Lymph node status is not part of the staging system for cervical cancer, but provides important information for prognosis and treatment. We investigated whether lymph node status can be predicted with proteomic profiling.Serum samples of 60 cervical cancer patients (FIGO I/II) were obtained before primary treatment. Samples were run through a HPLC depletion column, eliminating the 14 most abundant proteins ubiquitously present in serum. Unbound fractions were concentrated with spin filters. Fractions were spotted onto CM10 and IMAC30 surfaces and analyzed with surface-enhanced laser desorption time of flight (SELDI-TOF) mass spectrometry (MS). Unsupervised peak detection and peak clustering was performed using MASDA software. Leave-one-out (LOO) validation for weighted Least Squares Support Vector Machines (LSSVM) was used for prediction of lymph node involvement. Other outcomes were histological type, lymphvascular space involvement (LVSI) and recurrent disease.LSSVM models were able to determine LN status with a LOO area under the receiver operating characteristics curve (AUC) of 0.95, based on peaks with m/z values 2,698.9, 3,953.2, and 15,254.8. Furthermore, we were able to predict LVSI (AUC 0.81), to predict recurrence (AUC 0.92), and to differentiate between squamous carcinomas and adenocarcinomas (AUC 0.88), between squamous and adenosquamous carcinomas (AUC 0.85), and between adenocarcinomas and adenosquamous carcinomas (AUC 0.94).Potential markers related with lymph node involvement were detected, and protein/peptide profiling support differentiation between various subtypes of cervical cancer. However, identification of the potential biomarkers was hampered by the technical limitations of SELDI-TOF MS. Cervical cancer is the seventh most common cancer in both sexes combined and the third most common cancer in women. An estimated 530,000 women across the world were diagnosed with cervical cancer in 2008, accounting for nearly one in ten (9%) of all cancers diagnosed in women. The developing countries carry the biggest burden of cervical cancer, with more than 450,000 cases being diagnosed in 2008 .Lymph node (LN) status is not part of the staging system of the International Federation of Gynecology and Obstetrics (FIGO) for cervical cancer , but it In patients with early stage cervical cancer, the treatment of choice is either surgical, including radical hysterectomy and pelvic LN dissection, or chemoradiation. Combining both treatments leads to a higher morbidity, such as lymph edema and urological complications . SpecifiVarious proteomics techniques have been used to detect new biomarkers in gynaecological cancers with variable degrees of success . Over thWe investigated whether we could improve the prediction of LN involvement with SELDI-TOF MS proteomic profiling.Patient and tumour characteristics are represented in sTable In total 597 different peaks were detected in our panel of 60 samples: 284 peaks on CM10 and 313 on IMAC30. In Table The AUC values obtained by LOO internal validation with the optimal median and mean number of peaks across all LOO iterations are represented in Table LVSI was more difficult to predict. Although a median number of one peak was sufficient, the LOO AUC reached only a value of 0.81. A median number of 1 peak was needed to construct a model that was able to differentiate squamous carcinomas with adenocarcinomas (AUC 0.88), 4 peaks to differentiate between squamous and adenosquamous carcinomas (AUC 0.85), 1 peak to differentiate between adenocarcinomas and adenosquamous carcinomas (AUC 0.94), and 3 peaks to predict recurrence (AUC 0.92). The most frequently selected peaks for the different comparisons are represented in Table This study investigated whether we could improve the prediction of LN involvement with proteomic profiling. We used a combination of HPLC immunodepletion with SELDI-TOF MS to detect proteins that predict LN involvement. Using LSSVM models we were able to predict lymph node involvement with an AUC of 0.95. These findings suggest that serum biomarkers could help us identifying patients with LN metastases. Other outcomes, such as histological type (AUC = 0.85–0.94), lymph vascular space involvement (AUC = 0.81) and recurrence (AUC = 0.92), were also successful, however the number of patients in some of the subgroups was limited (e.g. adenosquamous subtype (n = 2)) making the results less reliable.The majority of serum proteins are high-abundance proteins, accounting for almost 99% of the total protein mass . Most ofUpon establishing the biomarker profiles for lymph node involvement in cervical cancers, it became interesting to identify the proteins behind the differentially expressed peaks. For the 15,254.8 peak detected on the IMAC30 chip, an approach was developed using immunodepletion and SDS-PAGE gel electrophoresis as initial separation steps. Unfortunately, due to the apparently very low concentration of this protein in serum, no Coomassie Blue band could be detected at the level of 15–16 kDa. For the two lower masses  an attempt was undertaken for direct identification from the corresponding SELDI target plate. This involved the use of a special SELDI Chip target adapter  to analyze the spots with a matrix-assisted laser desorption/ionization (MALDI)-TOF/TOF MS . Indeed, the TOF/TOF MS can induce fragmentation of selected masses, which is essential for their subsequent identification. However, SELDI-TOF MS is known for having a poor mass accuracy or reproducibility . This maet al.[et al.[When looking at the literature on SELDI-TOF experiments, it can be noticed that in only a minority of papers an identification was performed. Most of the papers mention that identification and validation of the newly discovered biomarkers is ongoing. However, follow-up papers on the identified proteins, or validation studies are rarely published. For example, SELDI-TOF MS was used to differentiate cervical cancer and normal cervix tissue in the study by Wong et al.. The autl.[et al. in whichl.[et al., but alsl.[et al.,25. ThisIn conclusion, the SELDI TOF MS approach has allowed to discover a set of proteomic profiles  that could help us in the diagnosis of LN metastases. However, the proteins/peptides concerned were not identified due to technical limitations of the SELDI-TOF MS technique.Serum samples of 60 cervical cancer patients were obtained before primary surgery. All patients were diagnosed with FIGO stage I or II cervical cancer. Prior to enrolment in the study, all patients were required to give fully informed consent. The protocol was approved by the Local Ethics Committee (reference: 3M040097/ML2524).For each of the 60 serum samples, immunodepletion was performed using a high capacity 4.6 × 100 mm multiple affinity removal system (MARS) column  in an Agilent 1200 high pressure liquid chromatography (HPLC) system . This column eliminates the 14 most abundant proteins ubiquitously present in serum: albumin, alpha1-acid glycoprotein, alpha2-macroglobulin, antitrypsin, apolipoprotein AI, apolipoprotein AII, complement C3, fibrinogen, haptoglobin, IgA, IgG, IgM, transferrin, and transthyretin. In brief, the serum samples were diluted four-fold with Buffer A , filtered through a 0.22 mm spin filter and 100 μl of the diluted serum was injected into the column in 100% Buffer A at a flow rate of 0.125 mL/min. After collection of the flow-through (i.e. depleted fraction) for 5.5 min, the column was washed and the bound (high abundance) proteins were eluted with 100% Buffer B  at a flow rate of 1 mL/min for 2.5 min. The column was re-equilibrated using 100% Buffer A. Protein elution was monitored at a wavelength of 280 nm during the chromatography fractionation process. Reproducibility and efficiency of MARS column was checked by inspecting the peak position and height of the flow trough and eluted proteins as well as the overlay of the first and last chromatogram of every column using pooled serum samples as controls.The collected flow-through fraction containing the low-abundant proteins was filtered using a 1,000 Da molecular weight Microsep spin filter  for the low molecular weight analysis and a 5,000 Da molecular weight Agilent spin filter  for the high molecular weight analysis. After a first filtration step at 7500 × g for 100 and 30 min for the 1,000 and 5,000 Da spin filter, respectively, a fixed amount of the SELDI-TOF MS binding buffer (CM10 and IMAC binding buffers: see below for specifications) was added and the filtration step was repeated. This last step (adding buffer + filtration) was repeated three times to perform a buffer exchange from Buffer A to the SELDI-TOF MS binding buffers. The samples were then stored at −80°C until further use.Fractions were analysed in duplicate on CM10 (weak cation exchanger) and copper-coated IMAC30  arrays . All samples were randomly assigned to the different spots. For the CM10 arrays, spots were pre-incubated twice with CM10 binding buffer  followed by application of 100 μl of the sample in the same binding buffer. For the IMAC30 arrays, spots were pre-incubated twice with 50 μl of 0.1 M copper sulphate for 5 min at room temperature followed by a wash step with 0.1 M sodium acetate buffer pH 4 for 5 min at room temperature. Spots were then pre-incubated twice with IMAC30 binding buffer  followed by application of 100 μl of the sample in the same binding buffer. Samples were incubated for 60 min at 4°C with shaking on a MicroMix . After three additional wash steps with the same binding buffer and two final washes with water, 2 × 1 μl of 20% α-cyano-4-hydroxy cinnamic acid (CHCA) or 100% sinapinic acid (SPA)  dissolved in 1% TFA/100% ACN were applied. CHCA was predominantly used to improve ionization for lower mass peaks  and SPA for the high mass peaks . Mass analysis was performed using SELDI-TOF MS  applying automated data collection protocols for a molecular weight of <10,000 Da (low molecular weight protocol) and for 10,000–100,000 Da (high molecular weight protocol). The following settings were used: (a) sampling rate 400 MHz; (b) 2 warming shots , 10 data shots per point and (c) total number of points evaluated equal to 12.5% of the spot surface. The low and high molecular weight protocols were further optimized in pilot studies (data not shown) to reach an optimal number of peaks and signal to noise (S/N) ratio (the maximum number of peaks at S/N > 2 and S/N > 5 were counted per laser intensity). For the low molecular weight protocol a laser intensity of 2,500 nJ; focus mass 5,000 Da; and matrix attenuation 500 Da was chosen. For the low molecular weight protocol a laser intensity of 2,500 nJ; focus mass 19,000 Da; and matrix attenuation 5,000 Da was chosen. Mass accuracy was calibrated externally using the all-in-one peptide and all-in-one protein standard according to the manufacturer’s instructions (Bio-Rad) for the low and high molecular weight analysis, respectively. A quality control sample (pooled serum) was analyzed weekly to validate the output of the system. Pooled serum samples were also used as positive controls (one spot on every chip was randomly assigned) and run with the same protocol as the weekly control samples. Data analysis of the control samples was performed with Shewhart control charts plots . The fulWith the aid of MASDA software the following additional preprocessing steps were performed ,28: (1) vs positive). Secondary outcomes were histological subtype, lymph-vascular space involvement (LVSI) and recurrent disease.Weighted Least Squares Support Vector Machine (LSSVM) in combination with leave-one-out (LOO) cross-validation was used to build classifiers ,30. For LN: Lymph node; FIGO: International Federation of Gynecology and Obstetrics; SELDI: Surface-enhanced laser desorption; TOF: Time of flight; MS: Mass spectrometry; MARS: Multiple affinity removal system; HPLC: High pressure liquid chromatography; CM10: Weak cation exchanger array; IMAC30: Immobilized metal affinity capture array; CHCA: α-cyano-4-hydroxy cinnamic acid; SPA: Sinapinic acid; S/N: Signal to noise ratio; LSSVM: Least Squares Support Vector Machine; LOO: Leave-one-out; AUC: Area under the curve; LVSI: Lymph-vascular space involvement; MALDI: Matrix-assisted laser desorption/ionization; CID: Collision induced dissociation; ca.: Carcinoma; SD: Standard deviation.The authors declare that they have no competing interests.TVG, EW, and IV were responsible for planning and designing the study. TVG and IC collected samples. TVG and IC developed the protocols. TVG and EW performed the experiments. AD en BDM performed the statistical analysis. TVG wrote the manuscript. All authors read and approved the final manuscript.This work was supported, in part, by the Belgian Federation against Cancer, a non-profit organization (SCIE2004-42), and the Research Foundation – Flanders (FWO) (G.0457.05).A complete list of differentially expressed peaks with corresponding m/z and p-values.Click here for file


Low bone mineral density (BMD) is a public health issue in Saudi Arabia. This study measured the prevalence and factors associated with low BMD in Saudi women in Riyadh, Saudi Arabia.A cross sectional study using two stage cluster sampling technique was conducted in Riyadh, 2009. Thirty clusters, each comprising of 300 houses were randomly chosen and from each cluster 38–40 households were selected to identify 1150 women of >40 years. Women were invited to primary health care center for filling of self-administered questionnaire (n = 1069) comprising of sociodemographic, health, diet and physical activity variables. 1008 women underwent screening for low BMD using the quantitative ultrasound technique. 535 (53%) women with positive screening test were referred to King Khalid Hospital for Dual X-ray Energy absorptiometry (DXA).362 women underwent DXA and 212 (39.6%) were screened low BMD either at lumbar spine or femur neck. Mean age of women was 55.26(±8.84) years. Multivariate logistic analysis found; being aged 61 to 70 years , no literacy  or primary education , history of fractures  and not drinking laban(diluted yogurt)  significantly associated with low BMD.Women with low level of education, who do not drink laban and had history of fractures were at high risk of low BMD. Low bone mineral density (BMD) manifesting as fragile bones mainly comprise of osteoporosis and osteopenia . Prevaleage, physical activity, hand grip, bone mineral density, dietary calcium intake, serum 25 (OH)D levels and history of falls) to contribute significantly to osteoporosis-related fractures [National Osteoporotic foundation, USA and the National consensus group on osteoporosis for the Middle East and North Africa has indicated; menopause, low physical activity, family history of fractures, personal history of fracture as an adult, cigarette smoking, alcohol drinking, thin build, use of oral gluco-corticosteroid therapy for 3 months or more or having history of rheumatoid arthritis, thyroid disease, liver disease as major risk factors for low BMD -9. In adractures .Low BMD leads to limitations in mobility along with high medical cost. To date, the gold standard technique for diagnosing low BMD is the dual energy x-ray absorptiometry (DXA) which has high predictive validity (sensitivity and specificity) . Number This was a community based household cross-sectional study conducted in Riyadh during April-May, 2009. Two stage cluster sampling technique was followed. Riyadh is divided into five administrative regions and one major primary health care center (PHCC) was selected from each region. The catchment population of PHCC served as a cluster. It is assumed that on an average there is one woman over forty years per Saudi family, therefore 230–240 households were randomly selected from each cluster to reach a sample size of 1150. Eligible women were invited to PHCC for filling of self-administered questionnaire and screening for low BMD using the Quantitative ultrasound (QUS) technique. Flow chart representing the enrollment and number of participants is given as Figure Questionnaire was designed based on previous identified factors and other biological plausible factors leading to low BMD. Information was collected on variables related to socio-demographic characteristics , sun exposure (time spent under the sun), present and past medical illnesses, obstetric & gynecological history , personal history of fracture as an adult on trivial (minor) fall, family history of fractures, cigarette smoking, intake of dietary items; low fat milk, low fat cheese, low fat laban (diluted yogurt), meat, fish, vegetables, fruits, tea, coffee and physical activity . All questions were phrased clearly for the participants to understand. Diet and physical activity was asked pertaining to last one week. Height and weight was measured through standard procedure. Those who had difficulty in reading or writing were interviewed by a trained research assistant.Saudi females, >40 years of age and living in Riyadh city were included in the study. Women who self reported secondary causes of low BMD, such as hyperthyroidism, hyperparathyroidism or liver disease were excluded from the study.QUS was done by scanning the calcaneum of the left foot using the Achilles machine . The Achilles device is a water-based system, using fluid coupled through transmission in a temperature-controlled water bath (37 °C). The cutoff point for positive screened case was t score < −1 at the calcaneum heel bone . Two QUSA workshop was conducted to train the research assistants on the use of Achilles machine. Study protocol was approved by the Institutional Review Board (IRB) of the King Saud University.Out of 535 referred women, 362 (68%) underwent DXA. BMD was measured at the anterio-posterior spine L1-4 and Dual Femur Total . The quality control procedure for the machine was carried out every morning according to manufacturer’s protocol. All patients had the test performed, processed and finalized for reporting on the same day. The automatic region of interest (ROI) was used in all procedures to calculate the BMD at lumbar spine and femoral neck. The manual adjustments in ROI were made for the lumbar spine when necessary, for example, in cases of severe scoliosis. In addition, women were asked to give their blood samples in the laboratory (KKUH) for measurement of serum calcium and 25(OH) vitamin D levels.The stiffness index measured using the QUS was converted to corresponding t-scores with cut off of ≤ −1 as positive for LBMD. Similarly, low BMD measured using DXA was defined according to WHO cut off of t score ≤ −1 (includevs 2.28 (±0.17)mmol/L and 42.20 (±28.43) vs 38.45 (±23.54)nmol/L respectively. Further division of vitamin D into ≥75, 74–50 and <50 nmo/L category found 30% vs 18%, 37% vs 20% and 136% vs 116% (p < 0.22) women in low BMD and normal BMD groups respectively.The socio demographic characteristic of women undergoing screening using the QUS are presented in Table low fat laban, green tea and Arabic coffee during last one week as significantly associated with low BMD  out of 362. Univariate analysis found age 60–70 years, education, personal history of fractures as an adult, husband occupation as an administrator and intake of dietary items; Out of 535 women, 362 visited the hospital for DXA and 173 were missing. Women who did not get their DXA done reported transportation and male members’ unavailability as main factor for their absence. We compared the sociodemographic, health, dietary and physical activity data between those who got DXA done and not done. We found no significant difference (p > 0.05) among any of the variables except for husbands occupation (p < 0.01) between the two groups (results not shown).In the final multivariate model, age 61 to 70 years (OR = 2.75 95%CI 1.32-1.48), no literacy , primary level of education (OR = 4.12 95%CI 2.05-8.29), history of personal fracture as an adult on trivial falls (OR = 2.20 95%CI 1.03-4.69) and not using laban in the diet (OR = 2.81 95%CI 1.47-5.37) were significantly associated with low BMD  . Milk aned milk, ,24 inclued milk, . Quasi-eed milk, ,27. Labaed milk, . Laban ied milk, , therefoed milk, . Future DXA. Recent studies found QUS to be a reliable screening instrument for low BMD/fractures and recommended its use in primary care settings [This study is novel as it has used QUS as baseline screening test to identify “at risk” women before referring them for a much costlier and radiation exposed investigation, settings ,31,32. UVitamin D deficiency is supposed to identify at risk population for low BMD  but our Study had some limitations. 173 32%) women did not undergo DXA testing. The main reason identified was limited mobility due to cultural factors. Although comparison of the characteristics of those tested and not tested with DXA revealed no significant difference except for husbands occupation, however, the groups may have differed on some characteristics that we did not ascertain. Several studies have taken cutoff point for QUS as t < −1; however, Sedat et al. recommended a different cut off [% women dIn conclusion, low BMD poses a high public health burden in Saudi Arabia which can be identified through screening and risk factors assessment at primary care level. Health education, regular screening and use of dietary products like laban (diluted yogurt) can help prevent low BMD in women.The authors declare that they have no competing interests.Dr. JAQ was involved in conceptualization and write of proposal, conduction of the study and write up of the manuscript. Drs. AK, ST, SS, AAS and FH were involved in data collection, data cleaning, analysis and write up phase. Dr SO facilitated the conduction of study in King Khalid Hospital and was involved in the manuscript write up. Drs MF and RS monitored and facilitated the data collection process and reviewed the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/15/5/prepub


Salmonella enterica serovar Choleraesuis, a host-adapted pathogen of swine, usually causes septicemia. Lactic acid bacteria (LAB) strains have been widely studied in recent years for their probiotic properties. In this study, a mouse infection model first screened for potential agents against infection, then a pig infection model evaluated effects of LAB strains and herbal plants against infection. Scutellariae radix (SR) and Gardeniae fructus (GF) showed abilities to reduce bacteria shedding and suppressing serum level of TNF-α induced by infection in swine. Bioactivities of SR and GF were enhanced by combining with LAB strains, which alone could speed up the bacteria elimination time in feces and boost immunity of infected pigs. Baicalein and genipin exhibited stronger cytotoxicity than baicalin and geniposide did, as well as prevent Salmonella from invading macrophages. Our study suggests LAB strains as exhibiting multiple functions: preventing infection, enhancing immunity to prepare host defenses against further infection, and adjusting intestinal microbes' enzymatic activity in order to convert herbal compounds to active compounds. The SR/GF-LAB strain mixture holds potential infection-prevention agents supplied as feed additives. Salmonella infection of humans and animals is still a severe health problem worldwide [S. enterica a key gastrointestinal pathogen responsible for food-borne diseases. In particular, S. enterica serovar Choleraesuis, a host-adapted pathogen of swine, sometimes causes necrotizing enterocolitis as well as septicemia characterized by hepatitis, pneumonia, and cerebral vasculitis [S. Choleraesuis also produces systemic infection in humans. It is a major serovar for salmonellosis transmitted from animals to humans [Salmonella infection. As with other bacterial pathogens, antibiotic resistance to Salmonella is a growing problem for eradication. Finding safe and efficient prevention to alleviate the need or even replace antibiotics for cleaning infection looms is a vital task.orldwide , S. entesculitis . S. Cholo humans . Yet antScutellariae radix (SR), Gardeniae fructus (GF), Houttuyniae herba, Taraxaci herba, Glycyrrhizae radix, Puerariae radix, and Rhizoma dioscoreae. LAB strains and potential herbal materials as pig feed supplements allowed the study of their preventive effects against Salmonella infection, thus offering an alternative way to improve immunity and digestive abilities of animals by using combinations of LAB strains and herbs as feed additives.Lactic acid bacteria (LAB) strains have been widely studied in recent years for their probiotic properties and are used as feed supplements , 5. A nuScutellariae radix (SR), Gardeniae fructus (GF), Taraxaci herba, Houttuyniae herba, Glycyrrhizae radix, Puerariae radix, and Rhizoma dioscoreae. Plant materials were finely powdered and extracted using distilled water at 100°C for 1 hr . With insoluble matter removed by filtration, filtrate was concentrated in vacuum and lyophilized to yield residue. Percentages of indicator compounds in each original herbal plant were confirmed by high-performance liquid chromatogram (HPLC) by Ko Da Pharmaceutical Co., Ltd. and listed in in vitro tests, baicalin and baicalein were purchased from Sigma-Aldrich Co. LLC ; geniposide and genipin were from Wako Pure Chemical Industries, Ltd.  and Challenge Bioproducts Co., Ltd. , respectively.All herbal materials were purchased from Ko Da Pharmaceutical Co., Ltd. at Taoyuan, Taiwan: S. Choleraesuis (ATCC 13312) reference strain was obtained from the American Type Culture Collection (ATCC). Strain SC37 was collected from a carrier pig to be used as inocula for infecting animals; is serotype identification applied as follows. Antiserum to detect O and H antigens was purchased from S&A Reagents Lab Ltd.  and from Denka Seiken Co., Ltd. , respectively. Isolate was analyzed according to Kaufmann-White scheme and serotyping protocols developed by Centers for Disease Control and Prevention  [600 nm of 0.8. After centrifugation at 8,000 ×g, bacterial pellet was resuspended in phosphate-buffered saline (PBS) and adjusted to final concentration of 1010 colony forming units (CFUs)/mL in PBS. Two lactic acid bacteria (LAB) strains, Lactobacillus acidophilus strain LAP5 [L. reuteri Pg4 [9 CFU/g. GA, USA) . Bacteriain LAP5  and L. rteri Pg4 , were isμL of bacterial suspension adjusted previously at concentration 106 CFU/mL was added to 100 μL of the herbal extracts or compounds, then an additional tube containing broth only used as a negative control was added. All test tubes and control were incubated at 37°C for 18–24 hr, after which the tube containing the lowest concentration of extract showing no visible growth was considered as MIC. Further, concentrations showing total inhibition of visual bacteria growth were identified. 50 μL of each culture broth transferred onto agar plates and incubated for 18–24 hr at 37°C. Complete absence of growth of bacterial colonies on the agar surface in the lowest concentration of sample was defined as minimum bactericidal concentration (MBC). Each assay in this experiment was replicated three times.Minimum inhibitory concentration (MIC) of extract or compound was tested by twofold serial dilution. Test extracts were first dissolved in PBS, then incorporated into Muller-Hinton broth to obtain concentration of 100 mg/mL and serially diluted to attain 50, 25, 12.5, 6.25, or 3.125 mg/mL. Test compounds were dissolved in dimethyl sulfoxide (DMSO) to obtain concentration of 20 mM and serially diluted to achieve 10, 5, 2.5, 1.25, and 0.625 mM. A volume of 100 10 CFU of strain SC37 per mouse. Clinical signs were monitored, body weight measured, and feces of each individual were collected daily for four days after challenge. Sera, intestines, livers, and spleens were collected from all groups on day 4 after challenge; all mice were humanely euthanized.Male BALB/c mice weighed about 20–22 g, obtained from the National Laboratory for Animal Breeding and Research Center, were maintained in specific pathogen-free (SPF) condition and used at 8–10 weeks of age. They were housed in an air-conditioned room at 25 ± 2°C with relative humidity 40–70% and 12-hour light/dark cycle and were fed with tap water and standard laboratory rodent diet. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of China Medical University ; experiments were performed according to the institutional ethical rules and laws. Mice were fed basal diet for a week before being used in the study. On treatment day 0, mice were randomly allocated into nine groups of ten mice. Herbal extract was dissolved in water and given orally at a dose of 5 mg/mice for seven days via stomach tube. Control and water groups were given the basal diet and were fed by water instead. After the last dose, herbal treated and watered mice were challenged orally with 106 CFU per gram. The dosage of herbs and Mix-LAB supplied in diet was based on previous studies [10 CFU strain SC37 per pig. Clinical signs were monitored for six days after challenge. During this time, pigs were fed with experimental diets. Body weight, body temperature, and feces of each pig were measured daily for six days. Sera and intestines were collected from all pigs on the sixth day after challenge; pigs were then humanely euthanized by electrocution.In all, 35 crossbreds weaned pigs weighing about 8–10 kg were obtained from the Animal Technology Institute Taiwan, maintained in SPF condition, and used at 28 days of age. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of National Pingtung University of Science and Technology ; experiments were performed according to the institutional ethical rules and laws. SPF pigs were housed in an environmentally controlled isolation facility with filtered air and supplied with sterilized food and water. They were fed basal diet for three days before study. On treatment day 0, pigs were randomly allocated into seven of five groups . Herbs were mixed in a diet with concentration 0.1%, and was Mix-LAB added to final amount of 10 studies , 17. BotSalmonella per gram in feces were determined. Aliquots (100 μL) of fecal suspensions were serially diluted in PBS, plated on duplicate Salmonella-Shigella (SS) agar plates , and incubated overnight at 37°C. Typical colonies were counted for plates containing between 30 and 300 colonies. S. Choleraesuis was confirmed by PCR assay, as described in a previous report [Fecal samples were collected at 1–6 days in pig trials after administering strain SC37, and numbers of s report .After the animals were sacrificed, their livers, spleens, intestines were aseptically removed, and blood was collected to determine bacteria load. Organs were homogenized in 1.0 mL of sterile saline with the aid of a tissue homogenizer maintained at 4°C, aliquots of homogenate processed for CFU counts.α (TNF-α), interferon-γ (IFN-γ), and interleukin-8 (IL-8) in peripheral blood were detected with an ELISA kit  according to instructions of manuals.Tumor necrosis factor-5 cells/well for 24 hr. Indicated concentrations of baicalin, baicalein, geniposide, or genipin were then added to cells, while only adding 0.1% DMSO in the control group, and grown at 37°C for 24 hr. To ascertain cell viability, trypan blue exclusion protocol was used. Briefly, about 10 μL of cell suspension in PBS (pH 7.4) was mixed with 10 μL of trypan blue; stained (dead cells) and unstained (live cells) were counted by a hemocytometer. Viability represented the percentage of cell survival after treatment.The murine monocyte/macrophage cell line RAW 264.7 (ATCC TIB-71) was obtained from ATCC; RAW264.7 cells were seeded onto 24-well plates at a density of 1 × 10μg/mL of gentamicin for 1.5 hr. Cell culture supernatants were removed gently; cells were washed with PBS, and osmotic lysis was performed to quantify total bacteria. For this purpose, sterile water was added to infected cells after washing, and cell lysates were resuspended with PBS, and bacterial numbers were derived by plating serial dilution on SS agar plates. Invasion activity was calculated as triplicate mean, and results were expressed as percent relative invasion of RAW264.7 cells were compared with infection group.RAW264.7 cells were treated with baicalin, baicalein, geniposide, or genipin for 0.5 hr before infection in antibiotic-free Dulbecco's Modified Eagle Medium (DMEM) supplemented by 10% fetal bovine serum (FBS), then cocultured in PBS-resuspended SC37 at multiples of infection (MOI) 10. Designated concentration of each indicator compound was then added to cells, while only adding 0.1% DMSO in the infection group. Cell-associated bacteria were quantified 0.5 hr after infection, then treated by 100 t-test, using SPSS software .Differences between treated and control groups in mean values were rated by Student's S. Choleraesuis infection, seven herbal plants were tested. S. Choleraesuis, ATCC 13312, and SC37; only SR displayed antibacterial activity against Salmonella strains tested. MBCs against reference strain and SC37 were 6.25 and 25 mg/mL, respectively, but bactericidal activities of other herbal extracts exceeded 50 mg/mL. SR showed moderate antibacterial activities against S. Choleraesuis, and the reference strain was more susceptible to SR than to clinical isolate.In order to screen for the herbs with bactericidal property against Salmonella shedding in feces caused by challenge (data not shown); only SR and GF could eliminate S. Choleraesuis in organs of infected mice and significantly decrease Salmonella load in spleens. GF treatment reduced amount of Salmonella in both spleen and intestine . Yet no herbal plant treatment could reduce level of ntestine . No bactin serum , but notBased on data in mice model, SR and GF had the best bioactivities in eliminating bacteria and suppressing inflammation induced by infection. Bioactivities of SR and GF were further evaluated by pig infection model; probiotics as feed supplements are commonly used in livestock diets. This study applied two LAB strains (Mix-LAB) for investigating their probiotic property along with herbs.Salmonella infection. We were not intent on producing server sickness in pigs. The infection did not affect the body weight or cause fever, but only induced diarrhea and mild inflammatory responses in pigs. Mix-LAB, SR, and GF supplements could all reduce the levels of diarrhea caused by infection. Furthermore, SR, SR + Mix-LAB, GF, and GF + Mix-LAB groups could also dramatically reduce the Salmonella shedding in feces (Salmonella fecal shedding in the group fed with SR + Mix-LAB and GF + Mix-LAB was even lower by 68% and 80% than that in the groups fed with SR and GF alone. SR, SR + Mix-LAB, GF, and GF + Mix-LAB could all decrease SC37-induced IL-8 levels in serum. And SR + Mix-LAB and GF + Mix-LAB could reduce both IL-8 and TNF-α expressions in serum, closer to the levels of control groups. Duration of Salmonella fecal shedding in pigs fed with Mix-LAB was shorter than that in the infection group. Still, as of day 6 after challenge, bacteria load in intestines of the Mix-LAB group tallied the lowest and was reduced by 97% compared with the infection group. Mix-LAB supplement elevated immune abilities in swine. Serum levels of TNF-α and IL-8 were still high in Mix-LAB group, even with most Salmonella shed by that time (After a 10-day supplement, pigs were challenged with the clinical isolate SC37. This study was designed to show the interaction and bioactivity of herbs and probiotics as feed supplements in preventing in feces  and intein feces . The amohat time .S. Choleraesuis strains were both 5 mM, whereas MBCs of genipin against test strains were both 10 mM; those of geniposide against S. Choleraesuis strains were above 10 mM (S. Choleraesuis activity than geniposide and genipin. Furthermore, RAW264.7 viability was inhibited by baicalin with IC50 of 475 μM and was unaffected by geniposide at concentrations less than 3 mM. By contrast, IC50 of baicalein and genipin dropped as low as 155 μM and 1.42 μM, respectively. Their aglycones exhibited stronger cytotoxicity.Based on the data above, SR and GF were bioactive, eliminating bacteria in intestines and suppressing inflammation induced by infection. Since both have shown aglycones as metabolites wrought by intestinal microbes , 20, we ve 10 mM . BaicaliSalmonella invasion of macrophages, even at concentration of 200 μM. However, 100 μM of baicalein and genipin could prevent 52% and 44% of bacteria invading cells, respectively (S. Choleraesuis invasion.Abilities of these compounds to prevent SC37 from invading macrophages were scrutinized. Neither baicalin nor geniposide could inhibit ectively , this deLactobacillus spp., Bifidobacterium spp., Enterococcus spp., and many other intestinal microbes are proposed as probiotics. LAB strains show beneficial effects in health promotion for a host [Salmonella colonization in mice and chickens [r a host . Some LAr a host –24. Mechr a host  and protr a host . Severalchickens , 23, 24.chickens , 26.S. Choleraesuis infection in swine. The mouse infection model screened for infection preventive agents; pig infection model evaluated effects of target herbs and LAB strains against infection. Seven herbal plants served to evaluate their efficacy in preventing Salmonella infection. They grow locally in Taiwan and hold antibacterial potential, seeing wide use in folk medicine. SR alone showed antibacterial activity in vitro against S. Choleraesuis. Mice are not common models to establish such infection. However, it is a relatively accurate way to evaluate bioactivity of herbs in vivo in order to screen for preventive agents. We managed to monitor several useful indicators affected by challenge, for example, Salmonella shedding in feces, Salmonella load in organs, and levels of serum IFN-γ. In mouse infection model, both SR and GF could eliminate S. Choleraesuis in spleens. GF could also decrease the bacteria load in intestines and infection-induced IFN-γ expression in serum. However, SR did not suppress the load of S. Choleraesuis in intestines; therefore, the serum levels of IFN-γ were not suppressed in serum significantly. Biotransformation in mice's intestines might not be active enough to process SR into active compounds, therefore, affecting the elimination of bacteria from intestines.This study aimed for a combination of LAB and herbal plants as feed additives to prevent in vitro [Salmonella activity.SR sees wide use in traditional Chinese medicine , 27, 28,in vitro , 37. GF in vitro . GF has in vitro , as wellin vitro , anti-inin vitro , 42, andin vitro . HoweverS. Choleraesuis already eliminated from the body. Results concurred with previous findings [SR and GF were proved to be able to reduce bacteria shedding and decrease inflammation induced by challenge in swine in a significant manner. Likewise, bioactivities of SR and GF were enhanced combining with Mix-LAB. In our pig infection experiments, Mix-LAB did boost immunity of infected pigs, even with findings , 7 and ifindings , 22, 24.in vitro. The aglycones revealed significant effects in bactericidal activity and cytotoxicity. Prior study demonstrated similar phenomena. It was reported that antimetastatic or anticarcinogenic activity of ginsenoside Rb1 could not show until biotransformation by intestinal bacteria [Salmonella fecal shedding and inflammation in the group fed with probiotics and herbs was even lower than those fed with herbs alone. In our study, LAB strains thus showed multiple functions for preventing infection, enhancing immunity to prepare host defense for infection, and adjusting enzymatic activity of intestinal microbes in order to convert bioactivity of compounds. Since SR and GF herbs grow in Taiwan, this study would also improve economic value of both plants as infection preventives for farm animals.For further identification of active compounds in herbs, bioactivities of indicator compounds baicalin and geniposide and their aglycones baicalein and genipin were studied bacteria –12. Baicbacteria . We atta


Acute respiratory tract infections are an important public health problem. Sensitive and rapid diagnostic techniques have been developed and are used in daily clinical practice. Here we evaluate the clinical relevance of detecting 20 common respiratory pathogens by molecular methods in a general pediatric clinic.Mycoplasma pneumoniae, using real-time polymerase chain reaction.Nasopharynx samples of children < 18 years of age with respiratory symptoms referred to a general pediatric clinic were tested for the presence of 19 viruses and Mycoplasma pneumoniae was detected, were younger  and more often hospitalized  than patients in whom no respiratory pathogen was detected. Also they were more likely to present with feeding problems, dyspnea, rhinorrhea and wheezing  than patients without a respiratory pathogen.Of 177 patients included in this retrospective study, 73% were positive for at least one virus. Respiratory syncytial virus (36.6%) and human rhinovirus (24%) were most frequently detected. Patients in whom a respiratory virus or In the majority of cases, clinicians did not change their antibiotic management after detecting a viral respiratory pathogen. No difference in mean Ct value was found between patients with one respiratory pathogen and those with >1 respiratory pathogen .Routine testing of common respiratory pathogens could lead to a better understanding of their role in disease in children with respiratory symptoms. Acute respiratory tract infections (ARTIs) are a significant cause of morbidity and account for a major percentage of mortality in early childhood worldwide. Though ARTIs can be caused by bacteria and fungi, viral infections seem to be responsible for most infections. Influenza A and B virus , respiratory syncytial virus (RSV), human parainfluenza viruses (HPIVs) and adenovirus (HAdV) are well recognized respiratory pathogens that account for 35% to 87% of ARTIs in children and cannot be distinguished on the basis of clinical presentation and symptoms -5.Mycoplasma pneumoniae, in children attending a general pediatric clinic and the influence of their detection in clinical decision making.Over the past two decades, molecular diagnostic techniques for the detection of respiratory pathogens have been developed, providing rapid results with an increased sensitivity ,4,6. In M. pneumoniae detection were included. Based on the Dutch health system, the majority of pediatric patients who visit the hospital are referred by their family doctor. Only a minority of patients who visit the emergency department are not referred.The study was conducted in St. Elisabeth Hospital, one of the largest non-university teaching hospitals in The Netherlands. All patients < 18 years of age who presented with signs and symptoms of upper or lower respiratory tract infection (RTI)  at the hospital emergency department, the pediatric outpatient clinic or admitted to the pediatric ward between January 2010 through December 2010 and in whom a nasopharyx sample was collected for respiratory virus and Relevant demographic and clinical data were extracted from hospital charts using standardized case record forms. Data collected included age, gender, underlying medical conditions, use of antibiotics prior to admission, use of antibiotics at admission, signs and symptoms at presentation, findings on physical examination, oxygen requirement, rate and duration of hospitalization, intensive care unit admission, complications, bacterial cultures and definitive clinical diagnosis.Both the data collection and analyses were conducted in a retrospective fashion from coded hospital medical records, for which according to the Dutch Medical Research Involving Human Subjects Act  no medical ethics review was required. This was confirmed by our institutional Ethics Review Board when we sought approval. The need for subject or parental informed consent was thus waived. To guarantee the privacy of study patients we coded all tested isolates according to the requirements of the National Privacy Regulations in The Netherlands.M. pneumoniae. (RT)-qPCR procedures were performed as described previously [All nasopharynx samples were tested with reverse transcriptase (RT) real-time PCR [(RT)-qPCR] using parallel qPCR assays for the following combinations of respiratory pathogens: 1) FLUAV/FLUBV, 2) RSV, 3) HPIV-(1–4), 4) HMPV/ human rhinovirus (HRV), 5) HCoV- HKU/NL63/OC43/229E, 6) HAdV, 7) HBoV, 8) KIPyV/WUPyV, 9) human enterovirus (HEV)/human parechovirus (HPeV), 10) eviously ,15.All samples had been spiked before extraction with internal control viruses, Phocine Herpes Virus , and Phocine Distemper Virus  to monitor efficient extraction and amplification. For each target a positive and negative control was used. (RT)-qPCR results were expressed in cycle threshold (Ct) values. Ct values are inversely correlated with viral load, i.e., low Ct values indicate high viral loads.Comparison among groups was performed using a chi-square test for categorical variables and for continuous variables a t-test or Mann–Whitney test was used with a significance level of p < 0.05. Ct values of each virus were compared between the various groups using a t-test. Whenever a given virus was not detected by (RT)-qPCR, the corresponding viral load was assigned a value of zero. Statistical analyses were conducted using PASW Statistics 18 .A total of 177 nasopharynx specimens of 177 patients were collected and analyzed. Ninety-eight (55.4%) patients were males and 79 (44.6%) were females. The median age was 8 months (range 3 days to 16 years). Thirty-five (19.8%) children had an underlying medical condition. Demographic characteristics of the patients are presented in Table M. pneumoniae was detected in 130 (73.4%) respiratory samples. Significantly more viruses were found in male  than in female  patients (p = 0.042). Except for FLUAV and HPIV-2, all other respiratory viruses and M. pneumoniae were detected. Most commonly detected were RSV in 64 (36.6%) and HRV in 42 (24.1%) samples. The detection rates of the other viruses were HEV in 8.5%, WUPyV in 5.6%, HPIV-1 in 5.1%, HAdV and HBoV in 4.5%, HCoV-OC43 in 4.0%, HPIV-3 in 2.8%, HPeV 2.3%, HCoV-HKU1, HMPV and M. pneumoniae in 1.7%, FLUBV in 1.1% of the samples, respectively. The detection rates for HCoV-NL63, HCoV-229E, KIPyV and HPIV-4 were <1%.At least one respiratory virus or M. pneumoniae were sporadically detected in this study.There was variability in seasonal detection of respiratory pathogens Figure . RSV, thM. pneumoniae were detected. Forty-seven (26.6%) respiratory samples had multiple (> 1) respiratory viruses; 38 (21.5%) samples contained 2 respiratory viruses, 8 (4.5%) samples contained 3 viruses, 1 sample contained 2 viruses and M. pneumoniae, and 1 respiratory sample contained 4 respiratory viruses. RSV and HRV were found in 27 (57.4%) and 28 (59.6%) of the 47 respiratory samples with multiple respiratory pathogens viruses, respectively and RSV and HRV were 12 (25.5%) times co-detected with each other. HCoV-HKU1, HCoV-229E, FLUBV and KIPyV were always co-detected.In total, 184 viruses and 3 Overall, Ct values between patients in whom only one respiratory pathogen was detected, and patients with > 1 pathogen did not differ significantly . The mean Ct values of the various respiratory pathogens are shown in Figure Patients in whom respiratory pathogens were detected, had a higher rate of hospitalization  in contrast to patients without respiratory pathogens, as presented in Table Patients with one respiratory pathogen detected had a mean hospital stay of 5.45 days in contrast to patients with more than one respiratory pathogen who had a mean hospital stay of 3.03 days (p = 0.024). There was no difference in pre-existent comorbidity, premature delivery and abnormalities on chest radiograph observed between these two groups. In addition, patients with more than 1 respiratory pathogen were more likely to present with the following symptoms when compared to those with 1 respiratory pathogen: vomiting, feeding problems and diarrhea . No difference in oxygen supply or admission to an intensive care unit was found.M. pneumoniae between patients ≤ 3 months and > 3 months of age . However, multiple respiratory pathogens were more often detected in patients ≤ 3 months .In a subgroup analysis we compared clinical and microbiological data of patients ≤ 3 months and those older. There were 68 patients ≤ 3 months of age (38.4%) and 109 (61.6%) > 3 months of age. Patients ≤ 3 months of age had a significantly higher rate  and duration  of hospitalization and more frequently rhinorrhea at presentation , than patients > 3 months of age. Patients > 3 months of age presented more often with fever  and had a higher level of C-reactive protein  than those ≤ 3 months of age. Four patients ≤ 3 months of age and none of those > 3 months suffered from respiratory insufficiency requiring intubation and mechanical ventilation in an intensive care unit. There was no significant difference in detection rate of respiratory viruses and/or M. pneumoniae, the detection of RSV was significantly associated with more abnormalities on chest radiograph (p = 0.015) and with more frequent observation of the following clinical symptoms; fever (p = 0.028), feeding problems, cough, dyspnea, rhinorrhea and wheezing . HRV was associated with general discomfort (p = 0.021) and rhinorrhea (p = 0.012) and HEV with fever (p = 0.045), dyspnea (p = 0.007), rhinorrhea (p = 0.014) and wheezing (p = 0.017). HAdV was associated with fever (p = 0.026), HBoV with feeding problems (p = 0.032) and HCoVs with rhinorrhea (p = 0.009).Compared to other viruses and Patients with respiratory pathogens detected, had more frequently an antibiotic treatment prescribed by the family physician before presentation to the hospital than patients without a positive respiratory (RT)-qPCR (p = 0.048).Of 139 patients with the clinical diagnosis of RTI, 120 (86.3%) were diagnosed with a viral RTI and 19 (13.7%) with a bacterial RTI. An antibiotic was prescribed to 29 (24.8%) patients with viral infection before the definite diagnosis was made, eventually 17 patients had a positive test result. In 10 (58.8%) of these patients detection of a viral pathogen in the nasopharynx specimen did not result in a switch in antibiotic policy of the pediatrician.Mycoplasma pneumoniae in symptomatic pediatric patients attending a pediatric outpatient clinic, emergency department or admitted to the pediatric ward of a large general hospital. At least one respiratory pathogen was detected in 73% of the enrolled patients. These findings are in support of the reports in the literature were viral detection rates of 47-95% are described in children. Possible explanations for the wide differences in detection rates in the literature include heterogeneity in study populations, differences in presenting respiratory symptoms (upper or lower respiratory symptoms), number of respiratory pathogens tested, method used for detection and genetic variability between populations [In this retrospective study we evaluated the diagnostic value and the clinical relevance of detecting 19 viruses and ulations -18. RSV Overall, in 27% of the patients, multiple respiratory viruses were found, comparable to other studies were the co-infection rate ranged from 17% to 41% . As expeThe recent introduction of more sensitive molecular methods in the clinical practice has increased the virus detection rates significantly compared to viral culture methods. This has, however, made the interpretation of positive test results more difficult, especially when multiple viruses are found with a low Ct value. Nevertheless, the detection of a virus by RT-qPCR does not necessarily means that it causes symptoms. The presence of a virus, particularly HRV, has to be interpreted carefully as viral RNA could be detected in nasal mucus for up to 5–6 weeks after infection and respiratory viruses are also detected in asymptomatic children ,24. For In literature conflicting results are reported regarding multiple viral co-infections and its association with the severity of acute disease. In this study, 66.6% of the HRV positive patients HRV was co-detected with another virus compared to 42% of the RSV cases. In addition, most samples with more than 1 virus, HRV and RSV were detected together, in 12 25.5%) of the 47 samples, respectively. Similar to our data, Aberle et al.  found HR.5% of thDetection of a virus in patients with respiratory symptoms would be expected to aid a clinician to refrain from the prescription and continuation of antibiotics. Unfortunately, as shown by the present study this is not the real life clinical practice as only in 40% the antibiotic treatment was ceased. The present findings confirm previous reports that have shown that detection of a viral respiratory pathogen in a large part of the pediatric patients did not influence a change in antibiotic management of individual patients ,28. ThisIn addition, in specific patient populations, e.g., after stem cell transplantation or with immunosuppression, identification of the viral pathogen can be important in predicting the clinical course, preventing graft-versus-host disease and graft rejection and, even lifesaving ,31.In conclusion, routine testing of common respiratory pathogens in children with ARTIs may lead to a better understanding of the role of viral pathogens in ARTIs and eventually to improvement of individual patient management. However, prospective studies are required to study the need of routinely using such tests in a general pediatric clinic.The authors declare that they have no competing interests.JR and CO conceived this project and participated in the study design. EH carried out the experiments. EH and RB collected patients’ data and wrote the manuscript. JR, CO and AB revised the manuscript. All authors read and approved the final manuscript.


Aspergillus fumigatus NTCC1222 showed the highest amylase activity (164.1 U/mL) in secondary screening under SSF conditions and was selected for further studies. The test strain showed maximum amylase production (341.7 U/mL) and supernatant protein concentration (9.7 mg/mL) for incubation period (6 days), temperature (35°C), initial pH (6.0), nutrient salt solution as moistening agent, and beef extract as nitrogen source. Pomegranate peel produced maximum amylase activity, but wheat bran (only slightly lesser amylase activity as compared to that of pomegranate peel) was chosen for further studies, keeping in mind the seasonal availability of pomegranate peel. TLC confirmed the amylase produced to be α-type and 60 kDa was the molecular weight of the partially purified amylase. The enzyme showed maximum enzyme activity at pH 6.0, temperature of 55°C, and incubation time of 60 minutes. UV (616.0 U/mL) and chemical (814.2 U/mL) mutation enhanced amylase activity as compared to wild test strain. The study indicates that Aspergillus fumigatus NTCC1222 can be an important source of amylase and the crude enzyme, hence obtained, can be cost effectively applied in multiple sections of textile wet processing.The study aimed at isolation and screening of fungal amylase producer, optimization of solid state fermentation conditions for maximum amylase production by the best amylase producer, and characterization of the crude amylases, so produced.  It is an exoacting enzyme that cleaves nonreducing ends of amylose, amylopectin, and glycogen molecule. Glucoamylase ) hydrolyses single glucose units from the nonreducing ends of amylose and amylopectin in stepwise manner. Amylases can be derived from various sources, such as plants, animals, and microorganisms, but microbial sources generally meet industrial demands [aw), and high osmotic pressure conditions make fungi most efficient for bioconversion of solid substrates [The chemical technology, in particular, has received the great attention due to its hazardous by-products contaminating and polluting the environment and also the high cost of energy inputs for large-scale product manufacture. Search for environmentally compatible and less energy intensive processes has made the biotechnological approach meaningful in the present context. In recent years, the potential of using microorganisms as sources of industrially relevant enzymes has stimulated our interest –3, where demands  as there demands –12. The bstrates  and thusbstrates –16.Although most researchers have used liquid culture, which allows greater control of culture conditions such as temperature and pH, SSF has gained huger interest in recent years due to advantages like high yield and high specificity, simple technique, and low moisture contents, which prevent bacterial contamination, capital investment, lower levels of catabolite repression, and better product recovery , 17. MorLignocellulosic substrates  were collected from the local market of Jalandhar, Punjab, India. They were first washed with cold water and subsequently with warm water to remove dirt and impurities. The washed substrates were then dried in sunlight. The dried substrates were grinded in a laboratory grinder and a particle size of 0.5 mm was selected for further studies.Fungal strains were isolated from soil samples from collected different sites of Jalandhar, Punjab, India, by sweeping off the debris from the top of the soil and scooping the sample of soil (about 100 grams) into a “Ziploc” bag. The soil samples were serially diluted, spread plated on starch agar (SAM) medium (fortified with 0.1 mg/mL streptomycin), and incubated at 25°C. After 3 days of incubation, the plates were stained with Lugol's iodine solution  and observed for amylase production, as indicated by the appearance of zone of clearance against dark (bluish-black) background.Stock cultures of purified isolates were maintained on PDA slants, incubated at 25°C for 24 hours, and subsequently stored at 4°C. The cultures were maintained as a suspension of spores and hyphal fragments in 15% (v/v) sterile glycerol at −20°C for long-term preservation.μ mole of reducing sugar per minute (U/mL), with glucose as standard, under the assay conditions described above. The supernatant protein concentration was also determined for the crude enzyme [Amylase positive fungal strains obtained, as detected after amylase plate assay, were further assayed to quantitatively determine actual amylase activities under solid state fermentation (SSF) conditions using wheat bran  as the se enzyme  at 660 nThe best fungal amylase producer was identified at Plant Pathology Laboratory, Forest Research Institute, Dehradun, India, on the basis of morphological and microscopic features and deposited at National Type Culture Collection (NTCC), Forest Research Institute, Dehradun, India.Starch hydrolysates were analyzed by thin layer chromatography (TLC) using silica gel plates, where sugar spots of hydrolysates were identified by comparing their Rf values with those similarly obtained for sugar spots of glucose and maltose as reference standards .The effect of incubation period on amylase activity was determined by incubating the inoculated flasks for variable incubation period (1 to 10 days) at pH 6.0 using wheat bran as carbon source and beef extract as the nitrogen source, at a temperature of 37°C. The wheat bran was moistened with NSS in the ratio of 1 : 3 as previously described. The enzyme was harvested after every 24 hours up to 10 days on incubation and amylase activity  and supeA total of 19 different substrates  were tested to see their effect on amylase production. The flasks were prepared, inoculated, and incubated as above and the amylase activity  and supeThe effect of nitrogen source  and moistening agents, namely NSS, distilled water, and tap water , 37 on aμg of reducing sugar liberated in one hour under standard assay conditions. Carboxymethyl cellulase (CMCase) activity was determined [μ mole of glucose released per minute [μm glucose per min. Xylanase activity was determined [μmol of xylose equivalents per minute under the assay conditions. Laccase activity was determined as per the method of Ride [The test fungal strain was subjected to optimized conditions of incubation temperature (37°C), incubation period (6 days), and initial pH (6.0), using NSS as the moistening agent, solid to liquid ratio (1 : 3), wheat bran as carbon source, and yeast extract as the nitrogen source under SSF. The amylase activity and supernatant protein concentration were then determined. The crude amylase preparation obtained from test fungi under optimized conditions of SSF was further tested for the array of enzymes present in the crude enzyme preparation that were produced under the above-mentioned optimized conditions. Filter paper assay (FPase assay) was carried  using fitermined  using car minute . Pectinar minute  using petermined  at 540 nThe crude enzyme was subjected to variable pH , incubation temperature , and incubation time , and the amylase activity was determined .2 in 3% KI) for 10 min.The molecular weight of alpha-amylase of test fungi was determined by subjecting the enzyme preparation to ammonium sulphate (0–40% and 40–70%)  precipitKm and Vmax using lineweaver burk plot by estimation of amylase activity [Kinetic studies were carried out for the said enzyme using different starch (5.0–75 mg/mL) concentrations for the determination of activity .2/S energy) for varying time intervals  and kept in the dark for the stabilization of thymine-thymine dimers [Plate cultures were exposed to UV radiations (254–260 nm) using UV lamp (1.6 × 102 J/me dimers . The amye dimers  of stable dimers –46 and tAll the cultures were replicated in triplicates and the results are mean standard deviation (±SD) of the value.Aspergillus fumigatus NTCC 1222 at Laboratory of Plant Pathology, Forest Research Institute, Dehradun, India, on the basis of morphological features and microscopic examination. Glucose and maltose were found to be the main products on subjecting the crude amylase of the test strain to TLC analysis, confirming that the amylase produced by the above organisms was of α-type.A total of 7 fungal strains were found to be positive for amylase production, as determined by measuring the width of the clear zone (zone of hydrolysis) formed around the fungal colonies . The funAspergillus sp. [As reported in llus sp. . The maxllus sp. . The resAs shown in Further, the effect of incubation temperature, pH, and incubation time was also studied. The maximum amylase activity (340.1 U/mL) was obtained at pH 6.0  when cruThe specific activity of the enzyme was 44.2 U/mg for crude enzyme, 309.3 U/mg after ammonium sulphate precipitation and 893.7 U/mg after dialysis. Thus, 7- and 20-fold purification was obtained by ammonium sulphate precipitation and dialysis, respectively. The molecular weight of amylase produced by the test strain was found to be 60 kDA by SDS-PAGE analysis. The amylase activity was confirmed by zymogram analysis as a pale yellow band in dark-colored gels .Km) was determined by Lineweaver-Burk plot using starch as a substrate  was found to be 2.04 μg/mL/min.The Michaelis-Menten constant (ubstrate . The KmA. fumigatus NTCC 1222 to UV treatment was optimized (data not given) and it was found that the UV-mutated strain of A. fumigatus NTCC 1222 showed an increase of 44.52% in amylase activity over amylase activity for the wild strain of the said fungus. The chemically (EMS-EtBr) mutated strain reported an increase of 58.03% in amylase activity as compared to wild strain of the said fungus. The UV-mutated strain showed an increase of 67.96% for total cellulase activity, an increase of 22.03% for CMCase activity, a decrease of 95.22% for pectinase activity and a decrease of increase of 10.0% for laccase activity, as compared to the respective enzymes obtained from wild strain of A. fumigatus NTCC 1222. For chemically-mutated strain, the total cellulase, CMCase, pectinase and laccase activities were respetively increased by 65.97%, decreased by 8.69%, decreased by 80.91%, decreased by 12.00%, as compared to respective enzymes obtained from wild strain of A. fumigatus NTCC 1222 , incubation temperature (35°C), and incubation pH (6.0), using nutrient salt solution (NSS) as the moistening agent, beef extract as the nitrogen source, and wheat bran as the solid substrate. The crude enzyme preparation, so obtained from the test strain under optimized conditions of SSF, was found to be thermoalkali stable, as it retained about 90% of its maximum activity at pH 8.0 and more than 50% of its maximum activity at a temperature of 75°C. UV-mutated and chemical-mutated strains were, respectively, found to exhibit an increase of 44.52 and 58.03% in amylase activity as compared to wild strain of Aspergillus fumigatus NTCC1222. The results are significant as they indicate that the enzyme will be active even under the presence of high temperatures and pH, thereby favoring its use under harsh conditions of textile wet processing. The crude amylase preparation is also found to contain important industrial accessory enzymes, which add to the value of the said enzyme preparation for industrial applications. Use of a single enzyme preparation in multiple steps of industrial processing has the potential to improve the economics of the overall process. Thus, the future work deals with application of the crude enzyme of Aspergillus fumigatus NTCC1222 in multiple sections of textile wet processing so as to explore cost-effective usage of enzymes.The present study attempts to explore the potential of indigenously isolated fungal strains to produce amylases using cheap lignocellulosic substrate for their potential application in textile industry. A total of 7 strains were isolated from soil samples collected from different locations of Jalandhar, Punjab, India. Out of a total of 7 fungal strains isolated from soil, 4 were found to be amylase positive and they showed variable amylase activities under conditions of SSF using wheat bran as the cheap lignocellulosic substrate.


Background: Exposure to arsenic via drinking water has been associated with adverse pregnancy outcomes and infant morbidity and mortality. Little is known, however, about the effects of arsenic on child growth.Objective: We assessed potential effects of early-life arsenic exposure on weight and length of children from birth to 2 years of age.Methods: We followed 2,372 infants born in a population-based intervention trial in rural Bangladesh. Exposure was assessed by arsenic concentrations in urine (U-As) of mothers  and children (18 months old). Child anthropometry was measured monthly in the first year and quarterly in the second. Linear regression models were used to examine associations of U-As (by quintiles) with child weight and length, adjusted for age, maternal body mass index, socioeconomic status, and sex (or stratified by sex).Results: Median (10th–90th percentiles) U-As concentrations were about 80 (25–400) µg/L in the mothers and 34 (12–159) µg/L in the children. Inverse associations of maternal U-As with child’s attained weight and length at 3–24 months were markedly attenuated after adjustment. However, associations of U-As at 18 months with weight and length at 18–24 months were more robust, particularly in girls. Compared with girls in the first quintile of U-As (< 16 µg/L), those in the fourth quintile (26–46 µg/L) were almost 300 g lighter and 0.7 cm shorter, and had adjusted odds ratios  for underweight and stunting of 1.57 (1.02–2.40) and 1.58 (1.05–2.37), respectively, at 21 months.Conclusions: Postnatal arsenic exposure was associated with lower body weight and length among girls, but not boys. Inorganic arsenic is found in drinking water based on groundwater in many countries, including Bangladesh. Chronic arsenic exposure is associated with multiple adverse health effects, including cancer [World Health Organization (WHO) 2001]. Arsenic easily crosses the placenta , and theThere is much less information on effects of arsenic on child growth. Two cross-sectional studies in Bangladesh reported associations of arsenic in drinking water with low body mass index (BMI)  and incrIn our ongoing mother–child cohort in rural Bangladesh, we observed that arsenic exposure during pregnancy was associated with lower birth weight . In the Study area and population. This study was conducted in Matlab, a rural area 53 km southeast of Dhaka. Matlab is the field site of the International Centre for Diarrhoeal Disease Research, Bangladesh  where a Health and Demographic Surveillance System (HDSS) has been in place for collecting vital information since 1966 . Farming is the main source of income, followed by fishing and trading , designed to investigate the effects of antenatal food and micronutrient supplementations on pregnancy outcomes and child health and development  with arsenic measurements at 18 months of age  were selected for this study. Arsenic exposure was estimated based on concentrations of arsenic metabolites in urine (U-As), which provide a measure of inorganic arsenic exposure from all sources. U-As measurements were available for 2,096 and 2,170 of the mothers at 8 and 30 weeks of pregnancy, respectively. The main reason for missing U-As data was a delay in initiation of urine sampling in early pregnancy.All infants born before the end of December 2003  as well as with reference materials  using hydride generation atomic absorption spectroscopy method . Childreaterials . Arsenicaterials .Anthropometry. Weight and length were measured at birth, every month in the first year of life and quarterly in the second  and length for age (LAZ), according to the WHO Multicenter Growth Reference Study child growth standards (WHO 2006b).Underweight was defined as WAZ < –2 and stunting as LAZ < –2. Maternal BMI (kilograms per meter squared) was calculated from maternal weight and height at enrollment.Weight and length measurements were converted to SES. We used a wealth index as a measure of SES, which was created based on information on household assets, including land, construction materials of the houses, household assets, number of certain clothes and shoes owned  and the attained weight or length of infants and young children . The models were then adjusted for child sex and maternal BMI and SES, which were found to influence the associations of U-As with both weight and length. Residual analysis indicated no major deviation from a linear pattern. As sensitivity analyses, we additionally adjusted for birth weight or length, and maternal U-As. We repeated the analyses stratified by child sex and SES (median split).Because we previously found that underweight and stunting were prevalent in this cohort , we evalp < 0.05 was considered statistically significant.Statistical analyses were carried out using SPSS version 18.0 . All tests were two-sided, and Ethical considerations. Mothers were informed about the study and their written consent was obtained at the time of enrollment during pregnancy. For 18-month tests on the children, written consent was obtained from parents or guardians after briefing them about the study. The project was approved by the ethical committees at ICDDR,B and Karolinska Institutet. Our parallel screening project for water arsenic  or did not  provide urine samples for arsenic measurements, and found no significant differences between groups (data not shown).We compared children with and without missing anthropometry data according to baseline characteristics  and found no significant differences between groups (data not shown). Similarly, we compared children of mothers who did (2 at enrollment (~ 8 weeks gestation), which indicates that they may have been undernourished. Median maternal U-As in pregnancy was about 80 µg/L, and median child U-As at 18 months of age was 34 µg/L.Infants were small at birth ; about 3Association of prenatal arsenic exposure with attained weight and length. Mean attained weight of the children generally decreased with increasing quintiles of maternal U-As, particularly at 30 weeks gestation (http://dx.doi.org/10.1289/ehp.103378)]. Overall results as well as results stratified by sex followed a similar pattern for children’s length (estation . With ths length .http://dx.doi.org/10.1289/ehp.103378)]. Additional adjustment by birth weight further changed the estimates (on average ~ 50%) (data not shown). Corresponding analyses with child length produced similar results  were inversely associated with attained child weight . When th results . Most of results .Association of postnatal arsenic exposure with subsequent attained weight and length. Mean body weight at all ages studied  decreased about 300 g from the first to the fourth quintile of child U-As at 18 months [see Supplemental http://dx.doi.org/10.1289/ehp.103378)]. In general, mean weight increased again at the highest quintile. When stratified by sex the overall pattern was observed in girls, but there was little evidence of a trend in boys. Similarly, child length decreased by about 1 cm from first to fourth quintile at all ages, but only in girls ]. Compared with girls in the lowest quintile of U-As (< 16 µg/L), those in the fourth quintile (46–96 µg/L) had an odds ratio (OR) for underweight  of 1.50 , 1.57 (1.02–2.40), and 1.15 (0.75–1.76) at 18, 21, and 24 months of age, respectively ]. Early childhood stunting is closely associated with poor cognitive and educational performance . The water used by the children in the fifth quintile of U-As had on average 1.6 mg iron/L . Thus, consumption of only 1 L/day will contribute significantly to the total iron intake. Interestingly, the prevalence of iron deficiency in the early pregnancy mothers was unexpectedly low  PDFClick here for additional data file.


Klinefelter syndrome (KS) is a frequent genetic disorder due to one or more supernumerary X chromosomes. KS is associated with an increased risk for venous thromboembolic events like deep venous thrombosis and pulmonary embolism. This paper describes a 37-year-old male patient with KS referred to our tertiary center with chronic thromboembolic pulmonary hypertension, and who was successfully treated by pulmonary endarterectomy. Klinefelter syndrome  is a complex genetic disorder with highly variable endocrinological, metabolic, morphological, and neurobehavioral manifestations of altered X-chromosomal gene expression. Recent studies estimate the prevalence of KS 1 : 640 , which mA 37-year-old male was referred to the CTEPH center of the Academic Medical Center of the University of Amsterdam for the analysis of suspected CTEPH. At the age of 30, he was diagnosed with KS by genetic counseling . One year later, following a high energetic trauma complicated with osteomyelitis of the right femur, he presented with acute onset dyspnea. By computed tomographic (CT) pulmonary angiography, acute bilateral PE was diagnosed as sequelae of a DVT of the right leg. The patient's family history was negative for VTE. Anticoagulant treatment was instituted with vitamin K antagonists for total duration of six months, whereupon he recovered promptly. N < 200 pmol/l, [N < 150%). CT angiography demonstrated large, organized thrombi in the left main pulmonary artery, as well as in the right upper lobe multiple webs (N < 40 mmHg). Left ventricular dimensions and function were normal. Right heart catheterisation demonstrated a pulmonary artery pressure of 59/29 mmHg, mean PAP of 43 mmHg (N < 25 mmHg), cardiac output of 6.0 L/min, pulmonary wedge pressure of 6 mmHg, mean right atrial pressure of 11 mmHg, and the calculated pulmonary vascular resistance (PVR) of 493 dynes·s·cm−5.Six years later, however, he started to suffer from slow onset dyspnea on exertion. Perfusion scintigraphy showed multiple segmental and subsegmental defects, consistent with possible pulmonary embolism. Since anticoagulant treatment for six months did not improve his complaints, he was referred to our hospital. At referral, the patient was in no respiratory distress at rest, with a peripheral oxygen saturation of 99%. He was mildly retarded and obese (body mass index 31.7), had a thickened neck, and a widened forehead with little hair growth. Systemic blood pressure was 120/80 mmHg. Cardiac and pulmonary examinations were normal, except for a split second heart tone. No peripheral oedema was observed. Laboratory tests were within normal range; NT pro-BNP:120 micrograms/mL . No coaple webs . Pulmonaple webs . ExercisThe patient was diagnosed with proximal CTEPH. His functional impairment was classified as New York Heart Association (NYHA) III/IV; that is marked limitation in activity due to symptoms, even during less-than-ordinary activity. Without treatment, he had an estimated 5-year survival of less than 30% . A multiKS is associated with substantial morbidity  and incrCTEPH results from incomplete resolution of the vascular obstruction caused by pulmonary thromboembolism . The incCTEPH manifested in this patient six years after the documented acute pulmonary embolism. Moreover, he had been fully asymptomatic for several years. Therefore, it is highly unlikely that it has been merely the consequence of this episode. Pengo et al. showed that CTEPH manifests itself within 2 years after an episode of pulmonary embolism . RetrospThromboembolic disease is frequently observed in KS , 12; howAs in VTE, elevation of PAI-1 activity is also considered to play a role in the pathogenesis of (postthrombotic) leg ulceration observed in KS patients , 30. PosIn conclusion, future studies are warranted to unravel the pathogenesis of VTE and the incidence of chronic thromboembolism in KS patients. However, given the increased incidence of VTE, in KS patients presenting with dyspnoea with or without a previous history of VTE, CTEPH must be considered. CTEPH is a life-threatening yet potentially curable form of pulmonary hypertension if diagnosed early in the course of the disease .


Background: Laparoscopic liver resection is growing in popularity, but the long-term outcome of patients undergoing laparoscopic liver resection for malignancy has not been established. This paper is a meta-analysis and compares the long-term survival of patients undergoing laparoscopic (LHep) versus open (OHep) liver resection for the treatment of malignant liver tumours.Methods: A PubMed database search identified comparative human studies analysing LHep versus OHep for malignant tumours. Clinical and survival parameters were extracted. The search was last conducted on 18 March 2012.Results: In total, 1002 patients in 15 studies were included (446 LHep and 556 OHep). A meta-analysis of overall survival showed no difference . Subset analyses of hepatocellular carcinoma (HCC) and colorectal metastases (CRM) were performed. There was no difference in the 1-, 3-, and 5-year survival for HCC or in the 1-year survival for CRM, however, a survival advantage was found for CRM at 3 years .Conclusions: Laparoscopic surgery should be considered an acceptable alternative for the treatment of malignant liver tumours. However, a laparoscopic hepatectomy remains one of the last frontiers to be fully implemented. The first laparoscopic hepatectomy, performed in 1992 by Gagner et al. found that a laparoscopic hepatectomy is not only comparable, but possibly superior to the traditional open procedure in the peri-and post-operative outcomes in hepatocellular carcinoma (HCC), where they report lower blood loss with no significant difference in terms of oncological resection margins. In this study, Zhou included 5-year survival data from four studies.5In 2009, 2804 laparoscopic liver cases were reported in a systematic review, which showed that peri-operative factors either showed no difference or favoured laparoscopy. The review also noted the oncological outcome of the laparoscopic was comparable to the open approach, however, only one study reported 5-year survival.The primary outcomes of the previous meta-analyses were peri-operative or immediate post-operative outcomes. What has yet to be confirmed is the long-term outcome of patients undergoing a laparoscopic (LHep) versus an open hepatectomy (OHep) for malignant solid liver tumours. Previous meta-analyses have touched on long-term outcome, but went unstressed by the authors or may have lacked the number of studies to make meaningful conclusions. This study will analyse the available current data to determine the two procedures’ long-term outcomes. The aim of this study is to perform a comprehensive meta-analysis of overall long-term survival of those undergoing a laparoscopic as compared with an OHep for malignant tumours.A PubMed database search was performed to identify comparative studies analysing laparoscopic versus open hepatectomies for malignant tumours. Medical Subject Heading keywords, ‘laparoscopy’ and ‘hepatectomy’ were used in combination with publication type ‘comparative study’ to search the database. A more extensive text word search in MEDLINE was conducted using combinations of the keywords, ‘laparoscopic,’ ‘laparoscopy,’ ‘minimally invasive,’ ‘hepatectomy,’ ‘hepatic lobectomy,’ ‘liver lobectomy,’ ‘hepatic segmentectomy,’ ‘liver segmentectomy,’ ‘liver sectionectomy,’ ‘hepatic sectionectomy,’ ‘hepatic resection,’ ‘liver resection,’ ‘liver surgery’ and ‘hepatic surgery.’ The search was restricted to only human studies. The most recent search was conducted on 18 March 2012.The meta-analysis was performed in line with previously established guidelines.Studies were included in the analysis if: (i) they were comparative human studies focusing on LHep versus OHep for solid tumours; and (ii) the 1-, 3-, or 5-year survival of patients with malignancies could be extracted or calculated.Criteria for exclusion from the analysis included studies that: (i) did not compare LHep and OHep, or where the operative approach was not clearly documented; (ii) there was an inability to extract essential data or conduct a statistical analysis; (iii) contained insufficient data on overall survival; or (iv) included repeat hepatectomies or hepatectomies performed prior to liver transplantations.All studies included were scored based on the Newcastle–Ottawa Scale (NOS) for assessing the quality of the studies. The NOS implements a ‘star system’, grading each study in three distinct areas: selection of the groups, comparability of those groups and the degree or quality to which the exposure or outcome of interest was expressed. A maximum total of nine stars can be allocated to one study, with ‘Selection’ a possible four, ‘Comparability’ a possible two and ‘Outcome’ a possible three. Tumour size was chosen as the most significant factor to be controlled in order to merit one star in ‘Comparability’.6The outcomes of interest encompassed operative parameters, post-operative parameters and long-term survivals. Operative parameters including operative time, blood loss and oncological margins were tabulated. Post-operative parameters included length of hospital stay and post-operative mortality. The analyses of overall survival for all malignancies were performed at 1-, 3-and 5-years. Two subgroup analyses for HCC and colorectal metastases (CRM) were also available for calculation of the overall 1-, 3-or 5-year survivals.I2  with 95% confidence intervals (CIs) were calculated using the DerSimonian–Laird random-effect model.The primary literature search garnered a total of 1091 abstracts. After review, abstracts were then narrowed down to 37 articles that compared LHep and OHep for solid liver tumours. Twenty studies were excluded from the analysis owing to the omission of survival data. Of the remaining 17 articles, 2 articlesThe articles reported a total 1002 patients: 446 of whom underwent LHep whereas 556 underwent OHep. Of those, 308 patients underwent LHep whereas 404 patients underwent OHep for HCC, while 94 patients versus 100 underwent LHep versus OHep for CRM, respectively. One study presented did not specify tumour type of 47 malignant open patients.P = 0.217). The other seven studies could not be included within the statistical analysis because of lack of detail. However, each of the studies individually determined that age and gender was not significantly different between LHep and OHep.In 11 studies, the two groups were matched for specific characteristics (see Table P = 0.951). The proportion of females undergoing LHep was 28.0% whereas OHep was 27.6%. All studies that recorded pre-operative tumour size reported that tumour size was not significantly different amongst the groups. Only eight studies could be included for the statistical analysis, which was not significantly different overall .A statistical analysis of gender in 14 studies was conducted, which overall was not significantly different  As tumour size was matched before treatment, no selection bias had taken place.et al. reported that tumour size was not significantly different. However, our own analysis shows a difference in Hu’s study between the two groups, with a mean difference of −2.00 cm (LHep versus OHep 95% CI −3.38 to −0.62). Another study, Ito et al.,Some of the studies in this meta-analysis presented unique concerns or findings with regard to tumour size. Castaing P = 0.048). Operative times were extracted from six studies and were included in the analysis.P = 0.395).Blood loss was extracted from five studies for statistical analysis.et al. chose to define positive margins as any margin less than 3 mm and reported a 10% positive margin for LHep and 26.8% for OHep.et al. reported a 41.2% positive margin for LHep and a 50% positive margin for OHep using a definition of less than 5 mm.Differences in the operative margins could not be analysed because of differences in the definitions of positive surgical margins and the variability of reporting. The most common definition of a positive margin was less than 1 mm of tumour-free parenchyma between the lesion and resection line. However, several previous studies included within this meta-analysis defined positive margins as greater than 1 mm. Sarpel Conversion to a laparotomy was necessary in 46 cases (4.2%) in 11 studies.28P-value.28Ten of the 15 studies demonstrated a significant difference in the length of hospital stay for patients, favouring LHep.The overall 30-day mortality was 1.4% (15 deaths), which included 3 (0.7%) deaths in the LHep group, and 9 (1.6%) deaths in the OHep group. Causes of death in the LHep group included clip failure from a portal vein branch,28P = 0.202)    = 12, P = 0.265; 3-years t = −0.405, d.f. = 12, P = 0.693; 5-years t = −1.49, d.f. = 8, P = 0.175; funnel plots not shown).All 15 studies were analysed for long-term survival for all malignancies. Using available data from 14 studies, LHep was shown to have a 92.6% survival at 1 year, whereas OHep was comparable at 90.51%  Fig. . Five-yeBecause of the various tumour types reported in the studies, subset analyses of the two most commonly encountered tumours, HCC and CRM, were performed.P = 0.422; 3-years: OR 0.95, 95% CI 0.64 to 1.39, P = 0.778; 5-years: OR 0.81, 95% CI 0.57 to 1.15, P = 0.239) .The survival of those undergoing LHep for HCC was 92.0%, 77.7% and 61.9% at 1-, 3-and 5-years. The survival of those undergoing OHep for HCC was similar: 91.3%, 76.5% and 56.5% at 1-, 3-and 5-years, respectively . . . Survival figures for CRM at 5 years were not analysed as only one study reported 5-year survival.22Although long-term data are limited, there is comparable survival at 1-year after LHep versus OHep for CRM (OR 0.55, 95% CI 0.17 to 1.80, et al. established that a laparoscopic procedure does not ‘adversely affect the oncologic outcome of the patient’.While the efficacy of a laparoscopic hepatectomy has been questioned in the past, this study demonstrates that LHep is comparable to OHep in treating malignancy. The success of LHep and the reservation with which the procedure has been viewed have spurred a collective need to examine the true benefits of a minimally invasive procedure. This meta-analysis sought to demonstrate that LHep is an alternative to OHep, a question that was previously explored regarding a laparoscopic resection for colon cancer. Similar to LHep, laparoscopic colectomy was under great scrutiny, with regard to long-term survival of patients, owing to a concern for local and trocar-site recurrences. In a breakthrough study, Fleshman et al. took a step towards validation of LHep as an alternative to OHep, but only looked at peri-operative outcomes without long-term survival.et al., this study found a lower blood loss for those undergoing LHep and no difference in operative time between the two groups. Owing to the difference in data reporting, not all data could be entered into a comprehensive statistical analysis. However, the objective of our meta-analysis to report on long-term survival was achieved.Similarly, in 2006, a meta-analysis from Simillis The aim in this meta-analysis was to investigate the use of the laparoscopic approach to liver resection for malignancy, focusing on long-term survival. The articles included within this analysis contained 10 studies reporting on only HCC and three studies that reported only CRM. The two other studies reported survival based on multiple types of hepatic malignancies.Within this study, we included a subset analysis of HCC and CRM. Of the patients undergoing a resection for HCC, 308 patients underwent LHep, whereas 404 patients underwent OHep. The survival of patients undergoing LHep for HCC was comparable to those undergoing OHep at 1, 3 and 5 years. Given these data and the benefit of the laparoscopic approach in cholecystectomy patients with cirrhosis,The gender distribution in the studies revealed a larger proportion of male patients, which is similar to that of the nation-wide patient population of nearly 3:1 male/female.19Several limitations of this study must be taken into account. The peri-and post-operative data were reported in several different ways, and therefore, could not all be included in the statistical analyses. A statistical analysis was not done for the oncological margin, owing to differences both in reporting and defining a positive margin.This meta-analysis has shown that LHep is comparable to OHep for all malignancies at 1-, 3-and 5-year overall survival, as well as for HCC. Although further study should be undertaken in those with CRM, the currently available data demonstrates that survival is comparable after LHep at 1 year and favourable at 3 years. Thus, LHep should be considered as an acceptable alternative for the treatment of malignant liver tumours.None declared.