Trypanosoma cruzi is the causative agent of Chagas disease, a parasitic infection endemic in Latin America. In T. cruzi the transport of polyamines is essential because this organism is unable to synthesize these compounds de novo. Therefore, the uptake of polyamines from the extracellular medium is critical for survival of the parasite. The anthracene-putrescine conjugate Ant4 was first designed as a polyamine transport probe in cancer cells. Ant4 was also found to inhibit the polyamine transport system and produced a strong trypanocidal effect in T. cruzi. Considering that Ant4 is not currently approved by the FDA, in this work we performed computer simulations to find trypanocidal drugs approved for use in humans that have structures and activities similar to Ant4. Through a similarity ligand-based virtual screening using Ant4 as reference molecule, four possible inhibitors of polyamine transport were found. Three of them, promazine, chlorpromazine, and clomipramine, showed to be effective inhibitors of putrescine uptake, and also revealed a high trypanocidal activity against T. cruzi amastigotes  and trypomastigotes  while in epimastigotes the IC50 were significantly higher . Finally, molecular docking simulations suggest that the interactions between the T. cruzi polyamine transporter TcPAT12 and all the identified inhibitors occur in the same region of the protein. However, this location is different from the site occupied by the natural substrates. The value of this effort is that repurposing known drugs in the treatment of other pathologies, especially neglected diseases such as Chagas disease, significantly decreases the time and economic cost of implementation. Trypanosoma cruzi is the causative agent of Chagas disease, a severe parasitic infection endemic in Latin America with ~7 million infected and more than 70 million people at risk, mostly living in conditions of extreme poverty T. cruzi –16.In cancer cells, high polyamine concentrations are required by every cell stage. Besides polyamine biosynthesis, cancer cells also utilize polyamine importers to augment their intracellular polyamine pools. These transporters can be targeted via the delivery of cytotoxic polyamine conjugates or via drugs which inhibit polyamine uptake . The co-50 of 20 μM, and apoptosis was the main mechanism of cell death. Ant4 was shown to inhibit putrescine transport and decreased its intracellular concentration  explored its ability to inhibit polyamine transport and affect cell viability. Ant4 induced cytotoxicity in the HL-60 cell line after only 24 h exposure with an ICntration . This coT. cruzi, Ant4 decreased the putrescine and spermidine transport in epimastigotes, the insect stage of the parasite, with IC50 values of 5.2 and 8.8 μM, respectively. Ant4 also showed a strong trypanocidal effect on trypomastigotes, the bloodstream stage of T. cruzi, with an IC50 of 460 nM, and a selectivity index of about 13. This trypanocidal effect of Ant4 is very promising since it is significantly higher than the observed for benznidazole, the drug currently used to treat Chagas disease. In addition, the combination of both drugs produced a significant increase on the trypanocidal effect compared with individual treatments  were cultured at 28°C in plastic flasks (25 cm2), containing BHT  medium supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, 100 μg/mL streptomycin and 20 μg/mL hemin. Vero cells (African green monkey kidney) were cultured in MEM medium supplemented with 10% heat inactivated FCS, 0.15% (w/v) NaHCO3, 100 U/mL penicillin and 100 U/mL streptomycin at 37°C in 5% CO2 atmosphere. Trypomastigotes and amastigotes of the Y strain were obtained from Vero infected cells as previously described  in the presence of different concentrations of the indicated drug. All the compounds to be tested were dissolved in water. Following incubation during 10 min at 28°C (epimastigotes) or 37°C (trypomastigotes), transport was stopped by adding 1 mL of ice-cold PBS. Cells were centrifuged as indicated above, and washed twice with ice-cold PBS. Cell pellets were resuspended in 0.2 mL of water and counted for radioactivity in UltimaGold XR liquid scintillation cocktail . Cell viability was assessed by direct microscopic examination. Non-specific uptake and carry over were assayed without incubation or incubated at 4°C . Parasites were resuspended in PBS (0.1 mL) and the assay started by the addition of 0.1 mL of the transport mixture containing [d at 4°C .T. cruzi epimastigotes were cultured as described above, in 24-wells plate at a start density of 106 cells/mL in BHT medium. Parasites were treated with different concentrations of each drug, and epimastigote proliferation was determined after 24 h. Trypanocidal activity in cell-derived trypomastigotes and amastigotes was performed using 106 cells/mL in 96-well plates which were incubated at 37°C for 24 h in the presence of the corresponding drug. Growth was determined by counting in a Neubauer chamber or via viability assays using “Cell Titer 96® Aqueous One Solution Cell Proliferation Assay (MTS)”  according to the manufacturer's instructions.4 cells/well) were incubated in 96-well plates with the indicated compound (or diluent only as a negative control) and maintained at 37°C for 24 h. At the end of treatment, cells were fixed for 15 min, and stained with 0.5% crystal violet. After washing with water and drying, the absorbance of stained cells was measured at 570 nm.Cytotoxicity against VERO cells was determined by the crystal violet staining assay. The cells (106 cells/ml) were incubated with the corresponding concentrations of each compound for 30 min at 28°C. Afterwards, the samples were settled for 20 min at room temperature onto poly-L-lysine coated coverslips and then fixed for 20 min with 2% paraformaldehyde in PBS. Slides were mounted using 80% glycerol in PBS and cells were observed under an Olympus BX60 fluorescence microscope. Images were recorded with an Olympus XM10 camera.Epimastigotes  compound. Structures  were obtained from the highly curated “Sweetlead” database of the world's approved medicines, illegal drugs, and isolates from traditional medicinal herbs . The scr4 as maximum number of generations. The figures of the docking poses where produced using Pymol  and the protein residues important for the interaction with the ligands were colored using Pymol according to the Eisenberg hydrophobicity scale . P-values of the comparisons were calculated using the extra sum of squares F-test.ICAnt4 is an experimental drug for cancer treatment not yet approved for use in humans, which also has a strong trypanocidal action . TherefoTo test the similarity screening predictions, the ability of PRM, CHL, LVM, and CLM to inhibit the polyamine uptake was analyzed. Transport assays were performed in epimastigote cells in the presence of putrescine or spermidine  and each putative inhibitor in a concentration range of 0–600 μM. Concentrations that inhibit the 50% of the transport activity (TIC50) were calculated. PRM, CHL, and CLM produced an inhibition of putrescine transport with calculated TIC50 values of 51.8 μM (±4.7), 22.6 μM (±1.3), and 35.3 μM (±2.6), respectively . HoweverT. cruzi. Epimastigotes were treated with each drug in concentrations between 0 and 500 μM and the trypanocidal effect was determined 24 h post-treatment. The calculated concentrations that inhibit the 50% of the parasite proliferation (IC50) were 34.7 μM (±2.3), 41.4 μM (±4.0), and 39.7 μM (±2.8) for PRM, CHL, and CLM, respectively ; corresponding to 3.4 μM (±0.5), 2.7 μM (±0.2), and 1.3 μM (±0.2) for trypomastigotes cells and 3.8 μM (±0.2); 1.9 μM (±0.1), and 2.9 μM (±0.1) for amastigote parasites  more susceptible than epimastigotes, the insect stage of the parasite. Additionally, the cytotoxicity of drugs was tested using mammalian Vero cells to determine the selectivity index (SI = IC50 on Vero cells/IC50 on trypomastigotes). Cells exposed to PRM, CHL, and CLM for 24 h in a concentration range from 0 to 300 μM showed SI values of 17.9 (IC50 62.8 μM ± 3.6), 9.4 (IC50 26.4 μM ± 0.3), and 38.2 (IC50 53.5 μM ± 2.1), respectively. These results indicate that the drugs are more selective against T. cruzi trypomastigotes than host cells.The trypanocidal activity was evaluated only for those drugs that inhibited polyamine transport activity, namely PRM, CHL, and CLM. Interestingly, all these compounds presented trypanocidal effect against the three stages of ectively . Next, tarasites . This meT. cruzi was also evaluated. PRM, CHL, and CLM were tested at concentrations similar to the TIC50 values calculated on epimastigotes. As occurred with epimastigotes, all the compounds inhibited the putrescine transport in trypomastigotes. The results showed that 50 μM PRM, 25 μM CHL, and 35μM CLM produced inhibition of 83% (±2.2), 84.3% (±5.9), and 79.2% (±11.4) respectively. In addition, in this stage of the parasite all drugs also inhibited the spermidine transport. At 25 μM the inhibitions observed were 64.6% (±2.7), 55.7% (±0.7), and 60.2% (±9.5) for PRM, CHL, and CLM, respectively. These results suggest that the compounds evaluated act through a polyamine transport inhibition mechanism in T. cruzi epimastigotes and trypomastigotes.In order to further test the correlation between the trypanocidal activity of PRM, CHL, and CLM and the polyamine transport, the inhibitory effect of the drugs on putrescine and spermidine uptake in the trypomastigote stage of T. cruzi polyamine permease TcPAT12  which has a strong activity on T. cruzi we performed a similarity virtual screening and identified three antipsychotic drugs; promazine, chlorpromazine, and clomipramine, which were repositioned as trypanocidal agents. All parasite stages tested for trypanocidal activity were affected by Ant4 and the tricyclic drugs herein reported and an increased sensitivity of the trypomastigote and amastigote stages was observed. It should be noted that psychiatric patients, who receive oral treatments with chlorpromazine or clomipramine within the reference doses, reach plasma concentrations in the range of the calculated IC50 for both drugs in the stages present in the mammalian host. In consequence these medicines are promising candidates to perform pre-clinical evaluations for the development of new therapeutic alternatives to treat Chagas disease.Tricyclic neuroleptic drugs, including clomipramine and chlorpromazine, were previously described as therapeutic agents for trypanosomiasis. The most studied mechanism of action was the inhibition of the trypanothione reductase; the enzyme responsible for the synthesis of the antioxidant compound trypanothione, the parasite analog of glutathione , 29. HowT. cruzi epimastigote and trypomastigote stages was previously observed by our group (T. cruzi life cycle is important since the trypomastigote is the most sensitive stage and the most relevant from a therapeutic point of view.The differential trypanocidal effect of polyamine transport inhibitors on ur group , 16 and in vitro. Moreover, isotretinoin showed a strong inhibition of trypomastigote burst from infected cells with a calculated IC50 of 130 nM and a selectivity index of 920 and is a promising drug for pre-clinical trials (Other trypanocidal drugs that inhibit the polyamine transport mediated by TcPAT12 were identified by virtual screening techniques. Isotretinoin, a popular drug used for acne treatment, showed the most stable predicted interaction with TcPAT12 and inhibited the polyamine transport l trials .T. cruzi epimastigotes suggesting distinct cellular targets besides polyamine transport. In human cancer cells it has been described that Ant4 anthracene moiety interact and induces DNA damage (Fluorescence microscopy studies also revealed the uptake and intracellular accumulation of Ant4 and chlorpromazine. Both compounds were internalized by A damage , howeverOur data suggests that both the putrescine moiety and the large hydrophobic ring of Ant4 contribute to its polyamine transport inhibition activity. From the perspective of designing new TcPAT12 inhibitors, the docking models suggest that these compounds should keep the putrescine motif, while retaining all protonable nitrogen as in the case of Ant4. Moreover, large hydrophobic rings should be present to anchor the drug into the hydrophobic pocket previously described. This work also suggests that halogen substitution on the hydrophobic moiety could provide higher binding affinity for the transporter.All these results highlight the importance of the polyamine transporter TcPAT12 as a therapeutic target for Chagas disease and drug repositioning as an adequate strategy for drug discovery to treat neglected diseases.All datasets generated for this study are included in the article/CR and CP conceived and designed the experiments. CR, MS, EV-V, and MM performed the experiments. CR, OP, MS, EV-V, MM, and CP analyzed and interpreted the data. CP and OP contributed reagents, materials, analysis tools, or data. CR, CP, and OP wrote the paper. CR, OP, MS, EV-V, MM, and CP revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Retrospective studies conducted in psychiatric wards have indicated a shorter duration of stay for depressed inpatients in bright compared to dim daylight-exposed rooms, pointing to a possible antidepressant effect of daylight conditions. Dynamic LED lighting, aiming to mimic daylight conditions, are currently been installed in several hospitals, but their feasibility is poorly investigated.To investigate the feasibility of these systems, we developed and installed a LED-lighting system in four rooms in a psychiatric inpatient ward. The system could function statically or dynamically regarding light intensity and colour temperature. The system consisted of (A) a large LED luminaire built into the window jamb mimicking sunlight reflections, (B) two LED light luminaires in the ceiling and (C) a LED reading luminaire. In the static mode, the systems provided constant light from A and B. In the dynamic mode, the system changed light intensity and colour temperature using A, B and C. Patients with unipolar or bipolar depression were randomised to dynamic or static LED lighting for 4 weeks, in addition to standard treatment. Primary outcome was the rate of patients discontinuing the trial due to discomfort from the lighting condition. Secondary outcomes were recruitment and dropout rates, visual comfort, depressive symptoms and suicidal ideation.No participants discontinued due to discomfort from the LED lighting. Recruitment rate was 39.8%, dropout from treatment rates were 56.3% in the dynamic group and 33.3% in the static group. 78.1% in the dynamic group were satisfied with the lighting compared with 71.8% in the static group. Discomfort from the light (glare) was reported by 11.5% in the dynamic group compared to 5.1% in the static group. Endpoint suicidal scores were 16.8 (10.4) in the dynamic and 16.3 (14.9) in the static group. The lighting system was 100% functional. The light sensor system proved unstable.Dropout from treatment was high primarily due to early discharge and with a lack of endpoint assessments. The feasibility study has influenced an upcoming large-scale dynamic lighting efficacy trial where we will use a shorter study period of 3 weeks and with more emphasis on endpoint assessments. The lighting was well tolerated in both groups, but some found intensity too low in the evening. Thus, we will use higher intensity blue-enriched light in the morning and higher intensity amber (blue-depleted) light in the evening in the upcoming study. The light sensor system needs to be improvedNCT03363529ClinicalTrials.gov:  The treatment of patients with severe depression, whether as part of a unipolar or bipolar disorder, still lacks efficacy. Patients are often discharged with residual depressive symptoms  making tCurrently, dynamic LED lighting is increasingly being installed in hospital settings attempting to mimic daylight conditions with temporally regulated intensity and spectral distribution. The aim is to strengthen circadian entrainment to stabilise the sleep-wake cycle, provide alertness and increase the antidepressant effect. However, the usability, acceptability, adverse effects and efficacy of these systems are poorly investigated.This study aims to investigate the feasibility of a dynamic versus a static lighting condition delivered to patients with depressive disorders as a pre-study to a planned larger efficacy study with dynamic lighting. In this study, we chose to include both patients with unipolar and bipolar depression because we wanted to know whether patients with bipolar depression would switch to a hypomanic or manic state. We also generally need more data on light treatment in bipolar patients as this condition has been studied less than unipolar depression.We developed a new general lighting system using dynamic LED lighting installed in four single-bed patient rooms in a psychiatric inpatient ward. The system consisted of (A) a large LED panel built into the window jamb to mimic sunlight reflections, (B) two dynamic LED light fixtures in the ceiling and (C) one dynamic LED reading luminaire by the bed. Patients with a current depressive episode as part of a unipolar or a bipolar depression were randomised, in a 3:2 manner to either dynamic LED-lighting intervention (dynamic) or static LED-lighting intervention (static), for 4 weeks in addition to standard treatment. The randomisation was done automatically within the OpenClinica electronic Case Report Form (eCRF) system after entering of all eligibility criteria, and randomisation was thus concealed from investigators. The OpenClinica eCRf system was operated by the Copenhagen Trial Unit (CTU). We opted for 15 participants as we anticipated that this number would give indications of major problems with the lighting system, trial design, and adverse events. The randomisation with more participants into the dynamic group was chosen to increase the likelihood of finding serious adverse events in this group where we expected that the brighter light and a luminaire that could not be turned off might cause problems for the participants.> 18 years, a major depressive episode as part of a unipolar or bipolar disorder according to the Diagnostic and Statistical Manual version IV (DSM-IV)  of 36.0 [26–56]/59.0 [34–70] years in the dynamic/static group with 66.7% females and 33.3% males in both groups. Median number (interquartile range IQR) of previous depressive episodes was 1.0 (0–2.0)/1.5 (0.5–19.0), and median duration of current depressive episode was 1.8 (1.0–8.5)/13.0 (4–41) months. In all, 20% (n = 6). Visual comfort results are shown in Table 17 scale showed moderate depression levels at baseline without any difference between groups during the 4-week period. The SIDAS scale showed low to moderate degrees of suicidal ideation in both groups with baseline SIDAS scores in the dynamic/static group of 15.5 (SD = 6.8)/16.6 (SD = 11.7) and endpoint scores of 16.8 (SD = 10.4)/16.3 at (SD = 14.9) (n = 4/4).Nine participants were randomised to the dynamic group and six to the static group in the period from December 12, 2017, to December 19, 2018. No participants discontinued the trial due to discomfort from the lighting condition. Recruitment rate was only 39.5% (15/38). Recruitment rate was low due to long staying non-eligible patients in one of the four test rooms. Dropout rates from treatment at week four were 55.6% (five out of nine) in the dynamic and 33.3% (two out of six) in the static group, primarily caused by early discharge (n = 9/6) in the dynamic/static groups and endpoint scores of 0 (SD = 0)/0.5 (SD = 1.0) (n = 4/4). The baseline MDI scores in the dynamic/static groups were 29.4 (SD = 12.8)/28.0 (SD = 15.4) (n = 5/8), and endpoint scores were 18.8 (SD = 10.9)/27.8 (SD = 13.6) (n = 4/4). The baseline WHO-5 scores (highest = best) in the dynamic/static groups were 29.5 (SD = 24.2)/21.6 (SD = 8.3) (n = 8/5) and endpoint scores were 40.0 (SD = 27.9)/37.0 (SD = 31.7 (n = 4/4). Adverse events from the UKU scale were few, including low levels of headache and light sensitivity. One participant in the dynamic group had a non-serious self-harm episode, and another participant in the dynamic group, when on leave, took an overdose of medication with no complications. The last incident was reported to the research ethics committee. Neither of the incidents was considered related to the LED lighting.The mean YMRS scores were low with baseline scores of 2.2 (SD = 3.4)/0 (SD = 0) /23:02 (SD = 52 min) during week 1 (mean of 7 days) and 23:59 (SD = 89 min)/23:29 (SD = 50 min) during week 4. Mean sleep offset in the dynamic/static groups was 6:45 (SD = 131 min)/7:18 (SD = 60 min) during week 1 and 7:46 (SD = 129 min)/7:16 (SD = 56 min) during week 4. Mean subjective sleep quality in the dynamic/static group  was 6.5 (SD = 3.1)/6.2 (SD = 2.6) during week 1 and 7.1 (SD = 2.5)/5.2 (SD = 2.0) during week 4. There was no difference between groups in number of awakenings. Mean PSQI global scores in the dynamic/static groups (lower score = best) was 10.6 (SD = 5.2)/8.4 (SD = 5.7) at baseline and 9.0 (SD = 7.0)/13.3 (SD = 2.2) at endpoint. Mean MEQ scores showed participants to be intermediate chronotypes with baseline scores in the dynamic/static groups of 48.8 (SD = 7.6)/46.2(SD = 12.0) and endpoint scores of 46.0 (SD = 8.0)/49.8 (SD = 9.2). The room occupancy diary showed that participants, during the 4 weeks period, were in their room in the dynamic/static group for 2.3 (SD = 1.5)/2.3 (SD = 1.4) hours in the morning, 2.6 (SD = 1.7)/3.3 (SD = 1.8) hours in the afternoon and 2.2 (SD = 1.6)/3.1 (SD = 3.8) hours in the evening, summing up to 7.1 (SD = 4.1)/8.7 (SD = 3.8) hours in total. Nearly all participants used their curtains at some time of the day (100%/93.3%). However, only 15.8% used curtains all day in the dynamic group compared to 35.7% in the static group.Luminaire log data were available for five participants in the dynamic group and four participants in the static group. The mean daytime use of the ceiling luminaires in the dynamic/static groups (07:00–23:00) was 9.2 (SD = 5.8)/3:6 (SD = 4.4) hours with a distribution of 33.3%/35.9% in the morning (7:00–12:00), 34.2%/31.8% in the afternoon (12:00–18:00) and 32.4%/32.2% in the evening (18:00–23:00). The mean nighttime (23:00–7:00) use of the ceiling luminaires in the dynamic/static groups was 81.9 (SD = 96.8)/0.7 (SD = 4.9) minutes. The mean daytime use of the reading luminaires in the dynamic/static group was 9.2 (SD = 5.4)/5.2 (SD = 4.5) hours, with a distribution of 31.0%/28.2% in the morning, 31.9%/24.6% in the afternoon and 37.2%/47.2% in the evening. The mean nighttime use of the reading luminaires in the dynamic/static groups was 86.7 (SD = 96.7)/33.5 (SD = 76.0) minutes. The LED-lighting system was 100% reliable. One participant experienced a 3-hour dark-out of the system due to a general electrical malfunction at the hospital. The light sensor system had too low sensitivity to measure the spectral distribution of room light reliable. Case files showed that participants in both groups were treated with antidepressants, mood stabilisers, antipsychotics, anxiolytics and sleeping medications.Figure It was possible to evaluate the feasibility of the LED lighting system and the study design. In this study, we chose the rate of discontinuation as the primary outcome because we were unsure of how patients would react to having light treatment built into their room. We feared that too high light intensity or glare would make patients leave their room, and we also were unsure how patients would react to the dynamic luminaire built into the window jamb that they could not turn off. However, the results showed no discontinuation due to discomfort from the lighting system, and the satisfaction with the dynamic system was equal or higher than with the static system. There were a low recruitment rate and large attrition rate, especially at week four. Most participants in both groups were satisfied with the lighting system. Depressive symptoms were moderate at baseline. The high dropout from treatment at endpoint made outcomes difficult to interpret. The high dropout was primarily due to early discharge. There was a moderate degree of suicidal ideation in both groups.Methodological strengths include a randomised design, reaching the sample size, a detailed assessment of feasibility measures and functionality of the lighting system.Methodological limitations include a low sample size with an inherently large risk of type II errors, large dropout and a malfunctioning lighting sensor system. The imbalance between groups in the mean length of current depressive episodes is likely an artefact of the low sample size.A larger number of light-equipped rooms would help attain a higher number of recruitments per month, making larger trials more feasible. The study period should be shortened to reduce dropout due to early discharge. The most important is to secure an endpoint assessment, even if the participants drop out due to early discharge or for other reasons. It may induce bias if trial participants are not assessed at follow-up, and we should aim at a 95% completion rate. This completion rate might be accomplished by informing in the oral presentation and in the written material that an endpoint assessment is important even if the participant is discharged. The visual comfort data indicated that light intensity was too low in the evening, and pointing to that in future trials, intensity of light can be increased. Higher light intensity should, however, contain very little blue light to avoid undue melatonin suppression that would disturb sleep. The light sensor system should be replaced with more sensitive and stable sensors. The study did show that it was possible to make a detailed assessment of patients regarding feasibility and depression and sleep outcomes. The study shows the importance of carrying out a feasibility trial to test the design and technical systems and to evaluate lighting tolerability. We are looking forward to implementing these results in a randomised clinical efficacy trial with dynamic LED lighting.This efficacy trial (now running) has ten rooms (out of 12 rooms) installed with a similar lighting system in a similar ward. We believe this will increase the number of inclusions. The efficacy trial aims at a total of 150 patients, with 75 in each group. This larger sample will increase the power to detect clinically meaningful differences between groups. We have decided to reduce the trial length from 4 to 3 weeks, thus reducing attrition due to early discharge. Furthermore, we have implemented a mandatory endpoint assessment at week 3 for all included patients even if they are discharged early or if the intervention is stopped for other reasons.


Intestinal parasitic infections (IPIs) are a major cause of morbidity among children in developing countries. Investigation about the etiological agents and socio-ecological pattern of the infection would help to design better preventive strategy. The previous studies reported high prevalence of IPIs among schoolchildren of Nepal. Though these data may be essential for the policymakers and researchers, in Kathmandu, the capital of Nepal it remains unexplored whether the types of school and socioeconomic status affect the IPIs or not. The present study is an extension of previous works to investigate causative agents and associated risk factors. We examined 508 stool samples of schoolchildren from two schools by formal-ether concentration technique and analyzed the data based on school types.Giardia duodenalis (32.7%) and Ascaris lumbricoides (21.8%) were the most commonly detected protozoan and helminth species respectively. Prevalence of IPIs was higher among children from public school (26.1%) than private school (12.1%). Higher infection rates were found among farmer’s children (29.0%) and Dalit children (36.2%). These findings reveal the different prevalence of IPIs among public and private schoolchildren and suggest the need of effective preventive measures.The overall IPIs rate was 19.9% (n = 101) with the dominance of protozoans (78.4%) over helminths (21.6%).  Intestinal parasitic infections (IPIs) are one of the common cause of diarrhea in developing countries. Globally 1.5 billion people are infected with soil-transmitted helminths, above 267 million preschool children and over 568 million school-age children live in intestinal helminths prevalent area , 4.Entamoeba histolytica/dispar (57.1%) and Ascaris lumbricoides (35.5%) infections [A. lumbricoides [Globally IPIs related morbidity has been considered as a major threat for public health. A prevalence of 85.7% has been reported in Ecuador for IPIs, with a majority of ricoides . Moreovericoides  showed tricoides  reportedThe objectives of this study are to compare the prevalence rate of IPIs among private and public schoolchildren of Kathmandu and to evaluate possible associations of IPIs patterns with demographic, socio-economic and behavioral factors. Our findings would be beneficial to implement possible preventive measures to control and cure the parasite associated infections among the dwellers.2 within a total area of 50.7 km2 [This cross-sectional type of study was carried out from March to September 2014. This study was done among public and private schoolchildren of Kathmandu, Nepal. Kathmandu Valley is situated at an average elevation of 1400 m (4600 ft) above the sea level and has an approximate population density of 4416 per km50.7 km2 .Among many public and private schools in Kathmandu, a public and a private school were randomly selected with a similar location. The total student population was 1027 and 673 in public and the private school, respectively. The sample size (508) was calculated using Cochran’s sample size formula for categorical data  and a toFor specimen collection, well labeled, clean, dry, disinfectant free, wide-mouthed plastic containers were distributed to the study population with instruction requesting them to bring about 10-g stool sample the next morning. The containers were labeled with children’s name, code number and date of collection. Stool samples were collected from each student along with demographic and behavioral data using a questionnaire.Collected stool samples were macroscopically examined for color, consistency, and presence of blood, mucus and fragmented or entire helminths, and were immediately fixed with the same volume of 10% formal saline. The fixed stool samples were transported to the Laboratory of  SICOST, Kathmandu, Nepal.About 3–4 mL of each formalin-fixed fecal sample were filtered using a cotton gauze in the test tube, mixed (3–4 mL) diethyl ether and vigorously shaken for 5 min, then centrifuged at 3000 rpm for 15 min. The sediment was collected, mounted with iodine solution and microscopically examined for fecal parasites  by using 10× and 40× objectives .Pearson’s Chi-Square test value was applied for statistical analysis of results using SPSS 16.0 data analysis software. The odds ratio for risk the factor was calculated using Microsoft Excel. p-values < 0.05 were considered as statistically significant.An overall IPIs prevalence of 19.9% (101/508) was found. Multi-parasite infections were detected in 25.7% (26/101) of the stool samples, 22.8% (23/101) was from public school and 2.9% (3/101) from private school (OR: 3.6).Giardia duodenalis 32.7% (33) was the most prevalent protozoan followed by Endolimax nana 31.7% (32), Entamoeba coli 22.8% (23), Entamoeba histolytica/dispar 20.8% (21) and Blastocystis hominis 2.0% (2). Among helminths, A. lumbricoides 21.8% (22) was the most prevalent species followed by Trichuris trichiura 4.0% (4) and Hymenolepis nana 2.0% (2)  in public schoolchildren and 12.1% (27/224) in private schoolchildren (p < 0.05) (OR = 2.6)  was the most prevalent species, followed by E. nana (31.7%) and E. coli (22.8%). Various reports from Nepal showed that G. duodenalis is a common intestinal parasite with prevalence rates ranging from 13.2 to 73.4% [G. duodenalis may depend on its capacity to resist normal level of chlorine treatment in drinking water [Similar to previous reports , 17, proto 73.4% –28. The ng water . A studyng water .Alike previous reports , 31–33 aIn addition, the prevalence of parasitic infections was statistically independent of gender, source of drinking water and family size. In the public school, most of the children drink water from common water sources whereas in private school, children only share their water bottle. Furthermore, the high susceptibility of parasitic infection among small aged 4–10 years) children reported in this study is in agreement with previous reports from Nigeria  and Nepa years chIn conclusion, IPIs is an important public health problem among schoolchildren in Kathmandu, Nepal. Our study revealed that the prevalence of IPIs in schoolchildren is significantly associated with types of school. Furthermore, protozoa were found more prevalent than helminths, which might be linked with drinking water. The socioeconomic status and occupation of parents, the age of children and ethnicity are significantly associated with parasitic infections. Health awareness program should provide especially to the parents from the Dalit ethnic group and farmers. Installation of mass water filter and chlorination of drinking water should be applied in both schools. Further studies equipped with advanced microscopic and molecular techniques would be helpful for a proper diagnosis to implement effective prophylaxis.Due to the limitation of time and resources this study was subjected to incorporate barely only one public and one private school for the comparative study.


Different genetic patterns have been demonstrated for narrowly distributed taxa, many of them linking rarity to evolutionary history. Quite a few species in young genera are endemics and have several populations that present low variability, sometimes attributed to geographical isolation or dispersion processes. Assessing the genetic diversity and structure of such species may be important for protecting them and understanding their diversification history. In this study, we used microsatellite markers and plastid sequences to characterize the levels of genetic variation and population structure of two endemic and restricted species that grow in isolated areas on the margin of the distribution of their respective genera. Plastid and nuclear diversities were very low and weakly structured in their populations. Evolutionary scenarios for both species are compatible with open-field expansions during the Pleistocene interglacial periods and genetic variability supports founder effects to explain diversification. At present, both species are suffering from habitat loss and changes in the environment can lead these species towards extinction. Petunia mantiqueirensis and Calibrachoa elegans) that grow in isolated areas on the margin of the distribution of their respective genera. Plastid and nuclear diversities were low and weakly structured in their populations. Evolutionary scenarios for both species are compatible with open-field expansions during the Pleistocene interglacial periods and genetic variability supports founder effects to explain diversification. Both species are suffering from habitat loss and changes in the environment can lead these species towards extinction.In our study, we used microsatellite markers and plastid sequences to characterize the levels of genetic variation and population structure of two endemic and restricted species ( Many rare or narrowly distributed species present low levels of genetic diversity . AdditioEcological and evolutionary processes define the distribution of species across space and time. The climatic changes that occurred during the Quaternary impacted the distribution and genetic diversity of most species through cycles of range contraction and expansion in the northern and southern hemispheres , and, moSome studies have focused on the evolutionary history of species adapted to open areas interspersed among regions of higher altitude in the southern section of the Atlantic Forest . The souPetunia and Calibrachoa share several evolutionary constraints and biological characteristics, many times occurring sympatrically, side by side. Based on a proposed origin in open fields , which is the northernmost distribution of the Petunia genus. This species is annual and herbaceous, with bee-pollinated flowers displaying a purple and short corolla tube . We sampled all known populations and adult individuals found in the same flowering season . Vouchers were deposited at the BHCB herbarium  and the geographical coordinates for all collection sites were obtained with a global positioning system (GPS) unit [see]. We collected three or four young leaves from each adult individual of both species while seeking to minimize damage to the plants; we dried leaves in silica gel and then pulverized them in liquid nitrogen for DNA extraction.These two species are classified as federally endangered according the Brazilian Red List  protocol , 1× buffer (Invitrogen), 0.2 mM each dNTP, 2 mM MgCl2, 0.2 µM of each primer and 10 ng of genomic DNA. The amplification conditions for trnH-psbA and trnS-trnG were conducted as described previously for Petunia species (P. mantiqueirensis as previously described (We estimated the genetic diversity based on the plastid sequences (cpDNA) of intergenic spacers,  species . SequencP. mantiqueirensis were genotyped with 12 microsatellite loci markers (SSR) previously developed for Petunia hybrida . We amplified SSR regions in a final volume reaction of 10 µL containing ~10 ng of genomic DNA as a template, 200 µM of each dNTP (Invitrogen), 1.7 pmol of fluorescently labelled M13(-21) primer, 3.5 pmol of reverse primer, 0.35 pmol of forward primer with a 5′-M13(-21) tail, 2.0 mM MgCl2 (Invitrogen), 0.5 U of Platinum Taq DNA polymerase (Invitrogen), and 1× Platinum Taq reaction buffer (Invitrogen). The PCR conditions were as follows: an initial denaturation at 94 °C for 3 min; 30–35 cycles of 94 °C for 20 s, 48–58 °C for 45 s and 72 °C for 1 min; and a final extension cycle at 72 °C for 10 min.To evaluate current variability in each population and species, individuals of GeneMarker 1.97 software  was utilized to determine the alleles. Additionally, all alleles were visually verified and scored. Repeat motifs, respective annealing temperatures and fluorescent dyes used in each fragment are presented in The forward primers were FAM-, NED-, HEX-, VIC- and PET-labelled. The amplified product was visualized on a 2.5 % ultra-resolution agarose gel stained with 2 µL 0.001 % GelRed™. The amplified DNA was denatured and size-fractionated employing capillary electrophoresis on an Applied Biosystems  with a LIZ (500) molecular size standard (Applied Biosystems). ChromasPro 1.7.5  and haplotype sequences were deposited at GenBank (available at https://www.ncbi.nlm.nih.gov/genbank/) under numbers: C. elegansJQ082471.1, JQ082474.1, KM982182.1, KM982262.1; P. mantiqueirensisAY772921.1, DQ792340.1. DNA sequences were aligned using MEGA7 software  and nucleotide (π) diversity indexes and the number of variable sites were calculated with Arlequin 3.5.1.2 , gene diversity (GD), allelic richness (AR) and inbreeding coefficient (FIS) for each locus. The frequencies of the null alleles, polymorphic index content (PIC), levels of observed (Ho) and expected (He) heterozygosity, and any significant deviations from the Hardy–Weinberg equilibrium (HWE), in order to evaluate the informativeness of the markers, were estimated using Cervus 3.0.3 software   approach (bc 2.1.0 . The prit (time since demographic change in scenarios 2 and 3); scenario 4: Ne4 (current Ne), Nb (Ne during bottleneck), Na4 (pre-bottleneck Ne), t2 and t1 . Demographic prior values are presented in 2 and t1, scenario 4), we first used as reference the maximum the influence of the Last Glacial Maximum  and as the minimum the first record of Araucaria forest expansion in the region . We employed Maxent 3.4.1 , Last Glacial Maximum  and Last Interglacial  conditions. Explanatory variables included a set of 19 bioclimatic Raster layers at a 30 arc-second resolution (ca. 1 km2 at the Equator) from the WorldClim website, version 1.4 (available at: http://www.worldclim.org/). The Raster package (http://www.R-project.org) was used to calculate Pearson’s correlation between variables. For P. mantiqueirensis, four variables  with pairwise Pearson correlation coefficients below 0.7 and with a high percentage of model contribution  were used to reconstruct the taxon distribution. For C. elegans, we employed seven variables .Ecological niche modelling (ENM) was used to compare niche preferences and potential range shifts that the species can suffer from in response to future climatic oscillations. Localities from the two species were obtained from field observations by the authors, SpeciesLink  long (471 bp corresponding to trnH-psbA and 650 bp to trnS-trnG). The sequences did not present any variable site, producing a single haplotype. The GC content was 28.1 % in trnH-psbA and 30.5 % in trnS-trnG. For C. elegans, the sequences of 81 individuals produced a concatenated alignment with 1191 bp (445 bp for trnH-psbA and 746 bp for trnS-trnG), leading to two different haplotypes. The GC content was 27.2 % in trnH-psbA and 30.7 % in trnS-trnG. Haplotype diversity in C. elegans was h = 0.0299 ± 0.0287, and nucleotide diversity was π = 0.004 ± 0.014 %. The frequency of each haplotype was CEh1 = 0.98 and CEh2 = 0.02; CEh2 was observed only at one collection site (Eleg1).The plastid intergenic spacers, P. mantiqueirensis were genotyped using 12 SSR loci, being 10 polymorphic and two monomorphic (PM110 and PM195) for this species. All loci were in linkage equilibrium . The 81 individuals of C. elegans were genotyped based on 14 SSR loci, from which five markers were polymorphic and nine monomorphic . All analyses were conducted based exclusively on polymorphic markers for each species, although the monomorphic loci are suggestive of low genetic variability and high homogeneity in these species as all of them are polymorphic among other Petunia and Calibrachoa species, respectively.The 38 individuals of Petunia mantiqueirensis presented a total of 39 alleles, with an average of 3.9 alleles per locus (2 to 7). The mean PIC value was 0.39 (0.05 to 0.75), and considering all loci and gene diversity, it was, on average, 0.36 (0.06 to 0.80). Allele richness was, on average, 3.33 (2.00 to 7.00). Six loci exhibited a significant deviation from HWE across all samples, three of them had a deficit of heterozygotes and the other three displayed an excess of heterozygotes. Tests for HWE indicated no significant FIS values for all loci and populations and the mean estimated frequency of null alleles was lower than 0.5 %  over all loci was 0.087 (P < 0.05). Five FST pairwise comparisons had significant genetic differentiation, ranging from 0.07 to 0.25 (P < 0.05). The Mant3 had significant differentiation from four other populations , as well as Mant4 vs. Mant1 (FST = 0.25) [see]. With the DAPC analysis, the individuals from a same collection site did not preferentially form groups and individuals from Mant4 and Mant7 were the most differentiated [see].The AMOVA indicated that the percentage of variation within populations (91 %) was higher than among populations (9 %), and the fixation index  and the PIC values for these markers ranged from 0.47 to 0.84 (average 0.63). The gene diversity was, on average, 0.54 (0.45 to 0.73) and allele richness 5.32 (3.06 to 8.42). All loci exhibited a significant departure from HWE across all samples with heterozygote deficits . Moreover, three of them had significant and positive FIS values, whereas one presented a significant and negative FIS value. The estimated frequency of null alleles was lower than 0.5 % in C. elegans (P. mantiqueirensis (FIS) [see]. Eleg2 had the highest average of FIS with one locus presenting significantly positive values. Furthermore, the populations had different numbers of loci with significant deviations from HWE, and all presented a deficit of heterozygotes at least in one locus. All populations also featured private alleles. Note that Eleg2 was the site with the lowest genetic diversity as measured by number of alleles (A) and allele richness (AR) [see]. When we consider the species’ global values  over all loci was 0.281. FST pairwise comparisons had significant and strong genetic differentiation in just the Eleg2 compared to the other sites . With the DAPC, individuals from Eleg2 were completely differentiated from the others, whereas a high superimposition was observed among the other collection sites [see].The AMOVA analysis revealed that the percentage of variation within populations (72 %) was higher than among populations (28 %) and the fixation index (P. mantiqueirensis and C. elegans using the ABC approach reflected that the most likely scenario for both species is that in which the population size in the past was much greater than in the present (C. elegans was very high according to the logistic approach (above 0.98), and a little smaller with the direct approach, above 0.90 (95 % CI = ~0.65–1.0), and extremely high for P. mantiqueirensis above 0.99 and 1.0, respectively. Confidence in the choice of these scenarios were high for the C. elegans and very high for P. mantiqueirensis, in which the predictive errors were 0.16 and 0.0, respectively [see]. The posterior model checking for scenario 2 in P. mantiqueirensis presented a good fit while for C. elegans the fit for one of the summary statistics was not good [see]. The posterior density estimates of the demographic parameters for scenario 2 are found in C. elegans while it is higher in t for P. mantiqueirensis[see]. The estimated time since the population reduction presented a wide confidence interval, the model time for P. mantiqueirensis was older than for C. elegans  and it was not younger than ~5 kya for either  curve’ (AUC)  all testent time , suitabl elegans  were furP. mantiqueirensis and C. elegans, which represent the northern limit of these genera’s distribution. Each one of these species grows in disjunct habitats from the rest of species in their respective genera, in micro-environments located at extremes of elevation  and C. heterophylla .The single haplotype found for Petunia mantiqueirensis features a strict relationship with its bee pollinator (Pseudagaspostemon fluminensis) that forages pollen on plants returning at the same plot between 5 and 13 days after the first visit . The maximum foraging distance for this genus of small and solitary bees is a maximum of 300 m  combined with the oscillations in the size of the species distribution areas based on the past modelling distribution may be a probable explanation for the lack of variation in C. elegans. Despite C. elegans being described as self-incompatible and bee-pollinated  to confirm the endangered status for these two species, iversity  resultiniversity . Our finP. secreta , the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Programa de Pós Graduação em Genética e Biologia Molecular da Universidade Federal do Rio Grande do Sul (PPGBM-UFRGS). A.B. was recipient of National Institutes for Science and Technology (INCT) in Ecology, Evolution and Biodiversity Conservation scholarship. G.M. was supported by PNPD-CAPES/PPGBot-UFRGS and C.T. was supported by PNPD-CAPES/PPGBM-UFRGS.A.B. and L.B.F. planned, designed and led the project; A.B., G.M., J.N.F. conducted the experiments; A.B, C.T., A.L.A.S., G.M. and S.L.B. ran the analyses; A.B. and L.B.F. wrote most of the text; L.B.F. provided reagents and equipment to develop the experiments. All authors contributed in the preparation of the study and have commented on and approved the final manuscript.The following additional information is available in the online version of this article—Table S1. Petunia mantiqueirensis and Calibrachoa elegans collection points.Table S2. Characteristics and PCR conditions of the microsatellite loci amplified for Petunia mantiqueirensis and Calibrachoa elegans.Table S3. Prior values (minimum and maximum with uniform distribution) for the parameters employed for the four demographic scenarios  analyses.Table S5. Geographic coordinates of Petunia mantiqueirensis and Calibrachoa elegans occurrence records used in the ENM.Table S6. Characterization of 10 microsatellites analysed on Petunia mantiqueirensis collection sites.Table S7. Pairwise FST values estimated for seven Petunia mantiqueirensis collection sites. (*) Significant values (P < 0.01).Table S8. Characterization of five microsatellites analysed on Calibrachoa elegans collection sites.Table S9. AICc scores and area under the receiver operating characteristic curve (AUC) values of the tested models.Table S10. Data availability: Individual allele per microsatellite loci.Figure S1. Clustering of individuals according to a discriminant analysis of principal components (DAPC) scatterplot performed based on SSR genotypes for: (A) seven populations of Petunia mantiqueirensis and (B) four populations of Calibrachoa elegans.plz037_suppl_Supplementary_MaterialClick here for additional data file.plz037_suppl_Supplementary_Table_S10Click here for additional data file.


Background: Addition of nimotuzumab to weekly cisplatin and radiation improves outcomes in head and neck cancer. HPV negative oropharyngeal cancer has unsatisfactory treatment outcomes and is a candidate for escalation of treatment. We wanted to determine whether the addition of nimotuzumab to cisplatin-radiation could improve outcomes in these poor-risk tumors.Methods: This was a subgroup analysis of a phase 3 randomized study. In this study, locally advanced head and neck cancer patients undergoing definitive chemoradiation were randomly allocated to weekly cisplatin (30 mg/m2 IV)- radiation (66–70 Gy) {CRT arm} or nimotuzumab (200 mg weekly) -weekly cisplatin (30 mg/m2)-radiation (66–70 Gy) {NCRT arm}. The data of HPV negative oropharyngeal cancer was extracted from the database of this study for the analysis. HPV testing was done with p16 immunohistochemistry (IHC) staining and reported according to the CAP criteria. The outcomes assessed were progression-free survival (PFS), disease-free survival (DFS), locoregional control, and overall survival (OS). Interaction test was performed between the study arms and HPV status prior to doing any HPV specific analysis for each of the studied outcomes. Kaplan Meier estimates for 2 year OS with 95%CI was calculated. The hazard ratio was obtained using COX regression analysis.p = 0.000), locoregional control (p = 0.007) and overall survival (p = 0.002) but not for DFS (p = 0.072). The 2- year PFS was 31.5% (95%CI 21.5–42) in CRT arm versus 57.2% (95%CI 45.8–67.1) in NCRT arm . The 2-year LRC was 41.4% (95%CI 29.8–52.6) in the CRT arm versus in 60.4% (95%CI 48.7–70.2) in the NCRT arm . The addition of nimotuzumab also lead to an improvement in 2-year OS from 39.0% (95%CI 28.4–49.6) to 57.6% (95%CI 46.3–67.4) .Results: We had 187 HPV negative oropharyngeal cancers, 91 in the CRT arm and 96 in NCRT arm. The interaction test was significant for PFS (Conclusions: The addition of nimotuzumab to weekly cisplatin-radiation improves outcomes inclusive of OS in HPV negative oropharyngeal cancers. Locally advanced oropharyngeal cancers possess a unique challenge. The anatomical location along with multiple physiological functions associated with this structure makes open surgical resection a difficult and moribund procedure . Hence iEfforts to improve outcomes in locally advanced oropharyngeal cancer have met with limited success , 6. ReceTaking this into consideration, we decided to perform a subgroup analysis of the HPV negative oropharyngeal cancer cohort, to study the absolute improvement in 2-year outcomes with the addition of nimotuzumab. We compared 2 year progression free survival (PFS), disease free survival (DFS), locoregional control (LRC) and overall survival (OS) between both arms.We had 536 patients in the study, out of which 269 had primary in the oropharynx. P16 testing was feasible in 212 patients and 187 patients were p16 negative. These 187 patients are included in the current analysis. There were 91 patients in the cisplatin radiotherapy (CRT) arm and 97 in nimotuzumab-cisplatin radiation (NCRT) arm. The baseline characteristics were balanced between the 2 arms .p = 0.000), LRC (p = 0.007) and OS (p = 0.002) but not for DFS (p = 0.072), suggesting a differential impact of the addition of nimotuzumab with respect to HPV status.The interaction test for HPV status (positive & negative) was significant for PFS  in the CRT and NCRT arm respectively. The 2 year PFS was 31.5% (95%CI 21.5–42) in CRT arm versus 57.2% (95%CI 45.8–67.1) in NCRT arm . The multivariate analysis for PFS is shown in P-Value = 0.002). The results of sensitivity analysis performed for PFS using a composite endpoint of progression or death was in line with the above-mentioned analysis. The unadjusted and adjusted hazard ratios were 0.62  and 0.62 , respectively. The site of failure was locoregional in 45 patients (49.5%), locoregional with distant failure in 3 patients (3.3%) and distant failure in 12 patients (13.2%) in CRT arm. The patients with corresponding sites of failure in the NCRT arm were 32 (33.3%), 6 (6.2%) and 5 (5.2%), respectively.At the time of data censoring 103 events had occurred, 60 in CRT arm and 43 in the NCRT arm. The median PFS was 12.9 months (95%CI 8.47–17.3) versus 35.3 months (95%CI 22.10-NA) . The 2-year LRC was 41.4% (95%CI 29.8–52.6) in CRT arm versus in 60.4% (95%CI 48.7–70.2) in NCRT arm  while the adjusted hazard ratio was 0.63 . These results were confirmed in the sensitivity analysis. The unadjusted and adjusted hazard ratios were 0.68  and 0.69  in favor of the cisplatin-nimotuzumab arm, with the use of composite endpoint of locoregional failure or death.The improvement in PFS with the addition of nimotuzumab is largely contributed by an improvement in locoregional control. The median time to locoregional failure was 17.3 months (95%CI 12.0–56.3) and 60.3 months (95%CI 22.6-NA) in cisplatin arm and cisplatin-nimotuzumab arm respectively . The addition of nimotuzumab also led to an improvement in 2 year OS from 39.0% (95%CI 28.4–49.6) to 57.6% (95%CI 46.3–67.4) .The median follow up was 38.5 months (95%CI 36.2–46.3). At the data cutoff, 105 deaths had happened; 57 in cisplatin arm and 48 in the cisplatin-nimotuzumab arm. The median OS was 19.0 months (95%CI 14.2–23.4) in cisplatin arm while it was 35.9 months (95%CI 22.8–53.7) in the cisplatin-nimotuzumab arm (.3–67.4) . The ass.3–67.4) , a diffeThe data for treatment compliance and radiation technique are shown in The clinical adverse events were captured in 180 patients while laboratory adverse events were captured in 182 patients. The adverse events details between the 2 arms are shown in Supplementary Table 2.The results of the current study clarify the importance of treatment intensification in HPV negative oropharyngeal cancers. Locoregional control, progression-free survival and overall survival were improved with the addition of nimotuzumab to cisplatin and radiation. The absolute improvement in the 2 years LRC and PFS were around 20–25 percent. The corresponding improvement in OS was 18 percent. These improvements, in accordance with the ESMO magnitude of clinical benefit scale for curative treatment would classify as “A” .As opposed to HPV related oropharyngeal cancer, HPV negative oropharyngeal cancers have worse prognosis. The median PFS, LRC, and OS in the current study are however, lower than reported in literature from the western world , 11, 12,Addition of nimotuzumab as a radiosensitizer to weekly cisplatin and addition of cetuximab to carboplatin 5-FU resulted in an improvement in outcomes over weekly cisplatin and carboplatin-5 FU in locally advanced head and neck cancers , 9. HoweThe current study is not without limitations. It is a subgroup analysis. However, an interaction test was performed prior to doing the subgroup analysis, to determine whether nimotuzumab had a differential impact or not. The study had used weekly cisplatin and not 3 weekly cisplatin, and so, the outcomes of the analysis are only applicable for weekly regimen.This was a phase 3 randomized superiority study conducted between 2012–2018. The study protocol was approved by the institutional ethics committee and the study was conducted in accordance with national  and international guidelines  on human research. The detailed inclusion-exclusion criteria with study protocol are already published . The stuThe standard arm received radical radiation to a dose of 66–70 Gy with conventional fractionation over 6–7 weeks. Altered fractionation schedules were allowed in both arms if the Biologically equivalent dose (BED) was around 70Gy10. The chemosensitizer used was weekly cisplatin –30 mg/m2. The dose of 30 mg/m2 was selected as it was considered as standard at our centre and had been proven effective compared to radical radiation in a randomized study . In the HPV testing was done using p16 IHC. The procedure for performing HPV testing has been already published in the literature . InterprAll patients, post consenting, underwent screening procedures, which were complete blood count, renal function tests, liver function test, viral serology, pure tone audiometry, dental examination, speech – swallowing function testing, nutritional status examination and evaluation by a social worker for compliance. All patients underwent an examination under anesthesia for mucosal disease assessment and a contrast enhanced computed tomography (CECT) neck with upper thorax for staging. In patients who had N2 nodes, the imaging used as positron emission tomography (PET). The radiation was administered either via conventional 2-D technique or 3-D technique. Use of intensity-modulated radiotherapy was permitted as per physician’s discretion. The patients were assessed each week during the treatment phase. Post-treatment completion at 8 weeks, patients were assessed in a joint clinic with a PET. Further follow up was according to the study protocol. The patients were followed up until death.The primary endpoint was PFS. It was defined as the time in months from randomization until progression. Progression was defined as per RECIST version 1.1. The secondary endpoints were LRC, DFS and OS. Locoregional control was defined as the time in months from randomization till loco-regional progression. Overall survival was defined as the time in months from randomization till death.P-value of 0.05 or below), which suggested that the addition of nimotuzumab had a differential impact on outcome in accordance with HPV status, further analysis was done on HPV negative patients.Descriptive statistics were performed. Nominal and ordinal data between both arms were compared using Fischer’s test. An interaction test was performed between HPV status and outcomes . Only if the interaction test was positive  were reported. The 95%CI was calculated in accordance with Brookmeyer and Crowley method. The unadjusted hazard ratio (HR) and adjusted HR (adjusted for prognostic factors) with its 95%CI interval were calculated using the COX regression analysis with Efron’s method of tie handling, with CRT arm being considered as reference. The assumptions of proportional hazard model were checked using Schoenfeld residuals and assumptions were met for PFS and LRC. However, the assumption was violated for OS. Hence mean overall survival between the 2 arms were calculated using the restricted mean survival method. The difference between the restricted means between the 2 arms with its 95%CI were calculated. A The addition of nimotuzumab to weekly cisplatin-radiation improves outcomes inclusive of OS in HPV negative oropharyngeal cancers and this regimen should be considered as standard if 3 weekly cisplatin 100 mg/m2 regimen is not used.


Mycoplasma pneumoniae using multilocus sequence typing. Of the 146 M. pneumoniae strains collected during the 5 consecutive outbreaks of M. pneumoniae pneumonia during 2000–2016 in South Korea, macrolide resistance increased from 0% in the first outbreak to 84.4% in the fifth. Among the 8 sequence types (STs) identified, ST3 (74.7%) was the most prevalent, followed by ST14 (15.1%). Macrolide-susceptible strains comprised 8 different STs, and all macrolide-resistant strains were ST3 (98.3%) except 1 with ST14. The proportion of macrolide-resistant strains in ST3 remained 2.2% (1/46) until the 2006–2007 outbreak and then markedly increased to 82.6% (19/23) during the 2010–2012 outbreak and 95.0% (38/40) during the 2014–2016 outbreak. The findings demonstrated that clonal expansion of ST3 M. pneumoniae was associated with the increase in macrolide resistance in South Korea.To investigate the genetic background for the emergence of macrolide resistance, we characterized the genetic features of  Mycoplasma pneumoniae is one of the major causes of community-acquired pneumonia in children and adolescents  scheme was first applied in M. pneumoniae strains between outbreaks, we conducted an MLST analysis of M. pneumoniae detected from children in whom community-acquired pneumonia was diagnosed during 5 consecutive epidemics of M. pneumoniae pneumonia during 2000–2016 in South Korea. We also sought to find the genetic background that may explain the emergence of macrolide resistance among M. pneumoniae strains.To clarify the genetic diversity of M. pneumoniae strains detected from children with pneumonia at 5 hospitals during 5 consecutive outbreaks of M. pneumoniae pneumonia in South Korea: 2000, 2003–2004, 2006–2007, 2010–2012, and 2014–2016. Epidemic periods were previously defined by an interval spanning an increase of >5 cases/2 months over a 4-month period to a decrease of <5 cases/2 months over a 4-month period in the primary site of this study (M. pneumoniae pneumonia was diagnosed using the following criteria: 1) the presence of rales on auscultation or infiltration of the lung demonstrated on chest radiograph and 2) a positive PCR result for M. pneumoniae or isolation of M. pneumoniae on culture. Five hospitals participated in this study: Seoul National University Children’s Hospital (Seoul), Seoul National University Bundang Hospital (Seongnam), Eulji Hospital (Seoul), Chungnam University Hospital (Daejeon), and Chonnam University Hospital (Gwangju). Two hospitals in Seoul and 1 hospital in Seongnam cover the Seoul metropolitan area, where almost half of the South Korea population resides. Daejeon is representative of central South Korea, and Gwangju represents the south. M. pneumoniae strains detected from community-acquired cases were included for further analysis. We excluded healthcare-associated infections and intrafamilial infections.This study comprised M. pneumoniae or from nasopharyngeal aspirates using an extraction kit , according to the manufacturer’s instructions. We amplified the p1 gene by PCR for the detection of M. pneumoniae. Starting in 2010, M. pneumoniae was cultivated using pleuropneumonia-like organism broth and agar for nasopharyngeal aspirates or pleural fluid obtained from the patient as previously described , and a corresponding sequence type (ST) was given for each sample (http://pubmlst.org/mpneumoniae/). We used eBURST version 3 software (http://eburst.mlst.net/) to estimate the relationships among the strains and to assign strains to a clonal complex (CC) . A linear-by-linear association model was used for Pearson’s χThe institutional review board of Seoul National University Hospital approved the study protocol (IRB no. H-1012–007–341). Informed consent was exempted because nasopharyngeal aspirates were obtained as a standard of patient care to identify the etiologic agents of acute lower respiratory tract infections.M. pneumoniae DNA samples. Samples included for each outbreak were selected as follows: 21 samples from 2000, 14 samples from 2003–2004, 29 samples from 2006–2007, 37 samples from 2010–2012, and 45 samples from 2014–2016. Until the 2006–2007 outbreak, DNA samples were directly collected from respiratory samples (64 samples), and DNA samples from the 2010–2012 outbreak were extracted from cultured M. pneumoniae (82 samples).Our study comprised 146 >5 years of age. The remaining 187 samples that were not selected for this study did not differ significantly from selected samples with respect to mean patient age and geographic region.For the samples before 2010, we included all the available specimens because of a limited number of archived samples relative to the 2010–2012 and 2014–2016 outbreaks. During the 2 outbreaks for which we have a larger number of samples, we selected samples to represent geographic region, month of isolation, and ages of patients. The proportions of selected samples were 28.5% (2010–2012) and 32.4% (2014–2016) of the archived samples. Of the study population, 56.1% were male. The mean age of children was 6.5 years; 7.4% were <2 years of age, 32.1% were 2–5 years of age, 60.5% were M. pneumoniae strains investigated, 59 (40.4%) strains expressed macrolide resistance associated with either an A2063G (58 [98.3%]) or A2064G (1 [2.7%]) mutation in the 23S rRNA gene. Differences in macrolide resistance were recognized in each of the outbreaks . P1 subtype 1 was the main subtype at 70.6% (60 strains), followed by subtype 2 with 29.4% (25 strains). P1 subtype 1 accounted for 85.7% of the 2000 outbreak, 42.9% in 2003–2004, 75.0% in 2006–2007, 71.9% in 2010–2012, and 50.0% in 2014–2016. P1 subtypes 2 and 2a were observed up until the 2003–2004 outbreak, and subtype variant 2c was observed from the 2006–2007 outbreak. P1 subtype variant 2c (13 [52.0%]) was most common within P1 subtype 2, followed by subtype variants 2a (7 [28.0%]), 2 (4 [16.0%]), and 2b (1 [4.0%]). Each ST was associated with a single P1 subtype or subtype variant, except for ST14 strains, which possessed both P1 subtype variants 2a and 2c.Macrolide-susceptible strains consisted of 8 different STs identified. Among 8 STs, ST3 (58/109 [53.2%]) and ST14 (1/22 [4.5%]) were the only ones that expressed macrolide resistance . One ST1M. pneumoniae is responsible for the increasing proportion of macrolide resistance.We found a correlation between the increasing proportion of macrolide resistance and the proportion of macrolide-resistant strains within ST3. All of the strains in ST3 were macrolide susceptible up ntil the 2003–2004 outbreak, and only 1 of the 22 strains in ST3 showed macrolide resistance in the 2006–2007 outbreak. The proportion of macrolide-resistant strains within ST3 dramatically increased to 82.6% (19/23) during the 2010–2012 outbreak and to 95.0% (38/40) during the 2014–2016 outbreak . These dTwo CCs were identified on eBURST analysis . CC1 conM. pneumoniae strains. Traditionally, diagnosis of M. pneumoniae pneumonia relied on the increase of mycoplasma antibody or the presence of IgM. However, with the emergence of macrolide resistance, it became important to know the presence of macrolide resistance of M. pneumoniae. Thus, direct detection of M. pneumoniae either by culture or by PCR is crucial for testing macrolide resistance. Although no treatment strategy for macrolide-resistant M. pneumoniae pneumonia has yet been established, alternative antimicrobial drugs (tetracyclines and fluoroquinolones) can be considered when patients remain febrile at least 48–72 hours after macrolide treatment  was related to changes in genetic backgrounds of M. pneumoniae is ≈820 kb and has up to 700 coding operons. On the basis of the data of the comparative analysis of 58 strains, the genome appears to be highly conservative among the strains  to determine the association of P1 subtypes with macrolide resistance  for characterization and classification. Waller et al. reported 7 different MLVA profiles associated with certain P1 subtypes from 12 M. pneumoniae strains during an outbreak in the United States , which was significantly higher than in other MLVA strains  to 9.51 DID (2014), with a peak of 11.99 DID in 2011. Therefore, the increase in macrolide consumption might explain in part the 17-year (2000–2016) change of macrolide resistance in M. pneumoniae. Nevertheless, the expansion of a single clone, which was demonstrated by our study, makes us assume another possible explanation. We think the macrolide-resistant clone, which we characterize as an ST3 clone by MLST, might have been introduced before the 2010–2012 outbreak and spread extensively among the community where the population density is high, supported by antimicrobial selective pressure.Antimicrobial pressure would have played a role to some extent because South Korea is a high antimicrobial drug use country. Trend analyses of the national data on antimicrobial drug consumption  demonstrated an increase in macrolide use in the community during 2005–2014  (Although various studies have investigated the genetic association of M. pneumoniae strains were collected from 5 hospitals, the data might not represent reality nationwide. However, this was the best possible multicenter-based study we could devise, given that no nationwide surveillance system exists. Second, despite our best efforts, the numbers of strains in the earlier outbreaks were smaller than those collected in the later outbreaks, when culture was performed prospectively. However, this study is of value because the distribution of STs in a certain region for a relatively long period of time is well described. The finding that an expansion of a single ST contributed to the increase in macrolide resistance is a potentially powerful insight for further research. Additional studies that investigate the epidemiology and mechanism of acquiring macrolide resistance will give further insight into better treatment strategies. In particular, further studies should be addressed to in silico methods for the analysis of genetic background that can explain macrolide resistance within ST3 strains. Recent advances in microbiology have made whole-genome sequencing a valuable investigation tool that can lead to the identification of a specific genotype associated with macrolide resistance or virulence of M. pneumoniae.Our study has several limitations. First, even though M. pneumoniae pneumonia that showed substantial increase in macrolide resistance in South Korea, all but 1 macrolide-resistant strain was ST3. These findings demonstrate that clonal expansion of an ST3 M. pneumoniae was associated with the increase in macrolide resistance in South Korea.In summary, we found that, during outbreaks of


Adalimumab is one of the top-selling drugs worldwide. Its imminent patent expiration has seen the emergence of numerous biosimilar agents. In this article, we recap the evidence from bio-originator trials in rheumatoid arthritis (RA) to provide context for a critical review of biosimilar trial data.Currently, three adalimumab biosimilars are approved in Europe and/or the USA: Amgen’s ABP 501 (AMJEVITA/Solymbic), Boehringer Ingelheim’s BI 695501 (Cyltezo) and Samsung Bioepis’s SB5 . All three agents met their pre-specified equivalence criteria. Subtle differences in adverse events and clinical responses between the reference and biosimilar products were noted.The introduction of adalimumab biosimilars will offer exciting opportunities in improving treatment access and increasing treatment options for RA and other licensed indications. Real-world data will further provide assurances on efficacy as well as safety. Rheumatoid arthritis (RA) is a chronic, autoimmune, systemic inflammatory disease. If inadequately treated, it can lead to irreversible joint damage and disability at significant costs to the individual as well as the wider economy. Life expectancy of patients with severe RA is reduced by 10 years on average , whilst With improved understanding of RA’s molecular pathophysiology, increasingly targeted treatments were developed in addition to conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) such as methotrexate (MTX). Around the turn of the century, biologic drugs were introduced that selectively targeted tumour necrosis factor (TNF). TNF inhibitors (TNFi), along with other biologic DMARDs (bDMARDs), formed an effective second-line for those with inadequate response to csDMARDs and dramatically improved mortality and outcomes for RA patients –7.In 2002, Humira, the originator adalimumab, became the third TNFi to be approved in the USA after infliximab and etanercept. It is the best-selling drug worldwide, with global sales of $18 billion in 2017 alone . It has The inevitable patent expiration for TNFi stimulated programmes to develop molecules that, whilst not identical to the originator, could be considered to be a biological equivalent of a ‘generic’. Such ‘biosimilars’ are defined as biological agents that are similar in terms of quality, safety and efficacy to the licensed reference product (RP) . BiosimiIn this article, we summarise the pharmacology and clinical efficacy of adalimumab to provide context for a critical review of evidence from randomised controlled trials of its biosimilars in RA. We focus on biosimilars that are approved by the European Medicines Agency (EMA) and/or the US Food and Drugs Administration (FDA).Adalimumab is a recombinant, fully human, IgG1 monoclonal antibody that is structurally and functionally indistinguishable from naturally occurring human IgG1  to adalimumab . ADAb caThe efficacy of adalimumab in both early and long-standing RA has been demonstrated by several phase III randomised controlled trials Table  and openAs monotherapy, adalimumab recipients had significantly better outcomes than placebo in terms of function, ACR and EULAR responses . The expAdalimumab monotherapy was not superior to MTX monotherapy in terms of ACR response or DAS28 remission over 2 years in the PREMIER study  . It was,Adalimumab combination therapy with MTX was superior to adalimumab or MTX monotherapies for all clinical and radiographic outcomes in early as well as established disease –19. ACR Other bDMARDs were similar in efficacy to adalimumab in head-to-head trials –24, netwBiosimilar approval does not require the manufacturer to re-establish efficacy but is instead based on the demonstration that there are no clinically meaningful differences from the RP , 31, 32.At the time of writing, three adalimumab biosimilars are approved in the EU and/or the USA: Amgen’s ABP 501, Boehringer Ingelheim’s BI 695501 and Samsung Bioepis’s SB5. In the following sections, we will review each in terms of clinical outcomes and trial characteristics. Other biosimilars will be mentioned in passing, including FKB327 by Fujifilm Kyowa Kirin Biologics which has phase III RA trial published in abstract form , 34; SanAmgen’s ABP 501 was the first adalimumab biosimilar to be approved by the FDA in 2016 (as AMJEVITA) and by the EMA in 2017 (as AMGEVITA/Solymbic). The manufacturer demonstrated that ABP 501 and the RP were highly similar in structure, function and pharmacokinetics (PK) –42. SubsThe RA trial was a randomised, double-blind, active comparator-controlled, 26-week equivalence study, comparing ABP 501 with US- and EU-sourced RP •. ParticThe primary endpoint was the risk ratio (RR) of achieving ACR20 at week 24, which was achieved in 74.6% of the ABP 501 group and 72.4% in the RP group )—numerically higher than response rates from pivotal RP trials. Results of the per-protocol set (PPS) analysis were similar (raw results were not provided). The ACR20 RR was 1.039 (90%CI 0.954 to 1.133), which was within the margin of 0.738 to 1.355 required to demonstrate equivalence. Equivalence was also demonstrated in patients with plaque psoriasis as monotherapy .In the RA study, the RR of ACR20 for ABP 501 was 1.421 (95% CI 1.086 to 1.860) after 2 weeks, suggesting superiority over the RP , 46. ThiThe proportions of binding ADAb (38.3 vs 38.2%) and neutralising ADAb (9.1 vs 11.1%) were similar for ABP 501 and the RP, respectively. More patients discontinued treatment (6.8 vs 4.6%) and the study (8.0 vs 4.2%) in the ABP 501 group; there were no specific AEs leading to withdrawal. Liver enzyme elevations occurred in 4.9% of the ABP 501 group and in 3.8% in the RP group. This signal for ABP 501 was also seen in the psoriasis study (5.9 vs 2.5%) . HoweverInjection site reactions were less common in the ABP 501 group (1.7 vs 5.2%). This was also seen in the psoriasis trial (1.3% v 3.8%) and explained as differences in the excipients rather than biosimilarity.Boehringer Ingelheim’s BI 695501 (Cyltezo) was approved by the EMA and FDA in 2017. It was shown to be similar to the RP in structure, function and PK , 48•. EqVOLTAIRE-RA was a randomised, double-blind, parallel-arm, 58-week study, comparing BI 695501 with US-sourced RP •. The trCo-primary endpoints were percentage of patients achieving ACR20 at 12 and 24 weeks (using FAS). At week 12, 67.0% of BI 695501 recipients and 61.1% of RP recipients achieved ACR20. The difference  was within − 12 to 15% set by the FDA to demonstrate equivalence; PPS results were similar . At week 24, 69.0 and 64.5% of BI 695501 and RP recipients achieved ACR20, respectively. This difference of 4.5% (90%CI − 3.4 to 12.5) was within ± 15% set by the EMA . Secondary endpoints  were also similar.There was a trend for superior ACR20 response for BI 695501 . At weekThere were no clinically meaningful differences in proportions of ADAb ; neutralising ADAb prevalence was also similar (raw results were not provided). The number of patients with at least one AE was 59.6% in the BI 695501 group and 60.0% in the RP group. Several safety outcomes were in favour of BI 695501: serious AEs (5.6 vs. 9.7%), injection site reactions (1.9 vs 5.7%), serious infections (0.6 vs 4.0%) and hypersensitivity reactions (2.8 vs 4.6%). However, three aspects of safety initially raised the concern about the safety profile of BI 695501. First, haematological disorders were more frequent in the continuous BI 695501 group than continuous RP group (5.2 vs 2.9%): anaemia  and ‘haemoglobin decreased’  were reported exclusively in the BI 695501 group. These AEs were mostly mild to moderate and did not lead to drug discontinuation. Furthermore, five of these patients had low haemoglobin at baseline. Second, bone fractures occurred exclusively in the BI 695501 group . This was also observed in the PK trial [Samsung Bioepis’s SB5  was approved by the EMA in 2017. Again, it was similar to the RP in structure, function and PK , 51•. ItThe primary endpoint was ACR20 response rate at week 24 using PPS, which was achieved by 72.4% of the SB5 and 72.2% of the RP group. The difference of 0.1% (95%CI − 7.8 to 8.1) was within ± 15% required to demonstrate equivalence. Results using FAS were similar  with a difference of 0.8% (95%CI - 7.0 to 8.6).ACR20 and ACR50 at week 52 showed a trend of superior response in both SB5 and SB5/RP-switch groups compared to the RP group, using the PPS  but not using FAS [The incidence of ADAb was similar between SB5 and RP groups . Overall, treatment emergent AEs occurred in 35.8% of the SB5 and 40.7% of the RP group; 10.1 and 11.7%, respectively, were considered related to the study drug. The number of injection site reactions was higher in RP recipients (32 reactions in 4 patients) compared to SB5 recipients (9 reactions in 8 patients) at week 52, although the proportions of patients were comparable . The rate of discontinuation due to AEs was lower in the SB5 than RP group .An interesting observation in biosimilar trials was that clinical responses in their RP groups were often different from responses reported in pivotal RP trials. For example in the EGALITY plaque psoriasis study, PASI75 response (75% improvement in Psoriasis Area and Severity Index) of the etanercept RP group (76%) was much higher than in previous RP studies (47 to 49%) –55. SimiUnlike the pivotal RP trials that were mostly conducted in North America, trials for SB5, ABP 501 and BI 695501 were predominantly conducted in Eastern European countries Table . These dn = 273) were rerandomised to switch to SB5 (n = 125) or to continue with the RP (n = 129) at week 24, until the end of the study at week 52. Switching had no impact on efficacy, safety or immunogenicity [n = 321) were rerandomised at week 24 to switch to BI 695501 (n = 147) or remain on the RP (n = 148) until week 42 [These subtle differences in trial characteristics, as well as minor discrepancies in rare safety signals, should be considered when drawing conclusions on the true biosimilarity of these agents. This has implications for switching between the RP and biosimilars. Unlike biosimilar infliximab and etanercept •, there genicity . An opengenicity , 64. In  RP n = 1 until weHowever, these RCTs were not powered to show the significance of differences in rare AEs. In addition, trials have stringent inclusion and exclusion criteria that may not reflect real-world patients, such as those with comorbidities. Therefore, pharmacovigilance studies are essential to gather clinical evidence of the benefits and risks of switching in all patients. We refer the reader to reference • for a mThe year 2018 will see several biosimilars of adalimumab emerge in clinical practices worldwide. The three EMA/FDA-approved agents discussed in this review each have robust RCT evidence that meet pre-defined equivalence criteria, although subtle differences in clinical responses and AEs between the reference and biosimilar products were noted. As with TNFi biosimilars already on the market, real-world data and pharmacovigilance studies are critical to developing long-term evidence to provide assurances on efficacy as well as safety. These adalimumab biosimilars, and many more in the pipeline, will offer exciting opportunities in improving treatment access and increasing treatment options worldwide.


The present work was undertaken to determine the possibility of using xanthan and guar gums as stabilizers to enhance the yogurt quality.Yogurt was manufactured from standardized milk  with the addition of 2% to 3% starter culture. Enzyme-hydrolyzed xanthan gum  and guar gum  were added to the yogurt as stabilizers. Prepared yogurt samples were kept at refrigeration temperature (4°C±2°C) for 21 days and various quality and sensory parameters were studied at regular intervals (7 days).Results showed that yogurt with 0.5% xanthan gum (T5) was best in terms of preventing syneresis and improving the viscosity, water holding capacity and texture of the product. Additionally, adding gums did not adversely affect the sensorial attributes of the product.Modified gums were found useful in augmenting yogurt quality and therefore addition of gums is highly recommended for manufacturing yogurt. Fermented foods have been extensively consumed around the globe because of their nutritional importance and improved sensory attributes. Traditionally available fermented products in the market include yogurt, cheese, kefir, nonalcoholic and alcoholic beverages, several types of breads and other bakery products, vinegar and different fermented vegetables . FermentYogurt is considered as the most common fermented dairy product and has been con sumed by a large group of people as a part of diet or refreshing drink. It is a multi-faceted colloidal arrangement developed due to the binding of water molecules with prolonged tiny structures of proteins . Yogurt Globally, the demand for different types of yogurts has been increased due to more concern about product quality and consumer’s satisfaction. This augmented demand for yogurt consumption has been ascribed to improved knowledge regarding the health assistances of yogurt, growing availability of fruit and flavored yogurts and the diverseness of product presentations. Furthermore, yogurt is thought to be healthy due to its probiotic effect. Yogurt bacteria are known as probiotics and possess various health-promoting characteristics including prevention from gastrointestinal disorders, enhancement of lactose digestion by mal-digesters, mitigation of cancer risks, lowering blood cholesterol levels, strengthening the immune system and aiding the body in protein, calcium and iron assimilation .Viscosity of the yogurt is generally influenced by homogeni zation process, heat treatment and yogurt processing conditions whereas syneresis usually onsets due to several factors such as high incubation temperature, low solid contents in the milk, excessive whey protein to casein ratio and physical mishandling of the product during processing, storage and transportation. The two major problems associated with yogurt are changes in the viscosity and leakage of whey proteins i.e., syneresis both of which negatively affect the yogurt quality. To overcome these glitches and to enhance the product functionality, the most common approach is the use of different stabilizers, ingredients added to the food products for smoothening and providing the uniform structure to the product. Additionally, stabilizers are also helpful in keeping the flavoring compounds in the dispersed form resulting in the maintenance of yogurt viscosity. Stabilizers also make strong networks with casein molecules which ultimately reduce the problem of syneresis and improve yogurt texture .Different types of stabilizers like starch, gelatin, pectin and hydrocolloidal gums (e.g. xanthan gum and guar gum) are used to enhance the quality of yogurt. The prime focus of incorporating stabilizers in the yogurt is to improve its characteristics such as appearance, stability, viscosity, mouthfeel and texture. Additionally, sensorial attributes of yogurt are also positively affected by adding different stabilizers. Amongst various types of stabilizers, gums are thought to be most appropriate because of their high gelation properties. Commonly used gums as stabilizers include guar gum, xanthan gum, carrageenan, locust bean gum, gum acacia, konjac and tara gum . Among tSeveral investigations have confirmed the potential of xan than gum and guar gum as stabilizers to improve yogurt stability and minimize the problem of syneresis . For insThe research work was conducted in Food Microbiology and Biotechnology Laboratory of National Institute of Food Science and Technology (NIFSAT), University of Agriculture, Faisalabad-Pakistan. Details of the work are provided in this section.Standardized pasteurized milk was procured from Nestle Pakistan (Pvt.) Ltd. Modified gums (xanthan and guar gum) and other chemicals needed to conduct various analyses were purchased from Sigma Aldrich , Oxoid , and Merck .0 = control (without gum addition); T1 = 0.1% enzyme hydrolyzed xanthan gum (EHXG); T2 = 0.5% EHXG; T3 = 1.0% EHXG; T4 = 0.1% enzyme hydrolyzed guar gum (EHGG); T5 = 0.5% EHGG; T6 = 1.0% EHGG. Yoghurt was prepared from standardized milk (3.5% fat) by adding 2.5% of starter culture  containing Lactobacillus bulgaricus and Streptococcus thermophilus. Pre-culture was prepared by dissolving 10 mg of freeze-dried culture in 50 mL milk followed by activation at 40°C for 30 minutes. Afterwards, the prepared cultured was used in yoghurt preparation at 2.5%.Xanthan gum and guar gum were added in different concentrations as stabilizers followed by yogurt preparation according to the flow diagram illustrated in The prepared yogurt was subjected to the following analyses on weekly basis for 21 days.The viscosity of yogurt was estimated by using Brookfield LVDVE-230  viscometer. Before viscosity determination, yogurt was stirred for 40 seconds. Afterword, viscosity was measured with the viscometer at 15°C±1°C using spindle number 4 (10 rpm). Viscometer reading was noted in centipoises (CPS) units and percent torque.The whey released by the yogurt samples was analyzed by taking 5 mL of yogurt followed by centrifugation at 5,000 rpm for 20 min at 4°C and separated whey was measured after 1 min. Amount of whey separation was expressed as volume of separated whey per 100 mL of yogurt.Digital pH meter was used for pH determination of yogurt samples according to the method given in AOAC . pH meteAcidity was determined by direct titration method described by AOAC . For thiTotal solid contents (TSCs) were determined by following the protocol described in AOAC . PurposeWater holding capacity (WHC) was determined by the method as described by Alvarez-Sabatel and coworkers . Twenty Textural analysis was performed on texture analyzer  using back extrusion plate Probe P-75 (75 mm Dia.) . TextureThe sensory evaluation of prepared yogurt samples was carried out by using 9-point hedonic scale  at different storage intervals . AccordiThe obtained data were subjected to statistical analysis using completely randomized design (CRD) under 2-factor factorial arrangement . All sta5 (0.5% EHGG) during storage  followed by T6 (1.0% EHGG) i.e. 2,710.5 cps, while the lowest value was recorded for T0 (control) i.e. 1,721.6 cps.Results regarding the viscosity determination of yogurt samples containing varied amounts of modified gums are presented in 2 (0.5% EHXG) secured minimum increase in syneresis percentage i.e. 38% to 62.42% followed by T5 (0.5% EHGG) i.e. 38% to 63% during the storage kinetics.It is evident from the inferences regarding syneresis of yogurt that the syneresis percentages varied in a significant manner due to treatments and storage time. Among all the treatments, an increasing trend in the syneresis percentages was observed from Day 0 (38%) to the 21th day of storage (71.42%) . The res5 (0.5% EHGG) i.e. 4.33, trailed by T4 (0.1% EHGG) i.e. 4.32 whereas the lowest mean pH value (3.82) was recorded in T1 (0.1% EHXG) and T3 (1.0% EHXG) collectively. Additionally, a decreasing pattern was observed in pH values (4.34 to 3.98) during the storage period of 21 days.3 (1.0% EHXG) and T6 (1.0% EHGG) while the lowest acidity value (0.98%) was acquired by T1 (0.1% EHXG) and T4 (0.1% EHGG).The results pertaining acidity analysis of yogurt containing modified gums are also depicted in 5 (0.5% EHGG) i.e. 11.97% followed by 11.96% in T6 (1.0% EHGG) at the end of storage period.5 (0.5% EHGG) had the maximum value for WHC (79.80%) at the start of the trial and remained highest at the termination of storage time (52.40%).The statistical data concerning WHC of yogurt samples indicated that WHC decreased during storage due to increase in acidity and syneresis. The results in 5 (0.5% EHGG) and T6 (1.0% EHGG) whereas lowest value (0.66) was observed in T0 (control).Data concerning the textural profile of yogurt containing modified gums revealed that both factors (treatments+storage time) imposed a significant effect on the textural attributes of yogurt . Results5 (0.5% EHGG) followed by T2 (0.5% EHXG) i.e. 28.27. On the other hand, the lowest consistency value (17.82) was attained by T0 (control). Similarly, cohesiveness results also showed that variations were highly significant among treatments and storage days. Highest mean value of cohesiveness was −0.40 in T5 (0.5% EHGG) trailed by T6 (1.0% EHGG) i.e. −0.39 and the lowest value (−0.26) was shown by T0 (control) and T1 (0.1% EHXG). Data regarding adhesiveness values depicted that highest retention of adhesiveness (3.14) was found for T5 (0.5% EHGG) followed by T4 (0.1% EHGG) whereas lowest value (1.89) was documented for T0 (control).Results regarding the variations in consistency of the stored yogurt are also presented in 0 (control), 7.37 in T1 (0.1% EHXG), 7.33 in T5 (0.5% EHGG), 6.66 in T2 (0.5% EHXG) and T6 (1.0% EHGG), 6.62 in T4 (0.1% EHGG) and 6.37 in T3 (1.0% EHXG). Flavor scores showed a decreasing trend during 21 days of storage and values decreased from 7.57 to 6.21 from 0 to 21st day of storage. Among the mean values of treatments, T5 (0.5% EHGG) had the highest score i.e. 7.33 followed by T1 (7.21) whereas T3 (1.0% EHXG) got the lowest score i.e. 6.41. Results also revealed that T5 (0.5% EHGG) showed better sensory scores as compared to the other treatments.Results regarding sensory parameters viz. appearance, flavor, body and texture mouth feel and overall acceptability, of yogurt samples are shown in 5 (0.5% EHGG) had highest score for mouthfeel i.e. 7.33 followed by 7.16 in T1 (0.1% EHXG), and lowest value (6.37) was attained by T3 (1.0% EHXG). Results also illustrated that color scores decreased during the storage time from 7.57 at 0 day to 6.21 at 21st day of storage. Mean values for treatments exhibited highest color score in T5 (7.33) followed by 6.83 in T6 (1.0% EHGG) while the lowest value was 6.41 for T3 (1.0% EHXG). Additionally, texture scores for different treatments of yogurt also decreased during storage ranging from 7.28 (Day 0) to 6.30 (Day 21). T5 (0.5% EHGG) showed greatest value i.e.7.33 followed by T1 (7.20) whereas lowest score was obtained by T4 (6.50). Results also indicated that overall acceptability of the product was also affected in a significant way by stabilizers during storage. Amongst treatments, highest mean value was observed in T5 (7.87) followed by 7.20 in T1 (0.1% EHXG) whereas lowest sensory score (6.37) was recorded T4 (0.1% EHGG) and T2 (0.5% EHXG).Statistical results regarding mouth feel depicted a highly significant effect of treatment and storage days on mouth feel of yogurt while the effect of their interaction (days×treatments) was non-significant. Sensory scores for mouth feel decreased during storage from 7.21 on 0 day to 6.33 on 21st day of storage. TUse of various stabilizers in cultured dairy products is quite important to control the problem of phase separation. Stabilizers also prevent syneresis and provide smooth mouth sensation by binding water. Some stabilizers also interact with proteins and increase hydration. In yogurt, stabilizers are generally used to increase viscosity, prevent syneresis and improve mouth-feel. Similarly, results of the present work show the role of hydrolyzed gums in improving the quality of yogurt. Findings of the current project demonstrated that viscosity was decreased with the storage period, but gum-containing yogurt showed higher viscosity as compared to the control group. The improved viscosity due to addition of stabilizers to yogurt is attributed to enhanced shear-thinning, time-dependency and viscoelasticity of the product. Polysaccharides also absorb water and swell which ultimately results in increasing the viscosity. However, a decrease in the values of viscosity with the passage of time was due to the development of syneresis during the storage period. The outcomes of the present investigation are supported by the findings of Ramasubramanian et al  who obseAdditionally, the augmented percentage s of syneresis were due to an increase in the acidity of yogurt with the passage of time which resulted in separation of whey proteins (serum). However, yogurt samples containing modified gums showed a smaller increase in syneresis percentages during the storage which can be described to the stabilizing potential of added gums. Stabilizers generally control the problem of syneresis in dairy products by binding the water molecules which reduces flow of water in the matrix space. The findings of present investigation are in accordance with the work of Galal et al  who demoA decreasing pattern was observed in pH values during the storage period which was attributed to the conversion of lactose into lactic acid with the passage of time. Consequences of the current project are in line with the findings of Mazloomi et al  who infeOutcomes of the present investigation also postulated that WHC values of all the yogurt samples were decreased during the storage but comparatively less reduction was noticed in samples containing gums. The current findings are supported by the results of Galal et al  who repoResults regarding sensory evaluation of yogurt samples de picted that sensory parameters viz. appearance, mouthfeel, flavor, color, texture and overall acceptability of the product were affected in a significant way by stabilizers during storage. Results showed that addition of gums as stabilizer increased the overall acceptability of yogurt. Findings of the present work are in harmony with Milani and Koocheki  who conc5), the maximum reduction in syneresis and retention of WGC of the yogurt was obtained as compared to other fractions and types of gum. These findings imply that use of hydrolyzed xanthan and guar gums could produce the yogurt with high quality and better sensory attributes. Hence, it can be concluded that use of xanthan and guar gums can be promoted at industrial level to improve the texture, firmness and viscosity of yogurt and to reduce the problem of syneresis during the marketing and storage of yogurt.The present work demonstrates that use of xanthan and guar gums as stabilizers is an effective approach in improving the yogurt quality by enhancing viscosity, WHC and textural profile of yogurt and combating the problem of syneresis. It was concluded from the study that EHGG at 1.0% showed best results for physicochemical, textural and sensory properties of yogurt. It was also observed that with the addition of 0.5% EHGG (T


There was a mistake in the original code evaluating the occupation number of polymers. That mistake resulted in overestimated values of occupation numbers as compared to values obtained after the mistake was corrected (compare original One correction should be made also in the main text on p. 320, where 14.0 vs. 7.8 should be replaced with 14.0 vs. 9.9.