobilizable plasmids), conjugative transposons, and efflux pumps [Resistant microbes strains that emerged owing to antibiotic misdose can tolerate the effect of antibiotics at inhibitory concentration levels . Consumpux pumps –38. The ux pumps .L. monocytogenes in the original environment, identification for one major virulence factor is a better choice. Among the various virulence factors, LLO (58 kDa hemolysin protein encoded by the hlyA gene) is a major virulence factor and pathogenic marker for detecting Listeria sp. The phylogenetic study of L. monocytogenes is essential to improve our understanding on how L. monocytogenes is transmitted from animals or the environment through food to humans. The results showed that all three isolates contaminating chicken meat in Surabaya formed distinctive lineages, as they were not included in 4 previously reported lineages [Vazquez-Boland et al.  proved tlineages .In the present study, the result of antibiotic resistance test showed that the three isolates from Surabaya, Indonesia, that contaminated chicken meat in supermarkets, mobile vendors, and traditional markets were resistant to some old-generation antibiotics such as sulfamethonazole-trimetophrim (SXT) and amoxyllin sulbactam (MAS), but they were still sensitive to new-generation antibiotics such as cefotaxime (CTX) and meropenem (MEM).L. monocytogenes infection on meningitis case in the experimental study; it was 10-fold more potent than ampicillin and ertapenem [Meropenem in monotherapy has been found active against intracellular rtapenem . Meropenrtapenem ; it coulrtapenem .L. monocytogenes [αby peripheral blood mononuclear cells; it is also suggested that it may modify the host immune response. Cefotaxime is a safe and effective antibiotic in treating meningitis bacterial infections, of which 80% of these microorganisms are resistant to ampicillin [Cefotaxime is a bactericidal derived from cephalosporin; it has a broad-spectrum activity against gram-positive microorganisms and exceptional activity against most gram-negative microorganisms by interfering with synthesis of their cell walls. It is widely considered to be the antibiotic of choice for the management of neonatal meningitis and sepsis caused by gram-negative bacteria including ytogenes . Cefotaxpicillin .Listeria sp. is generally still sensitive to all antibiotics [This result was different from previous studies showing that ibiotics . It cannL. monocytogenes in chicken meat in supermarkets, mobile vendors, and traditional markets in Surabaya indicates the potential listeriosis distribution. Moreover, the three isolates found were included as distinctive lineages and already resistant to old-generation antibiotics such as sulfamethonazole-trimetophrim (SXT) and amoxyllin sulbactam (MAS). Therefore, surveillance policies for potential food contamination and antibiotic sensitivity of L. monocytogenes are required, while also ensuring effective antibiotic treatment.The contamination of


MELD score in ALC patients and NAFLD fibrosis score (NFS) together with BARD score in the NAFLD group were also obtained. Results were compared between research and control groups. Then, a correlation between evaluated indices was performed in study groups. Receiver operating characteristic curves (ROCs) and area under the curve (AUC) values were applied to assess the diagnostic accuracy of measured indices. Significant increase in PDW and decrease in PCT in comparison to controls were noted in examined ALC  and NAFLD  patients. Decreased level of MPV was observed in NAFLD group . Additionally, PCT correlated with NFS (p < 0.0001). Evaluated PLT indices correlated with MELD score . They correlated with indirect and direct markers of liver fibrosis in the whole research group, too. PCT was the parameter with the greatest diagnostic accuracy in ALC patients ; in NAFLD group, it was MPV . PCT in ALC and MPV in NAFLD can be perceived as potential diagnostic markers.Platelet (PLT) indices have been proposed as potential markers in the assessment of liver fibrosis and exacerbation of liver failure. The aim of our study was to verify mean platelet volume (MPV), platelet distribution width (PDW), and plateletcrit (PCT) in alcohol-related liver cirrhosis (ALC) and nonalcoholic fatty liver disease (NAFLD) patients. One hundred forty-two patients with ALC, 92 with NAFLD, and 68 in control group were enrolled in this study. Hematological indices  and serological (indirect and direct) markers of liver fibrosis (AAR, APRI, FIB-4, GPR, PICP, PIIINP, TGF- Liver cirrhosis (LC), being a final stage of diverse chronic liver diseases (CLDs), constitutes a severe systemic condition with a broad range of life-threatening complications. Its manifestation usually remains asymptomatic until the decompensation of the disease, when a fibrotic cascade in hepatocytes cannot be reversed. Alcohol-related liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD) belong to the most common hepatic pathologies with a global burden. According to international data, ALD is responsible for more than one million deaths yearly, and NAFLD might concern nowadays even 35% humans all over the world. From a clinical point of view, an early diagnosis of fibrotic rebuilding within the liver is of crucial importance, because it improves the outcome of patients diagnosed with CLDs –4. LiverThe local ethics committee of the Medical University of Lublin approved the study (No. KE-0254/86/2016), and all patients signed an informed written consent in accordance with the Helsinki Declaration for the procedures they underwent.Three hundred and two persons were retrospectively enrolled in the study: 142 patients with ALC, 92 with NAFLD, and 68 healthy volunteers in control group. AST to ALT Ratio), APRI − ([AST/∗ULN]/PLT × [109/l]) × 100; ∗ULN - upper limit of normal (AST to PLT Ratio Index), FIB − 4 − (age × AST/PLT × [109/l]) × ALT1/2 (ang.fibrosis − 4), GPR − (GGT/ULN/PLT × [109/l]) × 100 (GGT to PLT Ratio). MELD (model of end stage liver disease) score was used in ALC patients, and NAFLD fibrosis score and BARD score were used in the NAFLD group: MELD − 3.8[∗Ln bilirubin (mg/dl)] + 11.2[Ln INR] + 9.6[Ln creatinine (mg/dl)] + 6.4. ∗Ln - natural logarithm, NAFLD fibrosis score (NFS) − (−1.675) + 0.037 × age (years) + 0.094 × BMI (kg/m2) + 1.13 × impaired fasting glucose (IFG)/diabetes  + 0.99 × AST/ALT − 0.013 × PLT (×109/l) − 0.66 × albumin (mg/dl), BARD score − AST/ALT ≥ 0.8 − 2 points, BMI ≥ 28 − 1 point; IFG/diabetes − 1 point; together 0-4 points. Among direct indices of liver fibrosis, procollagen I carboxyterminalpropeptide (PICP), procollagen III aminoterminalpropeptide (PIIINP), platelet-derived growth factor AB (PDGF-AB), transforming growth factor-α (TGF-α), and laminin were obtained. The measurement of PICP and PIIINP was performed with quantitative ELISA tests . The measurement of PDGF-AB and TGF-α was done with R&D Systems Quantikine ELISA Kits . Finally, the measurement of laminin was performed with Takara Laminin EIA Kit without sulphuric acid .Venous blood samples  were collected from the studied patients and controls . Ethylenediaminetetraacetic acid (EDTA) was used to obtain hematological parameters and citrate to assess clotting indices. Biochemical markers were measured from the remaining blood sample without anticoagulant. The blood was obtained after at least 12 hours of fasting. Hematological and biochemical parameters were obtained 4 hours after blood sample collection. The analysis of morphotic blood indices was done with automatic ADVIA 2120i analyzer, Siemens and biochemical markers with ADVIA 1800 analyzer, Siemens. Prothrombin time (PT) and its International Normalized Ratio (INR) were measured with ACL TOP 500 analyzer, Instrumentation Laboratory. The part of blood samples without an anticoagulant was centrifuged at speed 2000 × g for 10 minutes within 15 minutes from blood collection. Obtained serum was stored in 1 ml Eppendorf test tubes in the temperature of -80°Celsius until the measurement of direct markers of liver fibrosis with enzyme-linked immunosorbent assay (ELISA). Among morphotic parameters of the blood, MPV, PCT, and PDW were obtained. The assessment of indirect indices of liver fibrosis included the following: AAR − AST/ALT . The Mann–Whitney U test was used for between-group comparisons because of nonnormal distribution. Spearman correlation analyses were used to verify the correlations. All probability values were two-tailed, and a value of p less than 0.05 was considered statistically significant. ROC (receiver operating characteristic) curves and AUC (area under the curve) values were applied to assess the sensitivity and specificity of examined markers and to evaluate proposed cutoffs of measured indices in the course of ALC and NAFLD.Statistical analysis of the results was conducted using Statistica 13.0  for Windows system. The demographic data and results of laboratory tests were presented as the mean value ± SD, and Student's All ALC patients underwent esophagogastroduodenoscopy (EGD); in 126 persons, varices of the esophagus/stomach in the different stages were found. Ninety-two people were diagnosed with ascites, and 84 of them underwent paracentesis. p < 0.0001). Except for AAR, patients with NAFLD were found to have significantly higher values of all abovementioned indices in comparison to the control group (p < 0.0001). Among direct markers of liver fibrosis, median laminin value in the ALC group was significantly higher than in controls. Beside of PICP, medians of PIIINP, PDGF-AB, and TGF-α were significantly lower. Median values of TGF-α and laminin in NAFLD patients compared to controls turned out to be significantly lower.Median MPV value in ALC patients was in a normal range; median PDW value turned out to be too high and PCT to be too low. Except median of MPV which was too low, medians of PDW and PCT in the NAFLD group were in a normal range. Median MPV value in ALC patients was the only one that did not differ significantly from the control group; median of PDW was significantly higher, and PCT was lower in comparison to controls. Medians of all obtained indices in the NAFLD group differed significantly from the control group. Medians of MPV and PCT were significantly lower, and PDW was higher in comparison to controls. The analysis of AAR, APRI, FIB-4, and GPR revealed their significantly higher medians in ALC patients compared to controls  and PCT, negatively, in NAFLD patients. There was a strong negative relationship between PCT and NFS and a weaker one between PCT and laminin. Finally, PDW and PCT correlated with each other negatively in ALC and NAFLD patients.PLT indices correlated positively with indirect indices of liver fibrosis in the ALC group: MPV with APRI, FIB-4, and GPR and PDW with APRI and FIB-4. There were also negative relationships between PCT and both: APRI and FIB-4. Strong positive correlations were noted between PCT and direct markers of liver fibrosis: PDGF-AB and TGF-Diagnostic accuracy of examined PLT indices is shown in ROCs presenting examined platelet indices in ALC and NAFLD patients are presented in α in the ALC group and a negative one between PCT and laminin in NAFLD patients; to the best of our knowledge, these are the first findings reported in the course of CLDs, so far. The goal of our survey was not to compare a diagnostic accuracy of selected hematological indices between ALC and NAFLD patients. We tried to figure out whether an isolated liver steatosis might be affected by certain deviations in hematological indices. Our study evaluated the population of patients with NAFLD without the assessment of coexisting hepatitis in liver biopsy. A further direction should concern the differentiation of the patients with a simple steatosis and steatohepatitis in the context of hematological indices. It is worth highlighting the involvement of PLT markers in the course of portal hypertension (PH), as well. More and more available surveys are invented nowadays to reveal a relationship between PLTs, PLT indices in combination with various laboratory/imaging techniques, and the presence of esophageal varices (EVs) in cirrhotic patients [Platelet indices were shown to participate in the pathological appearance of cardiovascular diseases, cancer, or stroke. However, the number of studies devoted to possible linkage between hematological parameters and indirect and direct markers of liver fibrosis is not satisfactory, and the role of MPV, PDW, and PCT in liver diseases still remains unclear. Inflammatory process, splenomegaly, and secondary activation of the bone marrow are potential mechanisms responsible for the deviations in PLT indices in patients with liver disorders. Enhanced breakdown of PLTs due to hypersplenism together with increased release of interleukin-6 shortens PLT life cycle. In the consequence, the production of PLTs by the bone marrow rises, promoting the release of larger, reticulated PLTs into the bloodstream. Theoretically, aforementioned disturbances should be reflected by an increase in MPV, PCT, and PDW. According to available literature, higher level of MPV might be even perceived as a prognostic factor of advanced fibrosis in primary biliary cholangitis patients, marker of hepatocellular carcinoma, and a parameter predisposing to the transformation of simple steatosis into steatohepatitis in the course of NAFLD –20. The patients . A decrepatients . Consequpatients . Other ipatients . On the patients . We did patients . The compatients . Ahmed epatients . Anotherpatients . APRI, Apatients . We did A current survey revealed that PLT indices can be treated as potential prognostic markers in ALC and NAFLD patients, correlating significantly with MELD score, serological markers of liver fibrosis, and NFS. According to our observations, PCT in ALC and MPV in NAFLD turned out to be the most powerful markers.


Intervertebral disc degeneration (IVDD) is a major cause of low back pain. Although the mechanism of degeneration remains unclear, aging has been recognized as a key risk factor for IVDD. Most studies seeking to identify IVDD-associated molecular alterations in the context of human age-related IVDD have focused only on a limited number of proteins. Differential proteomic analysis is an ideal method for comprehensively screening altered protein profiles and identifying the potential pathways related to pathological processes such as disc degeneration.In this study, tandem mass tag (TMT) labeling was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for differential proteomic analysis of human fetal and geriatric lumbar disc nucleus pulposus (NP) tissue. Parallel reaction monitoring (PRM) and Western blotting (WB) techniques were used to identify target proteins. Bioinformatic analyses, including Gene Ontology (GO) annotation, domain annotation, pathway annotation, subcellular localization and functional enrichment analyses, were used to interpret the potential significance of the protein alterations in the mechanism of IVDD. Student’s t-tests and two-tailed Fisher’s exact tests were used for statistical analysis.Six hundred forty five proteins were significantly upregulated and 748 proteins were downregulated in the geriatric group compared with the fetal group. Twelve proteins were verified to have significant differences in abundance between geriatric and fetal NP tissue; most of these have not been previously identified as being associated with human IVDD. The potential significance of the differentially expressed proteins in age-related IVDD was analyzed from multiple perspectives, especially with regard to the association of the immunoinflammatory response with IVDD.Differential proteomic analysis was used as a comprehensive strategy for elucidating the protein alterations associated with age-related IVDD. The findings of this study will aid in the screening of new biomarkers and molecular targets for the diagnosis and therapy of IVDD. The results may also significantly enhance our understanding of the pathophysiological process and mechanism of age-related IVDD. Low back pain (LBP) severely affects human health in the modern world, placing enormous burdens on patients and society ; unfortuIVDD begins in the nucleus pulposus (NP), the core component of the disc . The anaIn this study, the differentially expressed proteins between fetal and geriatric lumbar disc NP tissues were screened and analyzed by TMT labeling combined with liquid chromatography (LC)-tandem MS (MS/MS). Parallel reaction monitoring (PRM)  and Westn = 5). The specimens were derived from fetuses whose mothers had to terminate their pregnancies due to severe disease or trauma. The fetuses were delivered to the laboratory within 4 h after induced labor. The gestational ages ranged from 31 to 35 weeks (average 33.2 ± 1.6 weeks), which is very close to full term, and the fetuses comprised 2 males and 3 females. L4/5 intervertebral disc NP samples from geriatric patients were collected during spinal fusion for chronic LBP to form the geriatric group (n = 5). Magnetic resonance imaging confirmed that the L4/5 intervertebral discs were degenerated but did not exhibit apparent protrusions or sequestrations. The degree of degeneration was Grade IV, as classified by the Pfirrmann grading system [Human fetal lumbar L4/5 intervertebral disc NP samples were collected to form the fetal group  were added. The supernatant was collected after ultrasonic processing and centrifugation. A BCA kit was used to determine the protein concentration .5 mM dithiothreitol (Sigma) was used to reduce the protein solution at 56 °C. 30 min later, at room temperature, 11 mM iodoacetamide (Sigma) was added to alkylate the solution for 15 min in the dark. Triethylammonium bicarbonate (TEAB) (100 mM) (Sigma) was used to dilute the urea concentration of the protein sample to less than 2 M. For the first digestion overnight, trypsin  was added to the sample . For the next 4 h, a second digestion was performed .The peptides were desalted with a Strata X C18 SPE column  and vacuum freeze-dried after digestion. After being dissolved in 0.5 M TEAB, the peptides were labeled with a TMT kit  according to the manufacturer’s protocol. Peptides derived from fetal NP samples were labeled with TMT tags of 126, 127 N, 127C, 128 N and 128C, and geriatric NP samples were labeled with TMT tags of 129 N, 129C, 130 N, 130C and 131. The labeled peptides were mixed, desalted and dried.The peptide mixtures were fractionated by high-pH reverse-phase HPLC using an Agilent 300Extend-C18 column. In brief, the peptides were first divided into 60 fractions with a linear gradient of acetonitrile  over 1 h. Then the peptides were combined into 18 fractions and vacuum dried. The detailed fraction sequence is shown in Additional file Tryptic peptides were dissolved in solvent A (2% acetonitrile and 0.1% formic acid) and separated via a gradient of solvent B (90% acetonitrile and 0.1% formic acid) as follows: 9 to 25% (26 min), 25 to 38% (8 min), increasing to 80% (3 min) and holding at 80% (3 min). Separation was performed in an EASY-nLC 1000 ultra-performance LC (UPLC) system (Thermo Fisher Scientific). The flow rate was 700 nL/min. After injection into a nanospray ionization (NSI) source, the peptides were analyzed by MS/MS using an Orbitrap Fusion (Thermo Fisher Scientific). The detailed parameter settings are shown in Additional file The raw MS/MS data were processed by MaxQuant search engine v.1.5.2.8) against the SwissProt human database . A contaminant database and a reverse decoy database were combined. The detailed parameter settings are shown in Additional file .5.2.8 agp-value of < 0.05 was considered to indicate significance. The URLs of the databases and software programs noted above are shown in Additional file GO annotation, domain annotation, pathway annotation and subcellular location analyses were performed. The UniProt-GOA database was used for investigating the GO-annotated proteome, via InterProScan software. The proteins were classified by their GO annotations into three categories: biological process, cellular component, and molecular function. The InterPro database was used to annotate the domains of the identified proteins. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was employed for protein pathway annotation. WoLF PSORT  was usedThe identified proteins were ranked in terms of their degree of change and classified based on their known functions individually. Target proteins were screened for further verification via PRM, a highly targeted MS method, based on their traits and the pathophysiological mechanisms of IVDD. In brief, after being dissolved in solvent A, the tryptic peptides were separated through a gradient of solvent B as follows: 7 to 25% (40 min), 25 to 35% 12 min), increasing to 80% (4 min) and holding at 80% (4 min). Separation was performed in the EASY-nLC 1000 UPLC system. The constant flow rate was 350 nL/min. The peptides were subjected to NSI source, followed by MS/MS in a Q Exactive™ Plus (Thermo Fisher Scientific) coupled online to the UPLC. Each sample was measured separately. The raw data were searched via Skyline (v.3.6) . The det2 min, inAnalysis of the results indicated that the abundance of LTA (protein accession number: P01374) was significantly increased in the geriatric group. We carried out WB to detect the expression of LTA for further verification. In brief, 25 μg of protein from each sample was separated by 12% SDS-PAGE. After transferring the proteins to PVDF membranes (Beyotime), the membranes were subjected to immunoblotting with anti-LTA antibodies  at 4 °C overnight. They were then incubated with HRP-conjugated secondary antibodies  after washing with PBST buffer. Protein expression was detected by enhanced chemiluminescence.p-value of < 0.05 and a fold change of > 1.50 or < 0.67 (1/1.50) were considered to indicate differential abundance.Statistical analysis was performed using SPSS Statistics 22.0. The average value of each identified protein with each group was calculated. Student’s t-tests and two-tailed Fisher’s exact tests were used to evaluate the significance of differences between the two groups. Only differences with a corrected The workflow for the proteomic analysis is shown in Fig. In this study, 3259 proteins were identified, among which 2691 were quantified. A quantitative geriatric/fetal ratio of > 1.50 indicated upregulation; a ratio of < 0.67 (1/1.50) indicated downregulation. Six hundred forty five proteins were upregulated, while 748 were downregulated. The MS information is shown in Additional file GO analysis links information between genes and gene products, such as proteins. In this study, the quantified differentially expressed proteins were examined based on their biological properties as identified by GO analysis. The upregulated and downregulated GO annotation classifications are shown in Additional file The subcellular localizations of differentially expressed proteins were predicted and classified by WoLF PSORT software Fig. . The uprp-values obtained in the enrichment test were converted to negative logarithm (−log10) values. A larger converted value indicates a more significant enrichment of that functional type.Enrichment analysis was performed to detect the functional trends of the altered proteins based on their annotations. The GO annotation enrichment analysis was performed on the subitems in the three level-1 categories. The main results of the GO annotation enrichment analysis are shown in Fig. Protein domains are local, compact structural units that cannot be further subdivided and are considered “evolutionary units” of protein sequences. The domain enrichment analysis results are shown in Fig. The KEGG is an information network of known molecular interactions that includes information on metabolism, genes, cellular processes, and diseases. The results showed that the upregulated proteins  were enriched primarily in the complement and coagulation cascades pathway hsa04610) and the cytokine-cytokine receptor interaction pathway (hsa04060). The downregulated proteins were enriched mainly in the ribosome pathway (hsa03010)  were enriched mainly in the ribosome pathway hsa03010) Fig. b. In add010 Fig. Eleven target proteins in human NP tissue that had not previously been clearly reported to be involved in IVDD were screened based on well-known theories and findings related to the pathophysiology of IVDD. PRM was used to further validate the protein alterations, and the main results are provided in Table p-value: 3.07E-06). Therefore, we performed WB for secondary verification. The WB results confirmed that the expression of LTA was significantly increased in the geriatric group, consistent with the quantitative proteomic analysis results  and decreased protein synthesis, thus accelerating IVDD . The enrIVDD is an age-related degenerative disease, and the degree of IVDD gradually increases with age . The numIntervertebral discs are immunologically privileged tissues . After bTen additional altered proteins were identified: HSPA8, MATN3, CLU, MFGE8, TNFAIP6, MXRA5, ECM1, SERPINE1, PDGFC and ST13. Most of these proteins have not been previously identified to be associated with IVDD in human. These proteins are involved in cell proliferation, cell migration, inflammatory responses, vascular proliferation, fibrosis, ECM degradation, autophagy, apoptosis, cell senescence, tissue remodeling, and other biological processes closely related to IVDD –37. The The lack of an absolutely healthy disc source is the most important restrictive factor in basic research on IVDD. Relatively healthy scoliotic/traumatic disc specimens obtained from young patients have often been selected as normal control specimens in previous studies , 40. HowIn conclusion, our differential proteomic analysis comprehensively profiled protein alterations that are potentially associated with age-related IVDD. The results provide a useful database for further investigations into the pathophysiological processes and molecular biological mechanisms of age-related IVDD, especially regarding the association of the immunoinflammatory response with IVDD. New potential biomarkers and molecular targets for the diagnosis and therapy of IVDD have been identified.Additional file 1. Parameter Settings. Detailed parameter settings for the HPLC fraction sequence, LC-MS/MS analysis, database search, PRM analysis, Skyline analysis and database URLs.Additional file 2. Quality control plots. The mass error and length distributions of peptides.Additional file 3. Information identified by MS.Additional file 4. GO annotation classification.Additional file 5. PRM fragment ion peak maps.


Most of the previous studies on diet and colorectal cancer were based on diets consumed during the 1990s.n = 475 581). Dietary intakes were re-measured in a large sub-sample (n = 175 402) who completed an online 24-hour dietary assessment during follow-up. Trends in risk across the baseline categories were calculated by assigning re-measured intakes to allow for measurement error and changes in intake over time.We used Cox-regression models to estimate adjusted hazard ratios for colorectal cancer by dietary factors in the UK Biobank study. Men and women aged 40–69 years at recruitment (2006–10) reported their diet on a short food-frequency questionnaire : 4–37] higher risk of colorectal cancer. Participants in the highest fifth of intake of fibre from bread and breakfast cereals had a 14% (95% CI: 2–24) lower risk of colorectal cancer. Alcohol was associated with an 8% (95% CI: 4–12) higher risk per 10 g/day higher intake. Fish, poultry, cheese, fruit, vegetables, tea and coffee were not associated with colorectal-cancer risk.Consumption of red and processed meat at an average level of 76 g/d that meets the current UK government recommendation (≤90 g/day) was associated with an increased risk of colorectal cancer. Alcohol was also associated with an increased risk of colorectal cancer, whereas fibre from bread and breakfast cereals was associated with a reduced risk. Previous studies have found an increased risk of colorectal cancer in those with high intakes of red and processed meat. Most previous studies collected information on dietary intakes during the 1990s or earlier and patterns in meat consumption have since changed.In addition, few studies have used re-measured intakes to reduce the impact of measurement error, and to quantify the amount of red and processed meat that is associated with an increased risk. Measurement error generally biases the associations towards the null value; the associations observed in previous studies that did not re-measure intakes may be underestimated.Our study found that people who were consuming red and processed meat four or more times per week, had a 20% increased risk of colorectal cancer compared with those who were consuming red and processed meat less than twice a week.,In October 2015, the International Agency for Research on Cancer (IARC), part of the World Health Organization (WHO), concluded that there was sufficient evidence to classify processed meat as carcinogenic to humans (Group 1) and red meat as probably carcinogenic to humans (Group 2A).In addition, tumour characteristicsn = 175 402) who completed at least one online 24-hour dietary assessment and these data can be used to correct for regression dilution and other forms of measurement error.UK Biobank is a prospective cohort study of half a million men and women recruited between 2006 and 2010, and provides the opportunity to investigate prominent hypotheses related to diet and colorectal cancer in a contemporary population-based cohort in the UK. We have previously shown that the dietary data collected from the UK Biobank short food-frequency touchscreen questionnaire, which generally asks about frequency of consumption of main foods and food groups, is highly reproducible.http://www.ukbiobank.ac.uk/wp-content/uploads/2011/11/UK-Biobank-Protocol.pdf). Potential participants were identified from National Health Service patient registers and invited to attend a local assessment centre. Permission for access to patient records for recruitment was approved by the National Information Governance Board for Health and Social Care in England and Wales, and the Community Health Index Advisory Group in Scotland. At the assessment centre, the participants completed a touchscreen questionnaire, which collected information on socio-demographic characteristics and diet, lifestyle, reproductive and environmental factors. Anthropometric measurements were taken using standardized procedures. The touchscreen questionnaire and other resources are shown on the UK Biobank website (http://www.ukbiobank.ac.uk/resources/). Since recruitment, participants have been followed for cancer incidence and mortality via electronic linkage with cancer and death registries. UK Biobank has ethical approval from the North West Multi-centre Research Ethics Committee. At the touchscreen, all participants gave informed consent using a signature-capture device. The UK Biobank dataset for this project included 502 619 participants. We excluded participants with prevalent cancer  at recruitment (n = 27 038), leaving 475 581 participants (219 329 men and 256 252 women) included in the study. The number of participants in each analysis of food or nutrient and colorectal-cancer risk varies slightly, due to missing information for the exposure of interest [participants who selected ‘do not know’ or ‘prefer not to answer’ for the question(s) used to define the main exposure of interest]; the total numbers of participants for each exposure of interest are included in UK Biobank is a prospective cohort study of half a million men and women aged 40–69 years recruited from across the UK between 2006 and 2010.IJE online. We also used the fibre score that we generated from the questions on fruit, vegetables, bread and breakfast cereal and described previouslyIJE online. After recruitment, participants who had provided UK Biobank with an e-mail address were, between February 2011 and June 2012, invited via e-mail once every 3–4 months for a total of four times to complete an online 24-hour dietary assessment. A large sub-sample of participants (n = 175 402) completed at least one online 24-hour dietary assessment during this period.We have previously described the dietary data collected in UK Biobank.n = 23) during follow-up (and no previous cancer diagnosis), colon cancer was assumed to be the first incident cancer in the sub-site analyses. For participants who had a diagnosis of proximal colon cancer and distal colon cancer on the same date (n = 10) during follow-up (and no previous cancer diagnosis), proximal colon cancer was assumed to be the first incident cancer in the sub-site analyses.Prevalent and incident cancer cases within the UK Biobank cohort were identified through linkage to cancer and death registries. Eligible participants contributed person-years from date of attendance at the assessment centre at recruitment until the date of first registration with cancer [except non-melanoma skin cancer (ICD-10 C44)], date of death or last date of follow-up , whichever was earliest. The endpoint for these analyses is first diagnosis of colorectal cancer (ICD-10 codes C18–C20) or primary underlying cause of death colorectal cancer, whichever was first. Proximal colon cancers were defined as those that occurred within the caecum, appendix, ascending colon, hepatic flexure, transverse colon and splenic flexure (C18.0–C18.5). Distal colon cancers were those that occurred within the descending (C18.6) and sigmoid (C18.7) colon. Overlapping (C18.8) and unspecified (C18.9) lesions of the colon were categorized as colon cancers. Cancer of the rectum included cancers occurring at the recto sigmoid junction (C19) and rectum (C20). For participants who had a diagnosis of colon cancer and rectal cancer on the same date . These re-measured intakes were used to estimate the trends in risk; this approach reduces the impact of regression dilution bias and other forms of measurement error.Cox-regression models using attained age as the underlying time variable were used to estimate hazard ratios (HRs) for incident colorectal cancer by the reported frequency of foods, food groups and fibre from the short food-frequency touchscreen questionnaire at recruitment. Minimally adjusted analyses were stratified by age , sex, geographical region (10 regions) and socio-economic status  or preferred not to answer the question about whether they had made any major changes to their diet in the past 5 years (n = 1399). We also ran the fully adjusted model, adjusting for body mass index (BMI)  instead of waist circumference. We also adjusted the result for total red and processed meat by each covariate separately, to examine which factors attenuated the minimally adjusted HR. To test for heterogeneity of the main associations by sex, we compared models with and without an interaction term for the main exposure (as the continuous trend variable) and sex and evaluated the significance using the likelihood ratio test. In an additional analysis, we further adjusted red meat for processed meat and vice versa using the fully adjusted model and fitting red meat and processed meat as continuous variables.We conducted a sensitivity analysis for colorectal cancer excluding participants who reported that they had made a major change in their diet in the past 5 years due to illness  and type of alcoholic drink . We also investigated the association between incident colorectal cancer and source of fibre . For this analysis, the ,For the main exposures, we also examined the associations for colon (ICD-10 code: C18) and rectal cancer (ICD-10 codes: C19–C20) separately and for proximal colon (ICD-10 codes: C18.0–C18.5) and distal colon (ICD-10 codes: C18.6–C18.7) separately. We also examined the associations between type of alcoholic drink and colon and rectal cancer separately because of the prior hypothesis that beer may increase the risk of rectal cancer.P-values were two-sided.Analyses were conducted in Stata version 14.1 . All IJE online, compare participant characteristics between those in the lowest and highest categories of reported consumption of fruit, vegetables, total fibre, alcohol, tea and coffee.Among 475 581 participants without prevalent cancer (excluding non-melanoma skin cancer) at recruitment, followed for an average of 5.7 years (maximum 8.6 years), 2609 cases of colorectal cancer occurred (1504 in men and 1105 in women). IJE online. In the multivariable adjusted model, the HR for incident colorectal cancer was 1.20 [95% confidence interval (CI): 1.04–1.37] for those who reported consuming an average of 76 g/day of red and processed meat compared with those who reported consuming an average of 21 g/day [and per 50-g/day increment was 1.17 (95% CI: 1.04–1.32)]. For red meat, the HR was 1.15 (0.98–1.36) for those who reported consuming an average of 54 g/day compared with those who reported consuming an average of 8 g/day [and per 50-g/day increment was 1.18 (1.00–1.39)] and, for processed meat, the HR was 1.19 (1.01–1.41) for those who reported consuming an average of 29 g/day compared with those who reported consuming an average of 5 g/day [and per 25-g/day increment was 1.19 (1.03–1.38)]. We further adjusted the red and processed-meat analyses for milk, cheese and fibre from bread and breakfast cereals and the results were unchanged [the HR for each 50-g/day increment in red and processed meat was 1.18 (1.05–1.33)] for each 50-g/day increment in red meat was 1.19 (1.00–1.41) and for each 25-g/day increment in processed meat was 1.20 (1.03–1.40).The multivariable associations between foods, food groups and fibre and colorectal-cancer risk are shown in The HR for each 25-g/day increment in fish was 0.96 (0.86–1.07). The HR for each 100-mL/day increment in milk intake was 0.93 (0.87–1.01); we further adjusted the milk analyses for red meat, processed meat and fibre from bread and breakfast cereals and the HR for each 100-mL/day increment was 0.94 (0.87–1.02). The HR for each 5-g/day increment in total fibre was 0.94 (0.86–1.03) and for each 10-g/day increment in alcohol was 1.08 (1.04–1.12). We further adjusted the alcohol analyses for red meat, processed meat, milk, cheese and fibre from bread and breakfast cereals and the HR for each 10-g/day increment was 1.06 (1.03–1.10). The intakes of poultry, cheese, fruit, vegetables, tea and coffee were not associated with colorectal cancer. When we excluded participants who reported a major change in their diet in the past 5 years due to illness, the HRs were of similar magnitude.When we adjusted for BMI instead of waist circumference, the results were very similar (data not shown). When we examined the effect of adjusting for each covariate sequentially , with a positive association seen in men [HR for each 50-g/day increment in red-meat intake = 1.39 (1.17–1.64)] and no association was seen in women [HR for each 50-g/day increment in red meat = 0.99 (0.83–1.19)]. There was also heterogeneity by sex for red meat (Pheterogeneity = 0.008) and for processed meat (Pheterogeneity = 0.022). There was also heterogeneity by sex for alcohol, with a positive association seen in men [HR for each 10-g/day increment in alcohol intake = 1.12 (1.08–1.17)] and no association seen in women . There was no heterogeneity by sex for the associations between fish, dairy milk, cheese, fruit, vegetables, fibre, tea or coffee and colorectal cancer.IJE online).When we examined oily fish and non-oily fish separately, there were no associations between oily fish [HR for each 25-g/day increment = 0.87 (0.74–1.02)] or non-oily fish [HR for each 25-g/day increment = 0.94 (0.76–1.17)] and colorectal cancer  (IJE online).When we examined the source of fibre, intake of fibre from bread and breakfast cereals was associated with a reduced risk of colorectal cancer . The HR for each 10-g/day increment of alcohol from wine was 1.05 (1.00–1.10) and for each 10-g/day increment of alcohol from spirits was 1.08 (0.90–1.31)  for colon cancer and 1.16 (1.07–1.27) for rectal cancer, but there was no heterogeneity (Pheterogeneity = 0.223) (IJE online).Ptrend = 0.020) greater risk of incident colorectal cancer. We also found that each 50-g/day increment in red-meat intake  greater risk of incident colorectal cancer. Each 10-g/day increment in alcohol intake [equivalent to about half a pint of 4.5% alcohol (by volume) beer] was associated with an 8%  greater risk of incident colorectal cancer. Intake of fibre from bread and breakfast cereals (but not fibre from vegetables or fruit) was also associated with a reduced risk.In this large contemporary prospective study of half a million men and women from the UK that included 2609 cases of incident colorectal cancer, we found that a 25-g/day increment in processed-meat intake  to fit the trends in risk per increment in mean intake. This approach reduces the impact of measurement error in the assessment of food intakes and measurement error tends to bias associations towards the null.The directions of our results for red and processed meat are in agreement with the latest systematic review from the WCRF Continuous Update Project, which summarized the evidence from available prospective studies for red and processed meat.We found an increased risk for greater alcohol intake, in line with the WCRF review.Interestingly, we found heterogeneity by sex for red and processed meat, red meat, processed meat and alcohol, with the association stronger in men and null in women. The explanation for these differences by sex is unclear and could be a chance finding. However, there is evidence that the association between other factors, such as BMIStrengths of this study are the large number of participants and the ability to adjust for known and potential confounders. We have previously reported good reproducibility of the short food-frequency touchscreen questionnaire.In conclusion, in this systematic analysis of a contemporary cohort of half a million men and women from the UK population, we found that consumption of red and processed meat and alcohol was associated with an increased risk of colorectal cancer. We also found that fibre from bread and breakfast cereals was associated with a reduced risk. The current recommendation by the UK Government Department of Health is that people should not eat more than 90 g of red and processed meat a day.This work was supported by Girdlers’ New Zealand Health Research Council Fellowship (K.E.B.), Cancer Research UK , the UK Medical Research Council  and the Wellcome Trust . The funders did not influence the design of the study, analysis or interpretation of the data, the writing of this report or the decision to publish.dyz064_Supplementary_DataClick here for additional data file.


This case series study examines the ergonomic risk of surgery using wearable sensor inertial measurement units to monitor the ergonomics of surgeons at work. Work-associated pain among surgeons has garnered less attention, despite the implications of practitioner injury and disability on the surgical workforce.3The health care workforce faces numerous occupational hazards, leading to rates of injury and absenteeism that exceed those of the construction and manufacturing sectors.4(p1011) However, research has suffered from the absence of an objective means to measure surgeons’ ergonomic stress. This study describes the ergonomic risks of surgery using wearable sensor inertial measurement units (IMUs) to monitor the ergonomics of surgeons at work and identifies risk factors for injury.Ergonomists have long recognized the potential hazards facing the surgeon; from the ergonomic standpoint, surgery has been described as “a mess.”6 The high-risk classification was based on occupational ergonomic research exposure-response analyses, which have shown clinically significant musculoskeletal disorders and/or discomfort associated with exposure to neck, torso, and arm postures in the high-risk categories. Objectively measured ergonomic risk was compared across procedure categories , adjunctive equipment , as well as surgeon characteristics . This study was approved by the Mayo Clinic institutional review board, and oral consent was obtained from all participants. The α level was set at 5%. Statistical analyses were performed using SPSS, version 26 (IBM), and statistical significance was set at a 2-tailed P < .05.Preoperatively, surgeons had 4 IMUs placed on their head, torso, and upper arms to measure deviations from neutral body position. The IMU sensors measure body-posture angles via the fusion of data from an accelerometer, magnetometer, and gyroscope contained within each sensor. After processing, ergonomic risk was assessed by calculating the percentage of time spent in a specified range of risk categories for each body segment, facilitating stratification into ergonomic risk categories  using a P < .001).Fifty-three surgeons  representing 12 surgical specialties underwent continuous IMU recording during 115 cases . OverallP < .001; without loupes, 58.1% [25.7%]; t110 = 5.11; P < .007; with headlamps, 79.9% [15.7%]; without headlamps, 62.2% [26.7%]; t110 = 2.42; P = .02). Risk factors for surgeon-reported pain included longer case length , increased years in practice , and use of loupes  and headlights . Surgeon self-reported pain was associated with ergonomic risk .The use of surgical loupes and headlamps were both independently associated with increased time in ergonomically unfavorable neck positions (mean [SD] with loupes, 85.2% [14.5%]; 4 This study demonstrates the utility of wearable technology as a means to assess surgeons’ intraoperative ergonomics and postural behavior, providing an evidence base and method for future objective research in this area. The limitations of the study include the sample size and selection bias inherent in selecting surgeon participants. Hopefully, this Research Letter encourages further investigation and induces surgeons to be mindful of their intraoperative ergonomics.The physical demands of performing surgery are real. A surgeon’s cervical spine, in particular, is at unacceptably elevated risk during many procedures. Poor ergonomics are a cause of chronic pain and disability for many surgeons, reducing career longevity and threatening the public’s access to surgical care.


Sonchus leptocephalus belongs to a core member of the woody Sonchus alliance endemic to the Macaronesian Islands in the Atlantic Ocean. The alliance has been subject to intensive investigations of adaptive radiation on oceanic islands. As an attempt to fully understand the patterns and processes of evolution in this group, we determined the complete chloroplast genome of S. leptocephalus endemic to the Canaries. It was 152,406 bp in length, comprising 84,331 bp of large single copy and 18,583 bp of small single copy separated by 24,746 bp of inverted repeats. A total of 130 genes were determined including 87 protein-coding genes, 6 ribosomal RNA, and 37 transfer RNA genes. Phylogenetic analysis confirmed its position within the woody Sonchus alliance. Sonchus leptocephalus, a core member of the woody Sonchus alliance, is a delicate shrub with small heads (15-20 florets per head) and finely dissected leaves, and grows commonly on walls, rocks and cliffs in the lower dry vegetation zones in Tenerife and Gran Canaria of the Canary archipelago  belongs to one core group having highly dissected leaves with small heads (capitula), which can be easily distinguished from other members of the alliance. Phylogenetic relationships among seven species of Taeckholmia sensu Boulos (S. arboreus ( = T. arborea), while the other includes the remaining species  approach and assembled the complete chloroplast genome of S. leptocephalus.Bramwell . It was a Boulos ; howeverAldridge . Of appru Boulos  are poorS. leptocephalus  was isolated from the leaves collected from Gran Canaria (28°08′31.7″N 15°35′30.5″W) using the DNeasy Plant Mini Kit . NGS was performed using the Illumina HiSeq platform at Macrogen Corporation . The sequence reads were assembled by de novo genomic assembler, Velvet 1.2.10 (Zerbino and Birney Lactuca sativa (DQ383816) as a reference.Total genomic DNA of S. leptocephalus were determined based on 17 representatives of the tribe Cichorieae (Asteraceae) after alignment using MAFFT v.7  was 152,406 bp in length, consisting of large single-copy region , small single-copy region , and a pair of inverted repeats . It contains 130 genes, including 87 protein-coding genes, six rRNA genes, and 37 tRNA genes. Based on whole cp genome sequences, the taxonomic position and evolutionary relationship of


Illness anxiety disorder (IAD) is defined in the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-V) as the preoccupation with having or acquiring a serious illness, in the absence of somatic symptoms . Patients with IAD experience persistent anxiety or fear of having or acquiring a serious illness, which adversely affects their daily life. They remain unsatisfied with their physician’s reassurances to the contrary, mainly because their distress is created by the anxiety of the meaning, significance, and cause of the complaints and not necessarily due to the physical presentations. IAD remains a huge burden on both the health facility and for the managing healthcare provider. In this report, we present the case of a patient with IAD, which has been managed for the past five years with recurrent visits to the physician with no resolution of signs and symptoms. Despite extensive medical workup over this period, which repeatedly showed normal test results, the patient continued to have anxiety over his ill health and complained of recurrent mild somatic symptoms. After his most recent appointment, he got very upset and booked a flight to his home country to have a second opinion to validate his illness. Physicians are encouraged to build a therapeutic alliance with patients with IAD, rather than ordering expensive or unnecessary diagnostic tests or treatment. Illness anxiety disorder (IAD), previously known as hypochondriasis, is defined in the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-V) as the preoccupation with having or acquiring a serious illness, in the absence of somatic symptoms  -2. PatieHistory of the presenting complaintThe patient was a 34-year-old male immigrant to the US from Asia who was admitted to the medical floor of a community hospital for the management of his medical problems. He had experienced a recurrent history of anxiety and mild somatic symptoms for the past five years. He reported sleeping difficulties, panic attacks, ruminative worries, muscle tension, body weakness, and chest discomfort. The patient had a prior psychiatric history and hospitalization for anxiety and depression. He had been admitted for anxiety and depression in a free-standing health facility five years ago and had been treated with medications. He had been prescribed sertraline and quetiapine but had self-discontinued because of tolerability issues. He had continued to be anxious with mild somatic symptoms. The patient denied any manic or psychotic symptoms or any neurological symptoms. The patient also denied any suicidal or homicidal ideations. He had no history of substance use or alcohol intake. He had a family history of anxiety disorder, described as an obsessive-compulsive disorder in his mother. In 2019, he had consulted a physician who had performed an extensive medical workup. All test results were unremarkable; he was reassured and his symptoms improved. During the two to three weeks prior to the current presentation, his anxiety and somatic symptoms had recurred and had subsequently worsened. He was then admitted for further investigation.ExaminationThe mental status examination revealed a young man who was neat and well-groomed. He was cooperative, calm, and made appropriate eye contact. His speech was normal. He described his mood as anxious and his affect was constricted. His thought process was normal and linear. There was no suicidal or homicidal ideation. No delusions or hallucinations were reported. He was awake, alert, and oriented to time, place, people, and events. His judgment and insight were fair.InvestigationHe underwent a 12-lead electrocardiogram, which showed a ventricular rate of 80 beats/minute (reference range: 60-100 beats/minute), atrial rate of 80 beats/minute (reference range: 60-100 beats/minute), PR interval of 148 milliseconds (reference range: 120-200 milliseconds), QRS duration of 94 milliseconds (reference range: 80-100 milliseconds), QT interval of 350 milliseconds (reference range: 360-430 milliseconds), and QTC calculation (Bazett) of 403 milliseconds (reference range: ≤440 milliseconds) with normal sinus rhythm.Other medical workups done revealed thyroid-stimulating hormone of 2.398 uIU/mL (reference range: 0.35-5.5 uIU/mL), hemoglobin A1c of 5.6% (reference range: <5.7%), and urinalysis showing no ketones or proteins. Lipid profile showed cholesterol of 182 milligrams per deciliter (reference range: <200 milligrams per deciliter), high-density lipoprotein of 39 milligrams per deciliter (reference range: <40 milligrams per deciliter), low-density lipoproteins of 103.8 milligrams per deciliter (reference range: <100 milligrams per deciliter), and a slightly elevated triglyceride of 196 milligrams per deciliter (reference range: <150 milligrams per deciliter). His basic metabolic panel was as follows: sodium of 140 millimoles per liter (reference range: 136-145 millimoles per liter), potassium of 4.1 millimoles per liter (reference range: 3.6-5.2 millimoles per liter), chloride of 104 millimoles per liter (reference range: 98-106 millimoles per liter), glucose of 93 milligrams per deciliter (reference range: 70-100 milligrams per deciliter), calcium of 9.3 milligrams per deciliter (reference range: 8.5-10.5 milligrams per deciliter), phosphorus of 4.0 milligrams per deciliter (reference range: 3.4-4.5 milligrams per deciliter), blood urea nitrogen of 9 milligrams per deciliter (reference range: 7-20 milligrams per deciliter), and creatinine of 1.0 milligrams per deciliter (reference range: 0.84-1.21 milligrams per deciliter). His complete blood count revealed a hematocrit of 48.9% (reference range: 38.3-48.6%), mean corpuscular volume of 84.7 femtoliters (reference range: 80-96 femtoliters), and red cell distribution width of 12.5% (reference range: 11.8-14.5%). All of these results were unremarkable. Also, an MRI of the brain without contrast was unremarkable. His vitamin B12 was unremarkable as well: 337 picograms per milliliter (reference range: 160-950 picograms per milliliter).Both the hospitalist and cardiologist cleared him for any medical issues. Despite this reassurance, he got very upset and booked a flight to his home country to have a second opinion to validate his illness.The cause of IAD is largely unknown. However, several risk factors have been implicated in the development of IAD. These include an underlying anxiety disorder, excess amount of time spent reviewing health-related materials , history of previous serious childhood illness or illness of the patient's caregiver, family history of anxiety, and discussions and experience that involve labeling normal body sensations as pathological ,3-5. OurIAD is a diagnosis of exclusion. It is important that a comprehensive examination and testing be conducted to exclude any organic disease before establishing a diagnosis of IAD ,4. PatiePatients with IAD are more often found in primary care centers than in mental health clinics, resulting in a delay in treatment and subsequent worsening of their psychiatric condition ,8. The pThe most important aspect in the management of IAD is for the physician to establish a longitudinal trust relationship with the patient . CommuniPsychotherapy is the first-line treatment for IAD and has been shown to reduce symptoms associated with the condition . This inPhysicians are encouraged to build a therapeutic alliance with patients with IAD as they work with them on their path back to health. This is essential to reduce the tremendous burden on the healthcare system caused by the wastage of physician resources and ordering of expensive or unnecessary investigations while caring for patients with IAD.