Heroin addiction is a growing concern, affecting the socioeconomic development of many countries. Little is known about transgenerational effects on phenotype changes due to heroin addiction. This study aims to investigate changes in level of anxiety and aggression up to four different generations of adult male rats due to paternal exposure to heroin.Male Sprague–Dawley rats were exposed with heroin intraperitoneally (i.p.) twice-daily for 14 days with increasing dosage regimen (F0-heroin). Male Sprague–Dawley rats (6-weeks-old) were divided into: (1) heroin exposed group (F0-heroin) and (2) control group treated with saline solution (F0-control). The dosage regime started with the lowest dose of 3 mg/kg per day of heroin followed by 1.5 mg/kg increments per day to a final dose of 13.5 mg/kg per day. Offspring were weaned on postnatal day 21. The adult male offspring from each generation were then mated with female-naïve rats after 2 weeks of heroin absence. Open field test and elevated plus maze test were used to study the anxiety level, whereas resident intruder test was used to evaluate aggression level in the addicted male rats and their offspring.Heroin exposure in male rats had resulted in smaller sizes of the litters compared to the control. We observed a higher anxiety level in the F1 and F2 progenies sired by the heroin exposed rats (F0) as compared to the control rats. Paternal heroin exposure also caused significantly more aggressive offspring in F1 compared to the control. The same pattern was also observed in the F2.Our results demonstrated that the progenies of F1 and F2 sustained higher levels of anxiety and aggression which are due to paternal heroin exposure. Substance abuse and addiction is one of the serious public health concerns around the world. As reported by The World Drug Report , the gloThe impact of various psychoactive substances on aggressive behavior had been studied previously, due to the documented rise in drug abusers involved in crime. Several drug related factors may contribute to this occurrence, e.g. pharmacological effects of the drug, substance-induced psychological or biological changes, and withdrawal effects . OccasioOver the last few decades, there have been several studies of how parental drug abuse may affect their progenies. While most of these studies focused on susceptibility to drug use, others have looked into behavioral, molecular and physiological changes in the offspring. Among them were studies looking at the impact of maternal opioid use on fetal development –10. ThesThis study aims to examine the effects of paternal exposure to heroin on anxiety and aggression in the offsprings up to three generations .A total of 64 male and 48 female Sprague–Dawley rats weighing 130–150 g were used. For F0, rats were divided into two experimental groups, with eight rats per group  group). Rats were allowed to acclimatize for 2 weeks prior to experimental procedure and were placed in individual cages. Throughout the experiment, rats were maintained in a temperature and humidity controlled environment (24 °C ± 3.55 ± 1 %) in a 12:12 h light: dark cycle (lights on at 7:00 a.m.) and were given fresh water and standard rat chow ad libitum. All experiments were performed in accordance with the guidelines and approval from the Animal Ethics Committee of Universiti Teknologi MARA (UiTM) (46/2014).Heroin hydrochloride was obtained from LIPOMED (Switzerland). Heroin of 1 mg/ml concentration was prepared daily before injection by dissolving in 0.9 % normal saline heated to 40–50 °C.Intraperitoneal injections with heroin were given to F0-heroin (F0-H) rats twice daily at 9:00 a.m. and 6:00 p.m., with a 27 gauge 1/2 inch (12 mm) needle for 14 consecutive days. A dose of 3 mg/kg on the first day and then increasing by 1.5 mg/kg per day to a final dose of 13.5 mg/kg per day were administered to the rats . EscalatF0 rats were mated with heroin-naïve female rats at 2 weeks post heroin regime Fig. . WithdraThe elevated plus maze was constructed of wood and consisted of two open arms, OA (50 × 10 cm) and closed arms, CA (50 × 10 × 40 cm) set in a plus shape. The maze was elevated 50 cm from the floor with an open roof. The activities of the rats were recorded by an overhead camera attached on the ceiling and scoring was analyzed using ANY-maze Video Tracking System software . The test started with the rats being placed at the center region, facing the OA and were allowed to explore the maze for 5 min. The number of entries to the OA  and the total time spent in these arms of the maze were taken as an anxiety index . To measure locomotion, the total number of entries for each arm was taken. An entry was counted when the four paws of the rats were placed in the arms. The maze was cleaned with 70 % ethanol between each trial.To further analyze the anxiety behavior, the animals were tested in the OF for 5 min. The test was carried out in an arena (50 × 50 × 30 cm) divided into 25 squares (10 × 10 cm). Each of the rats was placed in the center and was allowed to explore the arena for 5 min and the time spent in the center of the arena was recorded. After 5 min, the rat was then returned to its home cage and the open field was cleaned with 70 % ethyl alcohol and permitted to dry between tests.In this test, the resident male rats were housed in observation cages (80 × 55 × 50 cm) with a female rat as companion for a week before the test begins to facilitate territorial behavior. As territoriality is based on the presence of olfactory cues, the bedding of the cage was not changed during the week prior to testing. An hour before the test started, the companion female was removed. New male rat (intruder) was then introduced into the resident rat’s cage and the test was started. The resident’s reaction, i.e. non-social activities , social activities , and aggression , towards the intruder rat was recorded over 10 min. After the test was completed, the intruder rat was removed and the male resident was reunited with its female companion.t test. One-way ANOVA followed by Dunnett’s or Tukey’s post hoc tests were used for comparison within group for the variables studied in the behavioral test. Statistical significance were set at p < 0.05. Statistical parameters were determined using SPSS 20.0 .Data are presented as mean ± SEM normal distribution was tested using Kolmogorov–Smirnov test. The data were compared between groups in the same generation using student’s The effects of paternal heroin exposure in F0 were apparent in the mean litter size of the first generation Table . Number As depicted in Fig. p > 0.001). However, a significant difference was seen between the heroin exposed rats and its third generation (F3). This shows that the behavior pattern was passed down only to F1 and F2 but not to its subsequent generation.Effects of paternal heroin exposure which were investigated using elevated plus maze (EPM) test indicated no significant differences for the open arms (CA) entries between the heroin exposed rats with its offspring , implicating that transgenerational anxiety effects from the paternal to the descendants sustained over two generations.It was found that anxiety behavior between the F0-heroin and F1-heroin groups were similar  were then evaluated. Table It was then further evaluated whether the next generation would also be affected by the previously heroin exposed father. Rats in the second generation (F1-heroin) showed higher frequency in non-social and social activity than the controls. In addition, the F1-Heroin also exhibited an increase in aggression parameters observed. This behavioral pattern was also observed in the third generation, F2-Heroin. However, in F3 generation, lower aggression behavior was observed.As shown in Table Drug abuse is an increasing societal burden. While it has been well documented that maternal opioids abuse during pregnancy results in detrimental effects in the offspring, less is understood about the contribution of paternal drug abuse on the next generation. The present study focused on paternal effects of heroin exposure during adolescence towards progenies i.e. F1, F2, and F3 generations.The results of the present study demonstrated that heroin exposure produced adverse effect on the litter sizes (F1) as compared to saline administered group. The litter sizes of heroin treated rats were significantly lower than in control group (mean pup = 10). Our results were similar to other studies on fetal effects of opiate and other toxicants administration that caused abnormalities in their offspring such as decreased in the size of litters and lost in weight , 16–18. Research has shown that prenatal exposure to drugs of abuse can have long-term effects on the behavior of offspring . Most ofAnxiety is generally believed to be associated with aggressive behavior . Thus, wInterestingly, rats that displayed heightened anxiety-like behavior exhibited higher aggression level, as indicated by the reduced time spent in the open arms of the EPM test as well as decreased ambulatory activities in the OF test. The mechanisms of heroin exposure towards the evolutionary phase of the gestational period and offspring’s anxiety and aggression level remains unknown and needs further investigation. Many drugs including morphine may be mutagenic which often cause a minor but significant change in the physiological parameters, behaviors and neuroendocrine parameters .In previous studies, possible effects of addicted father towards children were less considered. Thus, the results of this current study can be one of the supporting data highlighting the effects of heroin addicted fathers on children. It can be postulated that epigenetic mechanisms could be involved in transgenerational effects of heroin exposed paternal rats.BDNF gene, which encodes brain-derived neurotrophic factor (BDNF), a protein that is involved in numerous neurological conditions such as schizophrenia, depression, epilepsy, Alzheimer’s disease, obesity and drug addiction at many stages of development [Epigenetic factors such as histone modifications (e.g. acetylation) have been implicated in the behavior of addiction . In someelopment .As yet, no study has tracked the epigenetic effects on behavior of descendants of heroin abused fathers through the generations. Research is underway to examine the epigenetic influences on longstanding aggression behavior as a result of transgenerational substance abuse.The idea that maternal heroin abuse can impact phenotype of subsequent generations is not new. Data from our studies however, highlighted the effects of paternal heroin exposure on progenies. Our findings have shown that heroin exposure can affect the levels of anxiety and aggression in the F0 generation. Of note, these effects were also observed in both the first (F1) and second (F2) generations, suggesting multigenerational and transgenerational effects triggered by adolescent exposure to heroin. Further investigations by our group are currently under way to understand the mechanisms involved in these observed effect.


This paper presents an experimental study on the nature of permeation properties and pore structure of concrete surface layers containing fly ash. Concretes containing different dosages of fly ash as a replacement for cement  were investigated. Concrete without any fly ash added was also employed as the reference specimen. Laboratory tests were conducted to determine the surface layer properties of concrete including chloride transport, apparent water permeability and pore structure. The results demonstrate that incorporation of fly ash, for the early test period, promotes the chloride ingress at the surface layer of concrete but substituting proportions of fly ash may have little impact on it. With the process of chloride immersion, the chloride concentration at the surface layer of concrete with or without fly ash was found to be nearly the same. In addition, it is suggested that the water permeability at the concrete surface area is closely related to the fly ash contents as well as the chloride exposure time. Pore structure was characterized by means of mercury intrusion porosimetry (MIP) test and the scanning electron microscopy (SEM) images. The modification of pore structure of concrete submersed in distilled water is determined by the pozzolanic reaction of fly ash and the calcium leaching effect. The pozzolanic reaction was more dominant at the immersion time of 180 days while the calcium leaching effect became more evident after 270 days. When concrete is in long-term contact with the potentially aggressive aqueous environments, chemical changes (often starts at surface area of the mixture) may take place ,2. It isIt is generally acknowledged that cement is an important and integral segment of concrete materials. However, the overproduction of cement introduces high hydration heat, high autogenous shrinkage and high cost . Fly ashet al. [Numerous investigations regarding the pore structure or permeability of fly ash concrete have been carried out by many researchers in the past. In a study undertaken by Zhuqing Yu , it was et al. . HoweverTherefore, the main objective of the present study is to explore the properties of the surface layer in concrete with certain proportions of fly ash replacement. Experimental results on chloride ion precipitation in the surface layer, water permeability coefficient and pore size distribution will be presented and discussed in subsequent sections.Contents of chloride ion in the surface layer of all concrete under saturated conditions for 30, 60, 90, 120 and 150 days are shown in etc. have been found to be beneficial in resisting the ingress of chloride ions into concrete because of the microstructure densification caused by the pozzolanic reaction or secondary hydration of these materials [Based on the previous study , the mosaterials . From thaterials ,29,30,31Surface permeability coefficient of CF0 and CF30 under ultrapure water is also provided in the following Based on the experimental data provided in 2 with SiO2 and Al2O3 from fly ash [2+ ions in the cement hydration system are attached and fixed to fly ash particles [2+ ions prolongs the nucleation of CH or CSH. Therefore, in the early ages, due to the low levels of the Ca2+ ions caused by fly ash replacement, the progress of cement hydration in this period is slow and incomplete. Moreover, the delay of hydration is founded to be even more remarkable with an increase in fly ash, which hinders the pozzolanic reaction due to the lack of hydrate product Ca(OH)2 [Generally speaking, inclusion of fly ash has the following main effects: micro-filler effect and pozzolanic reaction. Pozzolanic reaction occurs from the reaction of Ca(OH) fly ash . However fly ash . An explarticles . The red Ca(OH)2 .With the immersion process, secondary hydration of fly ash produces a large amount of CSH, which reduces the large capillary pores sizes and thus enhances the high resistance to water ingress. A series of papers have pointed out that the permeable nature of concrete goes hand in hand with the size of capillary pores . IncorpoResults of the pore size distribution for fly ash concrete that was immersed in the distilled water at 90 days is given in Microstructure of concrete CF15 at 90 days of immersion is presented in In experimental studies, the P.O 42.5 Portland cement which is produced by Starfish Onoda cement limited company of Shenzhen, China, was used. The chemical properties of this cement are given at 3. River sand was employed as the fine aggregate according to JGJ52-2006 [3.Course aggregate that was obtained from a quarry of AnTuo Mountain, Shenzhen was used in tests. The maximum and the minimum particle sizes of coarse aggregate are 20 mm and 5 mm, respectively. As a result of experiments, the unit weight of the coarse aggregate is 2700 kg/mJ52-2006  standardDistilled water was used in concrete production.Type I fly ash (FA) was obtained from a power station in Ma Bay of Shenzhen. The chemical composition of fly ash is also provided in Sodium chloride that has purity above 99% was used to perform chloride bath for the specimens.The mixture proportions of concrete are summarized in The concrete specimens with a size of 100 mm × 100 mm × 100 mm were demoulded after 24 h and cured at the room temperature 20 °C and 95% relative humidity until the test age. In order to have one-directional flow through the concrete, wax coating was applied to the side faces and the bottom face of the specimens before they were completely saturated in the 5% NaCl solution see . The chlAfter the concrete samples were exposed to NaCl solution for a given time, the chloride ion concentration accumulated in the surface layer was determined by methods of Chemical Analysis (CA) that corresponded to JTJ270-98 . The surAutoclam was used for testing water permeation of the concrete in this experiment. Autoclam tester is able to non-destructively assess the permeability, and the hydroscopicity of the surface area in concrete materials. The tester’s system dates back to the 1980s, and was proposed by Calm. Basheer  develope−7m3/min0.5, and three parallel samples were tested and averaged to get one surface permeability index.The detailed procedures of this test were in a sequence: The specimens were first immersed in the distilled water until the given period. The specimens were heated in an oven at 80 °C for 12 h prior to the test. After cooling down, the specimens were then performed the water permeability test with tester Autoclam at given intervals of time . In this test, each measure was performed on three specimens and the best result was thought to be chosen. When conducting the water permeability test, the surface of the concrete was adhered to cohesive bottom ring see , isolatiD is the pore diameter (μm), γ is the surface tension of mercury (mN/m), θ is the contact angle between mercury and the fly ash concrete (°) and P is the applied pressure (Mpa). Based on the general suggestions in the literature [Mercury intrusion porosimetry (MIP) is widely used to measure the porosity and pore size distribution of cement-based materials for years ,47. In tterature , the surterature , which iScanning Electron Microscopy (SEM) was performed in microscopes. Prior to imaging, the concrete specimens were first crushed into small pieces. These pieces were then placed in a vacuum under 50 °C until constant weight, and coated with a thin gold layer. With the help of SEM results and the MIP data, a more accurate interpretation of pore structure of fly ash concrete can be achieved.In the early immersion period, fly ash has a significant effect on the chloride precipitation in the surface layer. However, the dosage of fly ash has little impact on the chloride precipitation in the surface layer. With the increasing immersion period, amounts of chloride ion in the surface layer of different fly ash contents were nearly the same; Fly ash content has a significant influence on the water permeability of the surface layer. With the immersion process, the secondary hydration of fly ash enhances resistance of the concrete to water ingress;When a fly ash concrete is under water immersion, both the pozzolanic reaction of fly ash and the water dissolution influence the pore structure development. The pozzolanic reaction is more dominant at the time of 180 days while the water dissolution becomes more evident after 270 days.This study discusses an experimental program carried out to investigate the permeation properties and pore structure of the surface layer of concrete with partial fly ash substitution. The following conclusions can be drawn according to the results of this study:


The aim of this pilot study was to measure changes in physical activity level (PAL) variables, as well as sleep duration and efficiency in people with locally advanced rectal cancer (1) before and after neoadjuvant chemoradiotherapy (CRT) and (2) after participating in a pre-operative 6-week in-hospital exercise training programme, following neoadjuvant CRT prior to major surgery, compared to a usual care control group.We prospectively studied 39 consecutive participants . All participants completed standardised neoadjuvant CRT: 23 undertook a 6-week in-hospital exercise training programme following neoadjuvant CRT. These were compared to 16 contemporaneous non-randomised participants . All participants underwent a continuous 72-h period of PA monitoring by SenseWear biaxial accelerometer at baseline, immediately following neoadjuvant CRT (week 0), and at week 6 (following the exercise training programme).n = 33), there was a significant reduction from baseline (pre-CRT) to week 0 (post-CRT) in daily step count: median (IQR) 4966 (4435) vs. 3044 (3265); p < 0.0001, active energy expenditure (EE) : 264 (471) vs. 154 (164); p = 0.003, and metabolic equivalent (MET) (1.3 (0.6) vs. 1.2 (0.3); p = 0.010). There was a significant improvement in sleep efficiency (%) between week 0 and week 6 in the exercise group compared to the usual care control group (80 (13) vs. 78 (15) compared to (69 ((24) vs. 76 (20); p = 0.022), as well as in sleep duration and lying down time (p < 0.05) while those in active EE  (152 (154) vs. 434 (658) compared to (244 (198) vs. 392 (701) or in MET (1.3 (0.4) vs. 1.5 (0.5) compared to (1.1 (0.2) vs. 1.5 (0.5) were also of importance but did not reach statistical significance (p > 0.05). An apparent improvement in daily step count and overall PAL in the exercise group was not statistically significant.Of 39 recruited participants, 23 out of 23 (exercise) and 10 out of 16  completed the study. In all participants  contains supplementary material, which is available to authorized users. Cancer treatment reduces physical fitness, which appears to be worse in those receiving surgery and radiotherapy in combination with chemotherapy than in those receiving radiotherapy or surgery alone , although relationships of cause and effect are complex. Remaining physically active during and after cancer treatment improves cancer-related fatigue, psychological distress, quality of life, as well as overall survival and reduces the probability of cancer recurrence  stage >T3N+ magnetic resonance imaging (MRI) identified circumferential resection margin threatened cancer], the standard treatment is neoadjuvant CRT followed by surgery  was given twice daily on radiotherapy days. No participants received brachytherapy. At 9 weeks post-neoadjuvant CRT, participants were restaged using chest, abdomen, and pelvic CT and pelvic MRI. The colorectal Multi-Disciplinary Team was blind to PAL results and participant allocation.TNM staging involved flexible sigmoidoscopy for histological diagnosis, colonoscopy, chest, abdomen, and pelvis computer-aided tomography (CT), and 1.5 T pelvic magnetic resonance imaging (MRI). All participants underwent 5 weeks neoadjuvant CRT. Standardised radiotherapy consisted of 45 Gy in 25 fractions on weekdays using a 3D conformal technique with CT guidance. Oral capecitabine . The SenseWear Amrband Pro is a reliable estimation of resting EE and provides useful information on daily EE when compared to indirect calorimetry (cancer patients) . The SenseWear Pro can distinguish between lying down and sleep time by using algorithms that detect the characteristics combination of orientation, motion, temperature and skin conductivity with each state.This was a nested study of a clinical trial, which was powered to detect changes in objectively measured physical fitness , mean (SD) or median and inter-quartile range (IQR), depending on distribution, and categorical variables as frequency (%). The Shapiro-Wilk test for normality of distributions was applied. Descriptive statistics and univariate statistical comparisons of patient characteristics between the groups were undertaken: for continuous variables, a two-sample p < 0.05 was taken as statistically significant. All analyses were performed with the statistical software IBM SPSS Statistics Ver.22 .Generalised linear mixed models, with a repeated effect for the comparison between the consecutive visits, were used to obtain restricted maximum likelihood (REML) solutions with an unstructured type of the covariance matrix for all or selected measurements in the two groups. Least square means with 95% CIs were obtained. A total of 39 participants were recruited of whom 23 (exercise group) and 10  completed the study (6 participants withdrew consent (dropped out) in the usual care control group: 4 before baseline measurements and 2 during the study). There were significant baseline differences between groups in age, ASA and WHO performance status: the usual care control group were older with poorer subjective performance Table .Table 1Cp < 0.0001), active EE  (264 (471) vs. 154 (164); p < 0.005), and MET (1.3 (0.6) vs. 1.2 (0.3) p < 0.05; table p = 0.022), sleep duration (min) (190 (269) vs. 369 (81) compared to (265 (315) vs. 299 (39); p = 0.028) and lying down time (min) (360 (352) vs. 47 (476) compared to (541 (360) vs. 341 (372); p = 0.029, Table There was a significant reduction in daily step count between pre-neoadjuvant CRT (baseline) and post-neoadjuvant CRT (week 0) in all participants (4966 4435) vs. 3044 3265); 65; p < 05 vs. 304This pilot study shows that neoadjuvant CRT significantly reduced daily step count, active EE and MET in people with newly diagnosed locally advanced rectal cancer. Furthermore, neoadjuvant CRT had a generally negative effect on the other exploratory PA variables, although findings were not statistically significant. People who participated in the 6-week in-hospital exercise training programme, in the time interval following neoadjuvant CRT and prior to surgery, showed significant improvements in sleep efficiency, sleep duration and lying down time compared to the usual care control group. Furthermore, the exercise group showed an improvement in daily step count and active EE, although these findings did not reach statistical significance.It has been previously been reported that neoadjuvant chemo- and chemoradiotherapy significantly reduce physical fitness and this change is associated with post-operative complications and reduced 1-year survival in locally advanced rectal and upper gastrointestinal cancer  , the clearly defined exercise intervention and the standardised neoadjuvant CRT regime. PA was averaged over a 72-h period, measured in an objective manner using validated SenseWear activity monitors. Furthermore, participants in the exercise group did not wear the PA monitors during exercise sessions. Potential weaknesses of this study include its design as a relatively small pilot study, which was powered to detect changes in objectively measured physical fitness (West et al. Our study shows that neoadjuvant CRT significantly reduces MET score, active EE and daily step count in people with locally advanced rectal. People who participated in a 6-week in-hospital exercise training programme following neoadjuvant CRT showed a significant improvement in sleep efficiency, sleep duration and lying down time and an apparent improvement in daily step count and overall PAL compared to the usual care control group.


IgG4-related disease (IgG4-RD) is a multisystem inflammatory disorder. Early recognition of IgG4-RD is important to avoid permanent organ dysfunction and disability. Neurological involvement by IgG4-RD is relatively uncommon, but well recognised—hypertrophic pachymeningitis and hypophysitis are the most frequent manifestations. Although the nervous system may be involved in isolation, this more frequently occurs in conjunction with involvement of other systems. Elevated circulating levels of IgG4 are suggestive of the condition, but these are not pathognomonic and exclusion of other inflammatory disorders including vasculitis is required. Wherever possible, a tissue diagnosis should be established. The characteristic histopathological changes include a lymphoplasmacytoid infiltrate, storiform fibrosis and obliterative phlebitis. IgG4-RD typically responds well to treatment with glucocorticoids, although relapse is relatively common and treatment with a steroid-sparing agent or rituximab may be required. Improved understanding of the pathogenesis of IgG4-RD is likely to lead to the development of more specific disease treatments in the future. IgG4-related disease (IgG4-RD) is a systemic, immune-mediated fibro-inflammatory disease of unknown cause, characterised by unique pathological features involving a wide variety of organs ••, 3, 4.Once regarded as an isolated single-organ disease, IgG4-related disease is now recognised as a single multisystem disorder which can affect virtually any organ system Table  5•, 6].•, 6.5•, The organs commonly affected by IgG4-RD are the pancreas (autoimmune pancreatitis), bile ducts (sclerosing cholangitis), retroperitoneum , salivary glands  and lacrimal glands (dacryoadenitis). The nervous system is less commonly involved, although a variety of manifestations are recognised , 12.The first report of central nervous system (CNS) involvement by IgG4-RD occurred in the context of hypophysitis , but sinIn 1961, Sarles et al. raised the possibility that chronic inflammatory sclerosis of the pancreas was a distinct clinical entity . HoweverFew large-scale epidemiological studies of IgG4-RD have been performed, and the majority have focused on AIP in Japanese cohorts . In contIgG4 is one of the four human IgG subclasses and usually constitutes <5% of IgG in healthy individuals . Despite+ cytotoxic T lymphocytes (CTL) serving as effective antigen-presenting cells and/or through the secretion of B cell-derived growth factors -fluoro-2-deoxy-d-glucose positron emission tomography–computed tomography (FDG PET-CT) has emerged as a potentially useful tool in the diagnosis and monitoring of IgG4-RD [In recent years, 2-[ IgG4-RD , 124–128 IgG4-RD .max) of main involved organ ; SUVmax of submandibular glands (higher FDG uptake when compared with other diseases); and presence of multi-organ involvement. There was a substantial degree of overlap between the distribution of tissue involvement between IgG4-RD and other diseases, but in this study, FDG PET-CT had a diagnostic sensitivity of 85.7% and a specificity of 66.1% for IgG4-RD.Recently, Lee et al. assessed the diagnostic utility of FDG PET-CT in the differential diagnosis of IgG4-RD. The authors compared data from 28 IgG4-RD patients with 66 patients with other diseases  . StatistIt is also worth noting that gallium SPECT/CT may also have potential clinical utility in the diagnosis and monitoring of IgG4-RD, particularly given its lower cost and more widespread availability .Serum inflammatory markers such as the erythrocyte sedimentation rate and C-reactive protein may be modestly elevated in IgG4-RD . OccasioElevated serum IgG4 concentrations are characteristic of IgG4-RD. Nonetheless, serum IgG4 has shortcomings both as a diagnostic marker and in monitoring disease activity or predicting disease relapse. For diagnostic purposes, it is neither necessary nor sufficient to confirm a diagnosis of IgG4-RD on its own, although when present it is supportive. Serum IgG4 concentrations tend to be higher in patients with multi-organ involvement , 131–135A recent meta-analysis reported that high serum IgG4 (>135 mg/dL) has a pooled sensitivity of 87.2% and a specificity of 82.6% for the diagnosis of IgG4-RD, although significant heterogeneity was observed . DoublinThe ability of serum IgG4 levels to predict relapses and effectively monitor treatment response is controversial—for example, in the study reported by Kamisawa et al., the relapse rate was 30% in those with persistently elevated IgG4 levels and 10% in those with normal concentrations , 144.More recently, Wallace et al. showed that circulating plasmablasts are elevated in active IgG4-RD  and that plasmablast counts are a potentially useful biomarker for diagnosing IgG4-RD and assessing its response to treatment . MeasureCSF analysis is of particular importance with regard to IgG4-RD, not only because it offers the possibility to make a positive diagnosis of disease mimics and may give specific information about intrathecal synthesis of IgG4. CSF evaluations in patients with pachymeningitis secondary to IgG4-RD generally reveal clear fluid with normal glucose concentration, normal to mildly increased protein levels and a variable degree of lymphocytic pleocytosis , 145. Ho+ plasma cells (or an elevated IgG4/IgG ratio) in tissue [Definitive diagnosis of IgG4-RD requires an appropriate histological appearance with increased numbers of IgG4n tissue , 110. Thn tissue •, 110. In tissue •. The mon tissue , 84, 85.+ plasma cells/HPF or a ratio of IgG4+ plasma cells to IgG+ plasma cells higher than 40%, but individualised cutoffs based on the site of involvement may be required [+ plasma cells per HPF is reasonable [+ cells may be acceptable in cases with the characteristic morphologic features [+ plasma cells is not specific for IgG4-RD and should always be interpreted in light of the accompanying clinical, histological, radiological and serological findings [The inflammatory lesion frequently forms a tumefactive mass that may destroy the involved organ, and the inflammatory infiltrate is composed of a mixture of T and B lymphocytes. T lymphocytes predominate in the infiltrate and are usually present diffusely, whilst B cells tend to be located within lymphoid aggregates or even germinal centres •. Semiqurequired , 135. Foasonable . A lowerfeatures ••. IgG4-findings .Because of its rarity, treatment of neurological disease secondary to IgG4-RD has been extrapolated from the relatively limited evidence available for even the more common organ manifestations. No randomised clinical trials have been performed in IgG4-RD, and the best evidence for therapeutic options comes from systematic review of the literature  • and expNot every patient with IgG4-RD needs treatment, and given that some cases of spontaneous remissions have been reported, a ‘watchful-wait’ decision may be appropriate in some patients (e.g. asymptomatic lymphadenopathy) , 146–148Glucocorticoids are the first-line treatment, unless there is a contraindication ••, 147. Relapse rates after steroid withdrawal are high, and may also be significant during glucocorticoid taper or maintenance therapy , 152. A For remission induction of IgG4-RD, prednisolone at a dose of 30–40 mg/day is commonly instigated . However−1 day−1 for 2–4 weeks, followed by a tapering schedule over a period of 3–6 months down to a maintenance dose (2.5–5 mg/day), which is then maintained for up to 3 years. These recommendations are mostly based on a study conducted by Kamisawa et al. in AIP—23% of the patients on glucocorticoid maintenance treatment relapsed versus 34% of the patients off treatment [The dose and duration of ongoing glucocorticoid therapy are a matter of ongoing debate. For example, some clinicians from Asia recommend treatment with prednisolone 0.6 mg kgreatment . Relapsereatment , 157–159reatment ••. Therereatment , 159. Pareatment ••, 83.Azathioprine, mycophenolate mofetil, methotrexate (MTX), 6-mercaptopurine, tacrolimus and cyclophosphamide have been used as potential ‘steroid-sparing agents’ (SSA) or remission maintenance drugs , but their long-term efficacy requires further evaluation , 157–160For those patients with IgG4-RD that is either refractory to glucocorticoids or which relapses, B cell depletion with rituximab (RTX) appears to represent a promising treatment , 164–166The prospective trial was conducted in a high-risk group of patients , and the results are reassuring; 97% responded, with response generally observed after 2 weeks and sustained for 6 months. At 6 and 12 months after RTX, only 10% of the participants were on glucocorticoid treatment, thereby avoiding steroid-related adverse effects in a population of patients that may be particularly vulnerable. Retreatment with RTX for relapses was deemed necessary in 13% (4/30) during the 12-month period after enrolment. In the retrospective cohort, clinical response was seen in 95% of the patients, but 37% relapsed following successful treatment . PredictSerial treatments with RTX (e.g. 1 g every 6 months) may lead to progressive decline in serum IgG4 concentrations and better disease control, although this observation needs confirmation , 167 andApart from pharmacological therapy, some patients may need urgent surgical intervention (e.g. laminectomy) in the face of ongoing neurological symptoms (e.g. paraparesis), and this has been more frequently reported in spinal pachymeningitis. In fact, some of these patients have been treated with surgery alone, without concomitant glucocorticoid therapy , 168.To assess the response to treatment, there is general agreement that one should use a combination of clinical, radiological and serological findings ••. An IgIgG4-RD is a systemic disease of unknown cause that affects virtually every organ system, and neurological manifestations have been increasingly recognised and reported. The most common of these are hypophysitis and pachymeningitis, which may be life-threatening, although treatable. Therefore, the differential diagnosis of IgG4-RD must be considered when a patient presents with signs or symptoms suggestive of hypophysitis or pachymeningitis, and special attention should be placed to other clues that may suggest IgG4-RD . Glucocorticoids are usually effective in the induction of remission, although a number of patients relapse. In this regard, retreatment with glucocorticoids and/or a SSA or RTX is usually necessary, although the optimal treatment strategy remains uncertain. There is a pressing need for clinical trials to address this as the long-term side effects of glucocorticoids add significant morbidity. In the future, new insights derived from a more complete understanding of the pathogenesis of IgG4-RD may facilitate the development of more effective and better targeted pharmacological options.


Among porphyrin systems, this trisporphyrin have the strongest binding affinity for 1-(1-naphthyl)ethanol, which leads to the highest CD sensitivity.A zinc trisporphyrinate has been developed as a chirality sensor for chiral mono-alcohols. In its structure, there are two “spaces” surrounded by three porphyrin moieties, which allow guests to fill in. It has shown extremely high CD sensitivity for a chiral mono-alcohol with a naphthyl substituent, 1-(1-naphthyl)ethanol, at μM level, which is at least three orders of magnitude lower concentration than previous reports. A crystallographic study of the host-guest complex reveals the binding of 1-(1-naphthyl)ethanol to the zinc trisporphyrinate is greatly enhanced by multipoint interactions, such as coordination interactions, hydrogen bonding, π-π and CH···π interactions etc. Spectroscopic studies suggest the corresponding binding constant K Determination of their absolute configurations is crucial to understand their functions, and the rapid determination of absolute configuration and enantiomeric excess for chiral molecules are also important for high-throughput screening of chiral catalysts4. Among these alcohols, mono-alcohols are more difficult to investigate due to their limited binding site and the weak binding affinity to metal centres. In past years, many efforts have been devoted to developing highly sensitive systems for recognition of chiral mono-alcohols. Some of them require the mono-alcohols to be derivatized7. Obviously, without chemical derivation, the determination of absolute configurations will be more convenient and rapid, especially through CD measurements.Chiral alcohols are commonly encountered structural elements in pharmaceutical drugs, biologically active natural products and synthetic intermediate11. However, because of the weak binding affinity of alcohols to metal centres, very few studies were reported14. For example, Borovkov and co-workers reported the ethane-bridged bisporphyrin system. In that case, the short ethane-bridge causes the steric interactions between the 3,7-ethyl groups of the porphyrin and the substituents of the ligand, resulting in the induced supramolecular chirality12. Another more sensitive system is an ester-linked bisporphyrin developed by Takanami et al., which improves the detecting concentration of mono-alcohols to mM level through the simultaneous double coordination of the hydroxyl group to the two metals14.Because of the intensive absorption in the Soret band, metalloporphyrins have been widely studied as good candidates for chirality related researchAlthough considerable progress has been made with respect to the recognition of chiral mono-alcohols, the search for highly sensitive sensors still remains challenge. In order to improve the sensitivity, one important strategy is to increase the host-guest interactions. In view of molecular recognition, such a purpose can be achieved through multipoint host-guest interactions.16. In this trisporphyrin, the linking group is surrounded by three porphyrin moieties, which leads to the formation of two “spaces”, one above the linking phenyl and another below it. So this host has potential for chirality sensing of specific guests through multipoint interactions, such as coordination interactions, hydrogen bonding, π-π interactions and CH···π interactions etc., which will enhance host-guest interactions, and the disadvantage of mono-alcohols can be overcome.We have recently reported a trisporphyrin system as shown in Fig. 16, the crystal structure suggests the “space” formed by porphyrin moieties is a little big for 1-phenylethanol, and that “space” can accommodate one 1-phenylethanol and one H2O. A bulkier guest could match the “space” better. Herein, we examined several mono-alcohols. This Zn trisporphyrinate showed the highest sensitivity to 1-(1-naphthyl)ethanol (the guest 2), the one with a naphthyl substituent. To obtain good CD signals, the concentration of the guest 2 only need to be at μM level, which is at least three orders of magnitude smaller than previous reports. To our knowledge, this is the most CD sensitive porphyrin system for chiral mono-alcohols so far.In our previous study3-BTATPP] and the guest 2 were shown in Fig. 2 is 0.55 µM, there are remarkable bisignate CD signals in the Soret band region. As shown in Figure −5 M, the A value reaches 1054 cm−1M−1, the strongest CD value in the reported porphyrin systems for mono-alcohols. For 2S, the longer-wavelength peak of the Soret band was negative and the shorter-wavelength peak was positive. For 2R, the CD spectra showed similar shapes and intensities but opposite signs in the Soret band region. So this host can be used to determine the absolute configuration of 1-(1-naphthyl)ethanol. The spectra for other mono-alcohols are shown in the supporting information. The corresponding guest concentrations at which the Aobs values are about 100 cm−1M−1 are also listed in Table 2, the detecting concentration is three orders of magnitude lower than those reported by Takanami et al.14. We also noticed there were limitations for this trisporphyrin system. For example, the signs of these CD spectra for these mono-alcohols did not show clear patterns as literatures reported. And this system is only highly sensitive to the specific case of 1-(1-naphthyl)ethanol. Except the guest 2, the required concentrations for other guests are much higher, ranging from 10−3 to 10−4 M, which are similar to Takanami’s results.The CD spectra for the mixture of ·(2S)2. Each 2S adopts the “inside” binding mode. Besides the coordination interactions, hydrogen bonding and π-π interactions are also found between hosts and guests as shown in Fig. 2S and the linking phenyl ring. The centroid-centroid distances are 3.55 Å and 3.72 Å, the dihedral angles are 2.0° and 8.4°, respectively. The two guests fit the two “spaces” surrounded by three porphyrin moieties to form a “sandwich” like structure.In order to evaluate the first factor, the binding affinity, we did studies on the crystal structure and the binding constants. When large excess of the guest As expected, 1-(1-naphthyl)ethanol is bulkier than 1-phenylethanol, which makes it match the “space” better, and also causes other interactions between hosts and guests. Besides coordination interactions, π-π interactions and hydrogen bonding interactions, there are also C-H···π interactions as shown in Fig. 18. The crystal structure reveals there are multipoint interactions among the host and guests, which could increase the overall interactions, and thus increase the binding affinity. This is further confirmed by binding constant determination.The chirality of the crystal is confirmed by the solid CD spectrum in Figure 2 change from 0 to 3.1 × 10−6 M, the peak at 419 nm keep decreasing; (2) When the concentrations increase from 7.4 × 10−6 to 4.2 × 10−4 M, the peak at 419 nm starts to increase and a shoulder appears at 424 nm, then two isobestic points are observed at 415 and 431 nm. These spectral changes indicate there are the following two equilibriums in solution, where L present the guest. There could be three equilibriums since there are three Zn centres. But since the binding of the first and second guests blocks the “inside” coordination position of the third Zn, the third Zn has much weaker binding affinity than other two. Under our experimental conditions, the concentrations of the guest 2 is not high enough to cause the formation of the detectable amount of the 1:3 complex, and thus the third equilibrium can be ignored. Actually, the 1:2 complex in solution is confirmed by the Job’s plot based on the 1H NMR titrations at relative high concentrations (from 10−3 to 10−2 mol/L)  Figure . The pea19 was used to calculate the binding constants for the two equilibriums. More fitting details were provided in the supporting information  × 105 M−1 and 1.4 (±0.1) × 104 M−1. The fitting of the CD spectral changes  is actually longer than 5). More importantly, the π-π or C-H···π interactions are absent for the guest 5, which make its binding affinity much less than 2. For the case of 3, it is a tertiary alcohol, others are secondary alcohols, which probably causes its binding affinity much weaker than others.The binding constant K2 suggest that more host-guest complexes are formed than other mono-alcohols when their concentrations of guests (and hosts) are the same. To further evaluate the CD results, we have also to consider the second factor - the CD contribution of the corresponding host-guest complexes per unit.The high binding constants for the guest per unit could be roughly treated as Aobs/P1. As shown in Table −1M−1 for different mono-alcohols. Obviously, among them, the 1:1 complex for the guest 2 leads to the largest CD magnitude per unit. So for the same amount of 1:1 complex, the CD amplitude for 1-(1-naphthyl)ethanol is several times (below 10) as other alcohols.We can actually obtain some direct information about CD contributions from experimental data. CD signals are contributed from both 1:1 and 1:2 host-guest complexes. For the CD sensitivity, we are concerned about the CD signals at low guest concentrations. Under such a condition, the amount of 1:1 complexes are much more than 1:2 complex as shown in Table per unit, which leads to the highest CD sensitivity. If we consider the binding constants for 1-(1-naphthyl)ethanol is over 100 times larger than others and its CD contribution per unit is only a few times than others, the major factor causing the highest CD sensitivity for the guest 2 should be the strong bonding affinity, which is due to the strong host-guest interactions.The above studies suggest that this zinc trisporphyrinate has the strongest binding affinity to 1-(1-naphthyl)ethanol and the host-guest complex has the largest CD magnitude 2, which is crucial to chiral recognition of mono-alcohols. We could adjust the substituents in hosts to create the right “space” for the specific guest, which can improve the corresponding recognition ability and sensitivity. This type of hosts can be used as powerful tools for molecular recognition of some specific chiral molecules, and have also potential applications in catalytic synthesis. Further studies on other guests and derived hosts are under investigation.In conclusion, a zinc trisporphyrinate has shown high CD sensitivity at µM level for 1-(1-naphthyl)ethanol. The crystallographic study reveals that coordination interactions, hydrogen bonding, π-π and CH…π interactions in the host-guest complex cause strong host-guest interactions, which is confirmed by the large binding constant. Our studies suggest the “space” formed by porphyrin moieties is “recognized” best by the chiral mono-alcohol with the naphthyl substituent, the guest 2Cl2 and n-hexane were distilled over CaH2. Triethylamine (Et3N) was distilled over KOH and then used immediately. Zinc 5-(2-aminophenyl)-10,15,20-triphenyl-porphyrinate was prepared as described in the literature20. The porphyrin host [Zn3-BTATPP] was prepared according to the reported method15. Elemental analyses were performed on an Elementar Vario EL III analytical instrument. A Shimadzu UV-3150 spectrometer was used to record UV−vis spectra. A MT Model J-815 spectrophotometer was used to record CD spectra at 25 °C in 10% CH2Cl2/hexane .Reactions involving moisture sensitive reagents were carried out under a nitrogen atmosphere using standard vacuum line techniques and glassware that was flame-dried and cooled under nitrogen before use. CH2Cl2/hexane were added to the [Zn3-BTATPP] solutions at 25 °C. Then CD and UV-vis spectra were recorded. The corresponding CD spectra were normalized based on the [Zn3-BTATPP] concentration.Solutions of various guests in 10% CHAccording to the following method, the measurement of the solid CD spectrum was performed. A single crystal and some solid KBr were grinded under high pressure to get the KBr pellets (the thickness is 0.3 mm), and then CD spectrum was recorded.1H-NMR titration of [Zn3-BTATPP] and the chiral guest 2R. Upon complexation of 2R with [Zn3-BTATPP]1,HNMR spectra undergo changes in chemical shift (Δδ) due to the anisotropic effect of the porphyrin rings. These changes can be used to determine the composition of host-guest complexation in solution. The 1H-NMR titration [Zn3-BTATPP] with 2R  was dissolved in CDCl3 (0.50 mL), and the NMR spectrum was recorded . Following this, 0.3, 0.6, 1.0, 1.2, … up to 20 equivalents of 2R was added to the above solution; the NMR spectra was recorded after each addition.For each titration, 4.7 mg of [Zn3-BTATPP] protons  was multiplied by the changes in 1H-NMR chemical shift of [Zn3-BTATPP] (Δδp) for each titration data point. The resulting value was plotted against the molar fraction of [Zn3-BTATPP]. Peaking at 0.33 mol fraction corresponds to a 1:2 host-guest complex  of one of the [Znx Figure .3-BTATPP]  in toluene (4 mL) was mixed with the solution of S-1-(1-naphthyl)ethanol  in CH2Cl2 (1 mL), and the mixture was stirred for 3 min, then transferred into glass tubes (8 mm × 250 mm). n-Heptane was added as the nonsolvent at room temperature. After one months, purple crystals were then isolated . Anal. Calcd for C330H222N30O10Zn6: C, 76.92; H, 4.34; N, 8.10; Found: C, 74.89; H, 4.40; N, 8.83.The solution of ·(2S)2. Non-hydrogen atoms were refined anisotropically and all hydrogen atoms were included in calculated positions. In the structure, several highly disordered solvent molecules were present, which could not be modeled successfully and, therefore, SQUEEZE22 was used. The electron count within the interporphyrin voids was 86 e, which corresponds to one molecule of dichloromethane per [Zn3-BTATPP]·(2S)2. Data collection parameters are listed in Table A Bruker APEX-II CCD was used for data collection at 293(2) K, which is equipped with graphite monochromated Mo Kα (λ = 0.71073 nm). Direct methods were used to solve the structure, further refinements were done on Supporting information for Host-guest assembly for highly sensitive probing of a chiral mono-alcohol with a zinc trisporphyrinate


Extranodal spread (ENS) in patients with head and neck squamous cell carcinoma (HNSCC) can greatly influence the prognostic outcomes. However, the relative risks of ENS in the primary (1st) and secondary (2nd) metastatic nodes (mets) are not well documented. We retrospectively analyzed the hazard ratios (HRs) of ENS in the 1st and 2nd mets from 516 HNSCC patients who had undergone primary tumor excision. The impact of clinically and/or histologically confirmed ENS-positive mets on prognosis in terms of cancer-specific survival was analyzed. Cox proportional hazard regression analysis indicated that ENS-positive 1st met  and ENS-positive 2nd met  significantly and independently predicted poor prognosis; however, other variables including primary site, met size or numbers, and met location in the contralateral side of the primary lesion, did not. Cumulative incidence function and Cox analyses indicated that differences in ENS profiles of 1st and 2nd mets stratified HNSCC patients with varying risks of poor outcome; HRs relative to patients with ENS-positive 1st met (-)/ENS-positive 2nd met (-) were 4.02 , 8.29 , and 25.80  for patients with ENS-positive 1st met (+)/ENS-positive 2nd met (-), ENS-positive 1st met (-)/ENS-positive 2nd met (+), and ENS-positive 1st met (+)/ENS-positive 2nd met (+) patients, respectively. Kaplan-Meier analysis indicated that the 2nd met that appeared in the neck side with a history of 1st met and neck dissection had a higher risk of ENS than the 2nd met in the neck side without the history (p = 0.003). These results suggested that ENS is a dominant prognostic predictor of HNSCC patients, with double-positive ENS in the 1st and 2nd mets predicting the most devastating outcome. Metastasis to the regional lymph node has been considered a critical risk factor in patients with head and neck squamous cell carcinoma (HNSCC). A metastatic node (met) in the neck has been found to be closely related to the prognosis of HNSCC patients . HoweverThe development of secondary (2nd) met may occur after surgical excision of the primary lesions without any locoregional failure of the primary site or distant metastasis, which could further deteriorate the prognosis of patients. Also in such cases, the ENS would be an additional prognostic risk factor. Neck dissection (ND) may be performed for the primary (1st) met(s) that was histologically confirmed on biopsy specimens and/or was suspected based on imaging information without histological evidence. The lymphatic systems in the neck and the microenvironment of the regional lymph nodes may be altered after surgical interventions for the primary and neck lesions , 7, whicTherefore, it is very interesting to know the extent to which the neck disease-related factors, including 1st/2nd mets, ENS in the 1st/2nd mets, and ND can contribute to the prognosis of HNSCC patients who had undergone surgical excision of the primary lesions. Accordingly, in the present study, we retrospectively investigated the impact of ENS in the 1st and/or 2nd mets on the prognosis of HNSCC patients in terms of cancer-specific survival, by assessing the relative risk factors of the different profiles of 1st/2nd mets and ENS. It should be noted that, by including the 2nd met for analysis in this study, we excluded patients who developed locoregional failures in the primary site and/or distant metastases during the periods from the surgery up to the development of 2nd met, which did not fulfill the requirements of 2nd met. Thus, the risks obtained in the present study were minimally influenced by post-treatment conditions of the primary and distant metastatic lesions.Fig 1). Patients who had distant metastasis, preceding HNSCC history, overlapping malignancy in other parts of the body, or recurrence of the primary lesion, or had received pre-operative radiotherapy were excluded from the study cohort. The study protocol was approved by the ethics committee of Nagasaki University Hospital, and the requirement to obtain informed consent for the review of images and records was waved.We searched the clinical databases of the Nagasaki University Hospital and identified 548 patients  with HNSCC who underwent excision of the primary tumor with or without ND , and were examined preoperatively and postoperatively with computed tomography (CT) or magnetic resonance (MR) imaging for the primary and neck (metastatic node) lesions between 1994 and 2015 , n = 151] or non-metastatic  nodes were confirmed on the pathology report of the excised ND specimens. ND (-) patients (n = 232) were categorized in the 1st met (-) patient group. The secondary metastasis (2nd met) was defined as a metastatic node that occurred after the initial surgical interventions for the primary tumor without any preceding primary recurrence or distant metastasis during the follow-up period, and that was histologically and/or clinically confirmed. The median duration between excision of the primary tumors and the diagnosis of a 2nd met was 6 months . The median follow-up period of the 548 patients was 54 months (1–241 months).Fig 1).ENS-positive nodes of the 1st met were histologically (n = 54) diagnosed, and those of the 2nd met were histologically (n = 38) or clinically  diagnosed in 516  of the 548 HNSCC patients , lower gingiva (81/75), oral floor (50/46), hypopharynx (48/42), larynx (46/45), upper gingiva (42/40), oropharynx (35/31), buccal mucosa (24/24), ear (12/12), maxillary sinus (9/9), palate (9/8), lip (6/6), upper plus lower gingiva (4/4), and nasal cavity (2/2). The summary statistics of the 548 (Group I) and 516 (Group II) patients are shown in The 2nd met was identified based on histological results from biopsy specimen or specimen from the neck dissection for 2nd met, or on CT and/or MR imaging findings. A 2nd met with or without ENS was identified on CT and/or MR images as a nodal structure in the neck that fulfilled at least one of the following criteria: (a) non-enhanced focal defects in the nodal parenchyma, (b) apparent size-up on images during follow-up periods, or (c) imaging characteristics of ENS. An ENS-positive node was defined on CT and/or MR images as a nodal structure having irregular or interrupted enhancement at the perimeter on contrast-enhanced CT and/or MR images.Table 1). Cumulative incidences in different patient groups were compared using Gray’s test. The Bonferroni correction was used to adjust p-values in multiple comparisons.Cancer-specific survival of the patients was estimated using cumulative incidence function in the presence of competing risk such as death due to non-SCC tumor or non-cancer deaths . The Cox proportional hazard regression analysis was performed on the variables that were confirmed not to violate the proportional hazards (PH) assumption. Schoenfeld residuals were used for assessing the violation of PH assumption. We found that the PH assumption was not violated in all models.Influence of pre-existing 1st met and history of ND in the same neck side of 2nd met on cancer specific survival was estimated using Kaplan-Meier analysis, and log-rank test was applied to assess the significance in cancer-specific survival rates between groups.https://www.R-project.org/) was used for assessing PH violations and Cumulative incidence function.Cox proportional hazard regression analysis, and Kaplan-Meier analysis with log-rank test were performed using JMP Pro  software. R software version 3.4.0 .We first analyzed the prognostic importance of demographical and neck disease profiles of the 548 patients with HNSCC. Multivariable Cox proportional hazard regression analysis indicated that 1st or 2nd mets were significant and independent risk factors in HNSCC patients .; however, age, sex, primary site, and the history of ND were not . Furthermore, the prognosis was much worse for HNSCC patients with 1st and 2nd mets than patients with a single type of met alone .Consistent with these results, cumulative incidence function of the same 548 HNSCC patient cohort revealed that the prognosis of HNSCC patients with 1st or 2nd met was significantly worse than patients without nodal metastasis  (Table 3).The predictive ability of ENS in the 1st and 2nd mets for cancer-specific survival of HNSCC patients was investigated in 516 patients with histologically or clinically confirmed ENS-positive or ENS-negative mets. Multivariable Cox proportional hazard regression analysis indicated that ENS in the 1st met  and 2nd met  were both significant and independent predictors of poor prognosis, but the 1st met and 2nd met per se were not . However, the difference in survival between the groups with either type of ENS-positive met did not quite reach the significance level (p = 0.052). In contrast, patients with ENS double-positive mets [ENS-positive 1st met (+)/ENS-positive 2nd met (+)] had a significantly worse prognosis than patients with either type of ENS-positive met .Next, we investigated the relative risks of ENS that appeared solely in the 1st or 2nd met, or that appeared in both the 1st and 2nd mets. Cumulative incidence function revealed that the cancer-specific survival of ENS-positive 1st met (+)/ENS-positive 2nd met (-) or ENS-positive 1st met (-)/ENS-positive 2nd met (+) patients was significantly lower than that of patients without ENS  . Cox proportional hazard regression analysis showed that the relative HRs of ENS-positive 1st met (+)/ENS-positive 2nd met (-) and of ENS-positive 1st met (-)/ENS-positive 2nd met (+) patients were 4.02  and 8.29 , respectively. The relative ratio was further increased to 25.80  in patients with ENS double-positive mets.To further evaluate the relative importance of ENS-positive 1st and 2nd mets as a prognostic variable of HNSCC patients, we determined the relative HRs for patients with various ENS met profiles . This patient subcohort did not include any competing risks. Therefore, we used Kaplan-Meier analysis instead of the cumulative incidence function for assessing the survival probability. The analysis indicated that the survival probability of patients with 2nd met in the neck side where a preceding 1st met had occurred and the subsequent ND performed was significantly lower than that of patients with 2nd met in the neck without any history of 1st met .Lastly, we asked whether the history of 1st met and ND can affect the prognosis of patients with 2nd met. To this end, we compared the survival probability between patients with or without a history of 1st met in a patient group having histologically and/or clinically confirmed 2nd met(s)  showed significantly different occurrence rates of ENS-positive 2nd met in the neck with preceding 1st met and ND, from those with ND history alone, and those without any history of 1st met or ND .A further investigation using a limited study population of patients with ENS-positive or ENS-negative 2nd met  of 1st and 2nd mets. An ENS-positive 2nd met carried a similar prognostic risk level as ENS-positive 1st met; however, the double-positive ENS in the 1st and 2nd mets in the same neck indicated a much worse prognosis than the presence of either type of ENS-positive met. Furthermore, a preceding 1st met with an ND history was a risk factor of ENS-positive 2nd met in the same neck side.As mentioned in the methods section, the 2nd met was defined as one that occurred in the absence of primary tumor recurrence and distant metastasis. Therefore, patients having such histories were tentatively categorized into the 2nd met (-) group in the present study, and the assessment for risk factors was conducted under the assumption that the primary lesions were kept under control during the periods from the surgery up to the development of 2nd met. However, in previous studies, the prognostic outcomes had been greatly affected by the conditions of the primary lesions , 2. ThusThe prognostic importance of ENS has been controversial for patients with HNSCC. For example, de Juan et al. showed that knowing the the presence or absence of ENS in the ND improved the predictive ability of tumor-node-metastasis (TNM) classification in HNSCC patients . HoweverMultiple occurrences of ENS-positive 1st mets have been indicated as higher-risk factor in patients with HNSCC compared to a single occurrence of an ENS-positive met , 15. HowIt should also be noted that the preexisting 1st met with ND history could be a risk factor in patients with ENS-positive 2nd met. The significantly higher rates of ENS-positive 2nd met in the patient group with a preexisting 1st met(s) and ND history within the same neck sides than in the group without these neck profiles (94% vs. 62%) suggest that the neck with these histories might be prone to developing ENS-positive mets. At this time, it is not well understood how ENS occurs in mets. Sumi and Nakamura showed that fibrotic foci were found in the immediate vicinity of ruptured nodal capsules of SCC nodes in the neck, and that those areas often contained cancer cell nests . IntratuThis study is partly limited by its retrospective nature. First, surgical excision of the primary lesions without preoperative radiotherapy was one of the inclusion criteria, but protocols for post-operative chemoradiotherapy (radiation doses and chemotherapy protocols) and ND types  were not fully controlled for in the present study. Second, all the 1st mets were histologically confirmed, but 2nd mets were clinically diagnosed on CT/MR images in 16 of the 79 patients. Although CT/MR imaging diagnosis of mets with ENS produced acceptable results , 23, a v


Manufactured sand differs from natural sea and river dredged sand in its physical and mineralogical properties. These can be both beneficial and detrimental to the fresh and hardened properties of concrete. This paper presents the results of a laboratory study in which manufactured sand produced in an industry sized crushing plant was characterised with respect to its physical and mineralogical properties. The influence of these characteristics on concrete workability and strength, when manufactured sand completely replaced natural sand in concrete, was investigated and modelled using artificial neural networks (ANN). The results show that the manufactured sand concrete made in this study generally requires a higher water/cement (w/c) ratio for workability equal to that of natural sand concrete due to the higher angularity of the manufactured sand particles. Water reducing admixtures can be used to compensate for this if the manufactured sand does not contain clay particles. At the same w/c ratio, the compressive and flexural strength of manufactured sand concrete exceeds that of natural sand concrete. ANN proved a valuable and reliable method of predicting concrete strength and workability based on the properties of the fine aggregate (FA) and the concrete mix composition. In many countries sources of natural sand for use as an aggregate in construction are becoming scarce as sand pits are exhausted and environmental legislation prevents dredging ,2,3. ThiThe shape and texture of crusher dust depends mainly on (i) the type of crusher ,4; (ii) The typical particle size distribution of crusher dust rarely conforms to the requirements of national standards. This is mainly due to an excess (>20%) of fine particles passing the 63 µm sieve and a deficiency of particles in the size range 0.3 mm to 1 mm. Crusher dust can produce “harsh” mixes with bleeding problems if it is washed and screened to fall within the prescribed limits. Therefore, to minimise voids and reduce water demand in concrete, crusher dusts are blended with fine natural sand to aid workability and finishability . The parMany studies have examined the influence of the partial replacement of fine aggregate in concrete using crusher dusts or small crushed sand samples on concrete properties ,12,13. HSince manufactured sands possess different properties to natural sands it would be beneficial to be able to predict the properties of the resultant concrete without extensive laboratory testing. There have been numerous attempts to model the influence of the physical and chemical characteristics of aggregates on the fresh and hardened properties of concrete and provide concrete mix design procedures ,16. ThesSeveral models which evaluate particle packing in aggregate blends have been developed and investigated ,18,19,20A number of researchers have turned to ANN models for the prediction of concrete properties using mix composition parameters, for a variety of concrete types ,24,25,26The primary purpose of this paper is to present a method by which sands manufactured from crusher dusts may be characterised according to their physical and mineralogical properties and subsequently used as a 100% replacement for natural sand in concrete. The structure of the paper is as follows:In this study CEM I 52.5N cement  complying with BS EN 197-1:2011 with a chemical composition given in For this study limestone, granite, basalt and gritstone crusher dusts were processed in the V7 plant. The V7 crushing plant has the ability to produce different sand gradations. At least four manufactured sand gradations from each crusher dust were produced and tested. Also the 0/4 mm fractions of the crusher dusts (unprocessed) were included for comparison. Sea dredged natural sand complying with BS EN 12620:2002 was used as the control fine aggregate. versus flow time has been developed to identify the performance of various natural sands in concrete. The envelope is based on the experiences of the New Zealand authorities and is included in this paper for comparison with manufactured sands.As discussed earlier, the grading of the fine aggregate is an important factor that influences the performance of concrete, therefore all fine aggregates were tested for particle size distribution according to BS EN 933-1:1997. The shape and texture of the sand is more difficult to measure directly, hence the predominantly qualitative characterisation tests that are employed in the UK national standards. However, the New Zealand flow cone (NZFC) test (NZS 3111:1986) used in the study offers an indirect measure of shape and texture through measurement of (i) the flow time of fine aggregate through a funnel of known geometry and (ii) the uncompacted voids content of the fine aggregates once it is collected in a receiving chamber. The flow of the material is mostly affected by the shape and surface texture of the particles and the voids content is determined by the grading and shape of the particles [Also as highlighted in the introduction, the presence of deleterious particles like clays may have a detrimental effect on the water demand of the fresh concrete and the performance of the concrete in its hardened state. Therefore, a quick and effective method was required to screen the sands. Two methylene blue (MB) value tests were used; the British Standard test (BS EN 933-9:1999 on the 0/2 mm fraction) involving titration with an MB solution and a test developed by Grace Construction Products (ASTM WK36804) using a pre-calibrated colorimeter allowing a direct estimation of MB solution consumption. These tests will be referred to as the Methylene Blue Value (MBV) and the Grace Methylene Blue Value (GMBV), respectively in this paper. A sand equivalent (SE) test (BS EN 933-8:1999 on the 0/2 mm fraction) was also used, which evaluates the proportion of very fine and clay sized particles in the whole sample. Manufactured and crushed sands will usually have lower SE values than clean natural sands due to the dust of fracture created during the crushing process.Particle density and water absorption, which are functions of the mineralogical composition of the aggregate, were determined according to BS EN 1097-6:2000. The dry density measurement was used in the NZFC voids calculation, and the absorption capacity was used to adjust the water content of the different concrete mixes.The main characterisation parameters of a concrete mix are its workability and strength. The fresh concrete was therefore tested for slump according to BS EN 12350-2:2009. Furthermore, observations were made during mixing, placing and finishing of the concrete specimens as the slump test alone does not fully characterise the workability of the concrete mixes incorporating manufactured sands .cf’) at 1, 7 and 28 days according to BS EN 12390-3:2009 and flexural strength (tf) at 28 days according to BS EN 12390-5:2009. Standard laboratory moulds were used for the compressive and flexural strength tests. These had dimensions of 100 × 100 × 100 mm3 and 500 × 100 × 100 mm3 respectively, complying with BS EN 12390-1:2012 mould size requirements with regard to maximum aggregate size. It is acknowledged that using these sizes of specimens for concrete made with 20 mm coarse aggregate may have resulted in a slightly greater variability, and marginally lower strengths, than would have been obtained with larger specimens . This is due to the increased relative heterogeneity and the ‘so-called’ wall effect that occurs when the maximum aggregate size to specimen dimension ratio exceeds a certain limit (of approximately 0.2) [3 cubes and three 500 × 100 × 100 mm3 beams were cast. These were de-moulded after 16 h and placed in a water tank at a temperature of 20 ± 3 °C until the testing age was reached.Hardened concrete was tested for compressive strength (ely 0.2) . NeverthThe study was conducted in two phases. The first phase considered slump-controlled mixes without the addition of water reducing admixtures. A control mix with natural sand was made with a target S2 slump (50–90 mm) as designated by BS EN 206-1:2000. For the manufactured sands, the sands with a -B notation were mixed to achieve the same S2 slump as the control and the required water/cement ratio was recorded. That w/c ratio was then kept constant for the remaining gradings of the same quarry sand, recording the change in slump observed in each concrete mix. The second phase of the study comprised mixes made with a constant w/c ratio (w/c = 0.55) and the incorporation of a sufficient volume of a water reducing admixture to achieve an S2 slump. The water reducing admixture was added in increments of approximately 6 mL and slump tests were repeated until an S2 slump was recorded. The limestone sand mixes achieved an S2 slump at a w/c ratio of 0.55 without admixtures, therefore these were mixed at a lower w/c ratio of 0.50. In order to compare the fresh and hardened concrete properties between mixes, the replacement of the fine aggregate in each mix was performed on a weight by weight basis. This ensured that CA/FA and FA/cement ratios were kept constant. Furthermore, the entrained air content of the mixes was tested according to the pressure gauge method in BS EN 12350-7:2009.The mix composition is shown in The fines content of the manufactured sands ranged from 1% to 9% In comparison with the unprocessed crusher dust materials, the majority of the manufactured sands had a greater quantity of the 0.3 mm to 1 mm particles, as indicated by the steeper gradients of the grading curves in this region. The improvement in the particle size distribution in this range was not as pronounced in the limestone sands as in the other manufactured sands. As commented previously this particle size range is often deficient in crushed rock sands [All the manufactured sands used in this study fall within the New Zealand standard envelope for natural sands unlike their crusher dust counterparts, as observed in As shown in Assuming that the governing factor for the flow time measurement is the shape and surface texture of the fine aggregate then there is evidence of the KEMCO V7 process improving these characteristics as the flow time of the unprocessed crusher dust material was between 28 s and 37 s, whereas for all the manufactured sands it was in range of 21 s to 27 s. If processed sands with a particular mineralogy are considered, it can be seen that flow times were slightly reduced with an increase in fines content. This reduction was typically 1 s to 3 s for the -D gradings. It can therefore be inferred that a major part of the flow time reduction can be attributed to particle shape improvements due to processing.The un-compacted voids content of all the basalt and gritstone sands was below that of their feed material. However, the voids content of all the granite sands and the coarsest limestone sand (L-A) was greater than that of their corresponding crusher dusts. This can be attributed to the combined effects of changes in grading as well as the shape of the particular sands.MBV and SE tests were used to identify the presence of potentially deleterious particles, and in particular clays, in the fine aggregates under investigation. et al. [et al. . This coIt can be concluded that for parent rocks containing clay the manufactured sand will also include a proportion of clay in the fines, whereas if the parent rock is clean then the fines are the dust of fracture created during the processing.The water absorption (WA24) of the unprocessed crusher dusts was either higher or the same as that of the corresponding manufactured sands as shown in The compressive strength of the slump controlled mixes was predominantly governed by the w/c ratio, as observed in The granite, limestone and natural sands with low MB values had much lower water and water admixture demands to achieve the S2 slump than the gritstone and basalt sands with MBV above 1.73, as seen in 2. This suggests that the particular clays present in the gritstone and basalt sands do not negatively affect the 28 days strength at the same w/c ratio. However, the long term effects of MB value and clays on strength were not investigated in this study.These results suggest that the angular shape and rough surface texture of the manufactured sand contributed to the compressive and flexural strength of the mix by virtue of aggregate interlock and improved bond between the cement matrix and aggregate particles. Similar results have been obtained by other researchers ,8. At w/Some of the basalt and gritstone sands mixed at a w/c ratio of 0.55 did not achieve the S2 slump even with admixture dosages exceeding those recommended by the manufacturer. This suggests that if a workable concrete mix with reasonable w/c ratio is required then the presence of clay in the manufactured sand is the first and foremost limiting factor. However, if high w/c ratios are permissible for the given concrete application, then an increase in the w/c ratio can offset the negative effects of the presence of clay on the workability of the concrete.A general trend of decreasing slump with increasing fines content was observed in the majority of mixes made without the use of an admixture, with the exception of the B-B, L-C and GS-C mixes, which exhibited higher slump values. This may be explained by the lubricating effect of the increased fines content . HoweverDuring the mixing and casting stages it was observed that the natural sand mix was the easiest to handle and finish as expected due to the rounded and smooth fine aggregate. Mixes with -A notation were sometimes “harsh” and could only be finished with some effort due to the low amount of fines and lack of particles with diameters less than 1 mm. Mixes G-B, G-C, G-D, L-B and L-C with low MBV were found to be easy to handle and finish even though they had lower slump values than the -A mixes. Gritstone mixes at a w/c ratio of 0.67 were found to be easy to work and finish. The basalt mixes were quite cohesive but finished well. The B-D mix was found to be very cohesive and rapidly lost workability, which could be attributed to the absorption of water from the mix by clay present in the high fines content. If the gradings in No noteworthy correlation between compressive strength, flexural strength and the fines content or the grading of the fine aggregates was observed, suggesting that in the range 1%–9%, the fines do not significantly affect the compressive and flexural strength for a given mix composition, when admixtures are employed to enhance workability. The importance of considering other methods to address the lack of fines such as the use of filers and supplementary cementitious materials, as well as the influence of cement content on the performance of mixes containing manufactured sands, is fully acknowledged and is the subject of ongoing work. The results imply that higher levels of fines may be employed in concrete applications with associated reductions in the storage or disposal of the fines material. Additional benefits of enhanced durability due to the pore blocking nature of the fines material  may ANN modelling was selected for this study due to its ability to generalize multiple variable, non-linear, complex relationships and thereby predict results from a range of inputs with which they have been trained . In thisAs discussed, the properties of concrete depend on the properties of the aggregates and the mix composition. Since all of the mixes used in this study had the same amount of cement, FA and CA, the only variables were the w/c ratio and water reducing admixture dosage. These were therefore selected as the two input parameters that described the changes in the mix composition. There are three major fine aggregate properties that affect the workability and strength of the concrete, giving a further 8 input parameters: grading , shape and texture (as inferred by NZFC flow time and voids content), quality of fines .The model was developed using the data presented in this paper together with that from other similar mixes created in the laboratory during the project giving a total of 44 data entries. These were randomly divided into 35 training and 9 testing entries. jy is a neuron in the hidden layer, ix is the scaled input value, ijw is the connection weight, n is the number of inputs and b is a constant referred to as “bias” or “threshold” that is calculated during the network training similarly to connection weights. F is the sigmoid activation function obtained from Equation (2) which represents the non-linear behaviour of concrete. The output values kz are calculated using Equation (1) but by exchanging ix with jy and ijw with jkw.A back propagation algorithm was used to train the neural networks. A full description of the algorithm and ANN in general is provided by Fausett . Figure The neural networks were created with a single hidden layer as it has been previously demonstrated to successfully model concrete strength and workability ,35,36. TThe weights for each neuron were randomized before the network was trained. The initial learning rate was 0.7, which was decreased by 1% after 7 learning cycles. In addition, the initial momentum factor was 0.7, which was decreased by 1% after each 500 cycles. The online training mode was used where the weights were updated after each entry and the data was presented in a random order. The training of each network was halted after 5000 cycles. It was observed that the RMS had stabilized for all networks after approximately 2000–3000 learning cycles.In order to check the prediction capabilities of the models four concrete mixes were made with the same FA, CA and cement contents as detailed in 2 for the 8-6-1 strength model. Again, an underestimation (if any) in the predicted compressive strength is preferable to an overestimation, particularly in structural design applications.The predicted and actual slump and compressive strength values for the validation mixtures are shown in Numerical evaluation of the model can help to confirm that the ANN has actually learnt the underlying theoretical relationship. Fine aggregate parameters should be considered concurrently since it is difficult to identify the influence of any one input parameter on the workability and compressive strength of concrete due to the multi-variable, non-linear nature of the relationship between the variables. et al. [et al. [et al. . The flo [et al.  that if It can be concluded that ANN models can be used to estimate workability and compressive strength of concrete when the fine aggregate properties are used alongside the mix composition as ANN model input parameters. However, there are limitations of such models, the main being that they only perform well in the range of input and output variables with which they have been trained. It can also be concluded that the ANN models developed in this study are valid and the predictions generally correspond to the theoretical relationships between the mix composition, fine aggregate parameters and concrete properties. Thus, this type of model could be used to reduce the effort needed to develop working concrete mixes or to compare the performance of different fine aggregates through simple aggregate classification tests.The primary aim of this study was to present a method by which sands manufactured from crusher dusts may be characterised according to their physical and mineralogical properties and investigate their use in concrete as a 100% replacement for natural sand. The results from the experimental study presented in this paper demonstrated that the V7 crushing plant is capable of producing manufactured sands with similar particle size distributions irrespective of the parent rock mineralogy. A series of tests were proposed, which allowed indirect measurement of shape, surface texture, grading and the presence of deleterious fines, which were used to characterise the physical properties of natural sands, unprocessed crusher dusts and manufactured sands. From these tests it was apparent that manufactured sand particles of improved shape and grading compared to natural sand and unprocessed crusher dust were produced by the V7 crushing plant, along with a reduction in the quantity of clay particles in the fines portion compared to the feed material.Workable concretes were produced using manufactured sand as the sole fine aggregate at different w/c ratios. The presence of clays in manufactured sands can be a limiting factor in their use in concrete applications where high consistency and relatively low w/c ratios are specified. Nevertheless adequate concrete incorporating manufactured sand as sole fine aggregate can be made. Indeed, at the same w/c ratio, the compressive and flexural strengths of manufactured sand concretes were higher than those of their natural sand counterparts. This is believed to be due to the angular shape of the material, which has a positive effect on aggregate interlock and hence leads to improved bond between the cement and aggregate particles. The presence of clay did not affect the 28 days strength of concrete made at the same w/c ratio with differing manufactured sand mineralogies. Therefore, there is the potential for clay containing aggregates to be used in concrete that were previously discouraged. An optimum fines content of 7% was observed to aid the handling, placing and finishing of manufactured sand concrete. However, no significant trend between fines content and concrete compressive strength was observed in the 1% to 9% fines content range that was investigated. Thus, in order to maximise the efficient use of materials, higher fines content manufactured sand gradings could be employed when compressive strength is the control property.It has been shown that ANN models can be used to estimate the compressive strength and workability of concrete based on the fine aggregate properties and mix composition with reasonable accuracy. Such models, together with the characterisation tests described previously, could be employed by the construction industry when new fine aggregates sources enter the market. The use of the models would eliminate the need for extensive laboratory trials to select an appropriate mix design and establish the fresh and hardened properties of the resulting concrete.


The cultivation of mycobacteria often requires the use of several antibiotics to limit the growth of other rapidly growing micro-flora present in the growth medium. This antibiotic cocktail is one of the most expensive reagents required for mycobacterium culture. Here we present a customized antibiotics mix that is easy to prepare at a fraction of the cost of the commercially available antibiotic mixture that protects against transient flora, which are normally present in lungs, without affecting mycobacterial colony number. Mycobacteria are slow-growing organisms  and Trimethoprim  tablets were dispersed in 10 ml water and kept on rocker shaker for 15–20 mins, the mixture was then centrifuged at 600TM; Samarth Life Sciences Pvt. Ltd.), Amphotericin B  and Azlocilin  were weighed and dissolved in water. The CAM was prepared by mixing appropriate volumes of working stock solutions and was filtered through a 0.2-µm syringe filter.Required amounts shown in of PolymTM Mycobacteria 7H11 Agar  was suspended in 4,500 ml water containing 25 ml glycerol. The medium was swirled on a hot magnetic plate to obtain a smooth suspension and autoclaved at 121°C for 15 min.A total of 105 g Difcooleic acid,albumin,dextrose andcatalase).The medium was allowed to cool to 50–55°C in aseptic conditions. In the meantime, 25 g bovine serum albumin, 10 g dextrose, 15 mg catalase and 4.25 g sodium chloride  were dissolved in 500 ml water and filtered through a 0.2-µm filter. Next, 250 µl oleic acid  was then added aseptically. This mix is commonly known as OADC (TM MGITTM PANTATM (BD-Panta) antibiotic mixture  was then added to 5 l media. In another preparation of media, the BBLTM MGITTM PANTATM was replaced with the CAM, formulated as aforementioned.Ten vials of commercial BBLMycobacterium bovis (BCG) by the aerogenic route to establish around 1,000–2,000 bacilli of BCG in each mouse .At every time point of day 1, 7, 14 and 30 post-immunization, two immunized mice were euthanized by an overdose of Ketamine and Xylazine given intraperitoneally. Typically, 35 mg ketamine and 3.5mg xylazine in 350 µl saline per mouse was used to euthanize a mouse.Their lung and spleen were isolated aseptically and homogenized in 1 ml PBS using a tissue homogenizer  at 30,000 rpm for 40 s. The homogenized mix was diluted 5 times in PBS and 100 µl diluted mix was spread on the aforementioned agar plates in triplicate.At every time point the tissue homogenates were plated on media plates prepared using CAM and commercial BD-Panta antibiotic mixture.Plates were incubated at 37°C for 4 weeks.GraphPad Prism 7 software was used to calculate p-values by two-way ANOVA.After incubation, BCG colonies present on plates were counted, the results of which are shown inNumber of BCG bacilli colony forming units (CFUs) on each plate from each experimental groupThe data show CFUs on each plate along with calculated bacilli load of the tissue.Click here for additional data file.Copyright: © 2018 Kesarwani A et al.2018Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).th of the cost of commercially available antibiotic formulation for the purpose. This antibiotic mix is highly economical, easy to prepare and can significantly reduce the total cost involved in mycobacterium culture.The cost of above discussed customized antibiotics mix for preparing 5 l of agar media was around 80 INR (around 1.25 USD) which is almost 1/100The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2018 Kesarwani A et al.Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Dataset 1. Number of BCG bacilli colony forming units (CFUs) on each plate from each experimental group. The data show CFUs on each plate along with calculated bacilli load of the tissue.http://dx.doi.org/10.5256/f1000research.14467.d201182 , presently in use. They have performed whole experiments in a very simple way and succinctly described the methods with appropriate conclusions. Since the findings obtained using CAM were comparable to the commercially available PANTA, this can be really useful to researchers in TB field. Bypassing the routine culture and BACTEC methods authors tested CAM in animal based study, which gave ample scope to new mixture of antibiotics to eradicate any prevailing contamination carried forwarded through organ homogenate. However, a control carrying only organ homogenates on the culture media plates without any antibiotic mix would have given fair idea of prevailing microbial contamination and allowed authors to assess how effectively CAM exterminated the microbial flora under the experimental conditions used by authors.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This is a very simple paper based on common microbiological techniques. Authors have taken a few common antibiotics and made a cocktail which is cost-effective and gave results comparable to the existing commercially available antibiotics (PANTA). Experiments are planned well, appropriately described, and conclusions are justified. The comparable findings obtained using this highly cost-effective formulation relative to commercially available formulations can be of great help to researchers in TB field. To check the reliability of the new formulation, authors have conducted animal experiments and enumerated bacillary load in the infected organs such as lungs and spleen. Authors detected no significant difference between commercial and the new mixture of antibiotics on the growth potential of the BCG strains isolated from the infected lungs. The statistical analysis was done using two-tailed ANOVA, which is most appropriate.  While experiments are reliable and results are presented well, I would have liked a control wherein authors plate organ homogenates on the culture media plates without any antibiotic mix. This would have confirmed that the normal microflora present in these organs are interfering with the outgrowth of BCG cells under the experimental conditions used by the authors. This way authors can bolster the efficacy of the antibiotic combination in reducing contamination in the mycobacterial cultures.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.