Few patients with pancreatic cancer may be candidates for immediate surgical resection at the initial diagnosis. Even if patients with borderline resectable pancreatic cancer (BRPC), micrometastases may occur before surgery. Therefore, neoadjuvant therapy is vital for improved survival, which has been confirmed in previous studies that neoadjuvant chemotherapy with or without radiotherapy provides superior overall compared with upfront surgery. However, question of whether the addition of radiotherapy to neoadjuvant chemotherapy can improve prognosis compared with chemotherapy alone is a challenging matter. Moreover, most of previous studies only adopted conventional radiotherapy as the neoadjuvant modality though stereotactic body radiation therapy (SBRT) has been proven effective and commonly employed in pancreatic cancer. Also, no studies have evaluated the efficacy of S-1 as the neoadjuvant chemotherapy regimen for BRPC albeit similar prognosis has been found between S-1 and gemcitabine in advanced pancreatic cancer. Hence, the aim of this study is to investigate whether neoadjuvant chemotherapy plus SBRT results in better outcomes compared with neoadjuvant chemotherapy alone and also compare the efficacy of gemcitabine plus nab-paclitaxel with SBRT and S-1 plus nab-paclitaxel with SBRT.Patients with biopsy and radiographically confirmed BRPC, no prior treatment and severe morbidities are enrolled. They will be randomly allocated into three groups: neoadjuvant gemcitabine plus nab-paclitaxel, neoadjuvant gemcitabine plus nab-paclitaxel with SBRT and neoadjuvant S-1 plus nab-paclitaxel with SBRT. Standard doses of gemcitabine and nab-paclitaxel are used. The radiation dose of SBRT is 7.5-8Gy/f for 5 fractions. Surgical resection will be performed 3 weeks after SBRT. Artery first approach pancreaticoduodenectomy or radical antegrade modular pancreatosplenectomy will be used for the tumor in the head or body and tail of the pancreas, respectively. The primary endpoint is overall survival. The secondary outcomes are disease free survival, pathological complete response rate, R0 resection rate and incidence of adverse effects.If results show the survival benefits of neoadjuvant chemotherapy plus SBRT and similar outcomes between S-1 and gemcitabine, it may provide evidence of clinical practice of this modality for BRPC.NCT03777462).The study has been registered in ClinicalTrial.gov ( Pancreatic cancer has been the fourth cause of cancer mortality in the US with a 5-year survival rate of 3% . In the Though surgical resection is considered as the only curative intent treatment, the 5-year survival rate of patients with upfront surgery is less than 20% .Due to high intolerance and incidences of adverse effects of FOLFIRINOX in Chinese, other regimens with mild toxicities may be the alternatives. It has been proven that gemcitabine plus nab-paclitaxel contributed to increased survival in pancreatic cancer . And thiFurthermore, compared with conventional radiotherapy, the advantages of stereotactic body radiation therapy (SBRT), precise treatment delivery by motion compensation strategies with high doses to target volumes, abrupt dose fall-off outside target volumes, shorter durations and real-time tracking, has rendered SBRT as an appealing technique of radiotherapy, which has been widely employed for pancreatic cancer with high rates of local control and acceptable toxicities. Nevertheless, only conventional radiotherapy was employed in the neoadjuvant treatment in the previous studies.Therefore, the aim of the study is to compare and evaluate the efficacy of neoadjuvant gemcitabine plus nab-paclitaxel versus gemcitabine plus nab-paclitaxel with SBRT versus S-1 plus nab-paclitaxel with SBRT for BRPC.The primary objective is to compare the OS of patients receiving neoadjuvant gemcitabine plus nab-paclitaxel versus gemcitabine plus nab-paclitaxel with SBRT versus S-1 plus nab-paclitaxel with SBRT for BRPC. The secondary objective is to compare DFS, pathological complete response rates and adverse effects of the three groups.Neoadjuvant chemotherapy plus SBRT is superior to neoadjuvant chemotherapy alone regarding OS, while no significant difference is found between gemcitabine plus nab-paclitaxel with SBRT and S-1 plus nab-paclitaxel with SBRT.This is a randomized, prospective, single center and phase III trial. Patients aged above 18, with radiographically and biopsy proven BRPC and without any prior treatment will be screened for eligibility. Therefore, all potential patients would receive fine needle aspirations guided by endoscopic ultrasound. Details about the inclusion and exclusion criteria are shown in Table Eligible participants would receive personal interviews with physicians for a detailed explanation of the whole study and related treatment. It is mandatory to acquire the written informed consents before the study. Afterwards, patients are required to complete the pretreatment evaluations, including blood routine tests, liver and renal function tests, coagulation function tests, serum tumor marker CA19–9) tests, enhanced CT and MRI, circulating cell free DNA (cfDNA), circulating tumor cells (CTC), DNA sequences of the specimens from fine-needle aspirations and pancreatic cancer-derived organoid cultures with specimens. In each group, participants will be randomized with a 1:1:1 allocation to receive gemcitabine plus nab-paclitaxel or gemcitabine plus nab-paclitaxel and SBRT or S-1 plus nab-paclitaxel and SBRT. Randomizations will be performed by a computer-generated random numbers table. After completion of the whole chemotherapy, patients will first receive PET-CT to exclude distant metastases and then undergo SBRT. Those with distant metastases will be precluded from the study and receive chemotherapy continuously. Likewise, patients confirmed with distant metastases after chemotherapy and SBRT will receive other aggressive chemotherapy regimens rather than surgical resections . The study has also been registered in ClinicalTrial.gov (NCT03777462).2) or nab-paclitaxel (125 mg/m2) is initiated on day 1, 8 and 15 during each 4-week cycle, which will repeat for 3 cycles. S-1 is orally administrated at a dose of 80 mg/m2 for 18 days followed by a 10-day rest, which also continues for 3 cycles.All participants are allocated into the three groups randomly. Intravenous administration of gemcitabine  and laboratory tests . Imaging examinations will be performed 3 months, 6 months, 1 year and 2 years after surgery to evaluate efficacy. Post-operative complications would be evaluated 1 month and 3 months after surgical resection. Adverse effects of chemotherapy will be assessed according to The Common Terminology Criteria for Adverse Events version 4.03 (CTCAE v4.03). Radiation-induced acute toxicities are adverse effects that occur within 90 days after treatment, and determined by the Radiation Therapy Oncology Group, ‘Acute radiation morbidity scoring criteria’. If patients experience disease progression during follow-up, cfDNA and CTC would be performed to evaluate the tumor burden and identify genetic differences of the tumor cells before and after treatment as far as possible. Disease progression is determined by the Response Evaluation Criteria in Solid Tumors (RECIST) criteria (v1.1).The primary outcome is OS and 1-year and 2-year OS rate. OS is defined as the time interval from the time of surgical resection to the date of all-cause death or the last follow-up.The secondary outcomes are DFS, pathological complete response rate, R0 resection rate, incidence of adverse effects. DFS is determined as the time interval from the date of surgery to the confirmation of recurrence at any sites or death from any cause, if this occurs before disease progression, or the last follow-up. The definition of pathological complete response is ypT0N0M0, which is no evidence of invasive tumor in the pancreas and the draining lymph nodes at the time of surgery. R0 resection is defined as both clear macroscopic and microscopic margins (≥1 mm).Assuming, for the gemcitabine plus nab-paclitaxel group, a median 2-year OS rate of about 20%, we target a 10 and 15% relative improvement in 2-year OS rate for the group allocated to S-1 plus nab-paclitaxel with SBRT and gemcitabine plus nab-paclitaxel with SBRT over the gemcitabine plus nab-paclitaxel group, respectively. This would provide approximately 80% power with one-sided α of 0.025. Hence, the maximum number of required patients in each group is 73. Thereafter, when a 10% dropout rate is taken into account, the number of participants in each group is 81.P values will be reported using a significance level of 0.05.For the continuous variables, statistical normality will be assessed firstly. Comparisons between these main effects of the three groups will be then performed by analysis of variance or its’2 non-parametric equivalents as appropriate. Categorical data will be compared by Chi-square tests or Fisher’s Exact tests. The OS and DFS curves will be conducted by Kaplan-Meier method. Log-rank test will be employed for comparisons of OS and DFS. Proportional hazard Cox regression adjusted for variables will be used when necessary. Data analysis will be performed on both intention-to-treat and per-protocol basis. All It has been long suggested that upfront surgery is given first priority for patients with BRPC or resectable pancreatic cancer based on NCCN guidelines. However, recent studies have clarified the possibility of employment of neoadjuvant chemotherapy with or without radiotherapy, which may provide potential survival benefits. For resectable pancreatic cancer, systemic reviews indicated that neoadjuvant therapy was associated with improved OS –21. LikeAdditionally, in most of previous studies, conventional radiotherapy was used as the modality. Usually, radiation doses of 45-54Gy in 1.8–2.0Gy fractions are prescribed leading to a long treatment period, which may result in micrometastases during neoadjuvant therapy. For patients with BRPC, relatively small tumor volumes without obvious invasions of adjacent vessels may provide opportunities for dose-escalation. Hence, SBRT may be an appropriate choice due to its delivery of high biological effective doses within a short period and quick dose fall-off, which may not delay subsequent treatment and provide high pathological complete response rates with acceptable toxicities simultaneously, combined with chemotherapy. Though it might be implied from the previous studies that neoadjuvant chemotherapy with or without radiotherapy could be an alternative to the surgery-first paradigm for BRPC, potential benefits from neoadjuvant chemoradiotherapy still need to be confirmed in prospective studies. Therefore, the BRPCNCC-1 trial, a randomized and prospective study, investigates whether neoadjuvant chemotherapy plus SBRT can prolong survival for BRPC compared with neoadjuvant chemotherapy alone.Though S-1 has been confirmed effective in pancreatic cancer, especially for Asians, most of studies have only adopted S-1 in advanced pancreatic cancer. No studies have evaluated the efficacy of S-1 in neoadjuvant therapy. Besides, only FOLFIRINOX or gemcitabine plus nab-paclitaxel is recommended in guidelines. As a result, it is necessary to assess whether neoadjuvant S-1 plus nab-paclitaxel with SBRT could be an alternative to neoadjuvant gemcitabine plus nab-paclitaxel with SBRT regarding OS.Another concern about the trial is the definition of resectability status. Recently, a new international consensus on the definition and criteria of BRPC has been proposed . It has In summary, this pilot study, BRPCNCC-1, will investigate whether the addition of SBRT to neoadjuvant chemotherapy results in superior survival compared with neoadjuvant chemotherapy alone and compare the efficacy of S-1 plus nab-paclitaxel combined with SBRT and gemcitabine plus nab-paclitaxel combined with SBRT. The results may provide sufficient evidence for clinical practice of neoadjuvant therapy for BRPC.


Nanocomposite dental resins with 0, 2, 5, and 10 wt % methacryl polyhedral oligomeric silsesquioxane (POSS) as filler in the resin matrix were prepared by a light curing method.The atomic force microscopy (AFM), fourier transform infrared spectroscopy (FTIR), nanoindentation, and nanoscratch tests were carried out to study the effect of POSS contents on the compatibility, double bond conversion, volumetric shrinkage, hardness, modulus, and resistance of the dental resins. POSS was very uniformly dispersed and showed a good compatibility with the matrix. The double bond conversion increased and the volume reduced with the addition of POSS. As the POSS addition increased, the mechanical properties increased initially. Small addition of POSS remarkably enhanced the hardness and scratch resistance of the resin matrix. Increasingly, conventional alloys in the current dental field have been replaced by resin materials due to their advantages such as operation technique and aesthetic quality ,2. Nanof3/2)n, where n is the number of the silicon atoms of the cage (1–3 nm in size) surrounded by organic corner groups R. There are very different types of POSS according to the various R organic groups [Hybrid organic-inorganic nanocomposites, which incorporate polyhedral oligomeric silsesquioxane (POSS) into polymeric matrices including dental resins, have received a considerable amount of attention. Polyhedral oligomeric silsesquioxane (POSS) is an organic-inorganic hybrid nanomaterial. Its formula is , FTIR, nanoindentation, and nanoscratch tests were performed, and the effects of the contents of POSS on the compatibility, double bond conversion, volumetric shrinkage, hardness, and resistance were studied.Bisphenol A glycerolate dimethacrylate (Bis-GMA) and Tri(ethylenglycol) dimethacrylate (TEGDMA) 98% were purchased from Aldrich Chemical Co. China . Camphorquinone  was used as a visible light photo-initiator and was selected with its co-initiator 2-(dimethylamino) ethylmethacrylate  for this research. The nano filler, methacryl POSS, was purchased from Hybrid Plastics .A resin matrix solution containing Bis-GMA, TEGDMA, CQ, and DMAEMA was prepared by mixing in a light resistance environment after sufficient mixing. Then different percentages methacryl POSS, according to the quantities reported in The whole process should be performed under vacuum conditions. The mixtures went through a light curing process which lasted for 40 s at room temperature in stainless steel molds. After being soaked in distilled water at 37 °C for 24 h, they were placed in configured artificial saliva (according to ISOTR1021) at 36.5 °C for 4 more weeks. Finally, the dimensions of the specimens were measured accurately before testing.AFM topography images were obtained in Peak Force Tapping on the Bruker instruments . The scan rate was set to be 1 Hz and imaging was carried out on Multimode 8.−1, with a resolution of 4 cm−1. FTIR spectrometer  was used to carry out the Fourier transform infrared (FTIR) spectroscopy analysis. The scan range was from 4000 to 400 cm−1 and normalized against the carbonyl C=O peak at 1720 cm−1 according to the Equation 1.−1, which are characteristic of methacrylate C=C and carbonyl, respectively. The degree of double bond conversion (DC) was monitored by FTIR. The DC was calculated from the methacrylate C=C peak at 1636 cmm0 is the mass of the empty bottle. Here, m1 is the mass of the bottle full of water at 20 °C; m2 is the mass of the bottle full of solution at 20 °C; m3 is the mass of the bottle with specimen and water; ms is the mass of the specimen; and The densities of both uncured and cured resin samples were measured by a pycnometer to determine the polymerization shrinkage according to the Archimedes’ principle. The volumetric shrinkage was calculated using the following equation:−1 was ensured by the loading speed. The results of 8 samples were averaged to get the most accurate results.According to the Oliver-Pharr method ,37, a naThe resistance of the POSS-reinforced resin was evaluated by nanoscratch testing using a Nano Indenter G200 instrument . The method employed was the “G-Series single direction wear test”. The authors followed a test procedure similar as published elsewhere ,39. The It was found during the tests that a higher percentage of the nano filler resulted in the deterioration of the transparency. However, this led to a high viscosity of the sample. Deterioration in the transparency thus led to a less light radiation fluence and a lower curing level of composites.The morphologies of a composite with the size of 2 μm × 2 μm, containing 0, 2, 5, and 10 wt % POSS, are shown in the AFM images in POSS showed a good compatibility with the matrix and could be uniformly dispersed at a low percentage of addition. However, once the POSS content reached 10 wt %, the aggregation of nanoparticles can be observed in the composite resin due to the self-assembly of POSS . When th−1 was attributed to the characteristic absorption peaks of the C=C bond. When the curing happened, the methacrylate double bonds partly converted and the resins polymerized [−1 became evidently weaker.The FTIR spectra of composite resins with different POSS contents after curing are shown in ymerized . The intThe values of double bond conversion of composite resins with different POSS contents are shown in In this subsection, the shrinkage of composite resins synthesized were studied. The value of volumetric shrinkage of the resin matrix was 10.65%. As the percentage of POSS increased, the volume shrinkage of composites became lower. The values were about 9.07%, 9.85%, and 7.81% for the additions of 2%, 5%, and 10%, respectively.In our previous work , the denBesides, our previous experimental research and the study of Fong also investigated the volume shrinkage of the composite resins with an additional 60 and 70 wt % of BG powder ,33. The The hardness and resistance are two of the most important properties for dental restorations. In this study, the resistance of the POSS dental composites was evaluated by the scratch depth. The scratch depth can be used to evaluate the deformation resistance and hardness of the samples at a specific load ,47. The The testing results of the hardness, elastic modulus, and the average scratch depth are listed in As the POSS addition increased, the mechanical properties increased initially. Once it exceeded 5 wt %, the mechanical properties fell down. A small addition of POSS remarkably enhanced the hardness and the scratch resistance of the resin matrix. These properties showed the similar variation trend according to the percentage of POSS, which indicated that it had the same influence on the composites’ matrix. POSS, serving as a nucleating agent, attracts matrix chains, which increases the rigidity of the systems and enhances the mechanical properties. While a larger content of POSS perhaps results in a large reduction of the crosslinks among the matrix chains owing to the aggregation of POSS, it enables the polymer matrix to form plastic flow easily and reduces the mechanical properties ,42,48. T2, POSS is a unique kind of filler with a hard inorganic Si–O cage core surrounded by soft organic chains. The overall performance of POSS derives from the combined action of hard “core” and soft “shell” [Compared with SiO “shell” . When th “shell” ,51. If t “shell” ,53. The  “shell” ,55. Compared with the values of the hardness in our previous work , it is fIn this paper, POSS is shown to be very compatible with the matrix. Weight fractions of 2%, 5%, and 10% of POSS in the resin matrix lead to an increase of the double bond conversion to about 55% when the curing time of each sample is 40 s. The values of volumetric shrinkage are about 9.07%, 9.85%, and 7.81%, respectively, at the above weight fractions. POSS limits the change in the free volume of the matrix during the reaction, but the volume of the nanocage of POSS itself does not change after the reaction. Thus, the shrinkage of resins with incorporated POSS decreases.As the content of POSS increases, the mechanical properties increase initially. The addition of a small amount of POSS remarkably enhances the hardness and the scratch resistance of the resin matrix. Once it exceeds 5 wt %, the mechanical properties deteriorate. POSS, serving as a nucleating agent, attracts matrix chains and thus increases the rigidity of the systems and enhances the mechanical properties. However, a larger content of POSS may result in a large reduction of the crosslinks among matrix chains owing to the aggregation of POSS.


This study evaluated the effect of recent ACA changes to Medicare Home Health Care (HHC) reimbursements on the mix of agencies and staff in the HHC market. We used Provider of Services (POS) data and logistic regression, to determine which agency characteristics were associated with the likelihood of exiting the HHC market and likelihood of decreasing staff before  and after  implementation of the ACA-mandated reimbursement cuts. Free standing agencies had 1.35 times the odds of exiting from the HHC market post ACA cuts. There were no differences in the odds of exiting the HHC market between for-profit and non-profit agencies. Agencies in the New York, Atlanta, and Chicago regions had a greater likelihood of exiting the HHC market post ACA cuts. Small agencies had two times the odds of exiting (aOR= 2.09) and agencies with one or more branch had less than half the odds of exiting (aOR= 0.46) from the HHC market. The average number of all staff was similar before and after the ACA cuts; however, office staff and home health aides experienced the greatest decrease in number. Agencies that were for-profit, free-standing, small, and/or with one or more branch were more likely to decrease staff post the ACA cuts. Agencies in the New York, Atlanta, Chicago, Dallas and Kansas regions were more likely to decrease staff. Overall, the reimbursement cut effects varied by geographic region and had greater impact on more vulnerable agencies and staff that were non-skilled.


In this data article, the splitting tensile strengths of bamboo reinforced lateritic concrete with varying bamboo culm sizes are presented and compared with bamboo reinforced pure concrete (i.e. no lateritic replacement for fine aggregate). 25% of fine aggregate was replaced with laterite and concrete cylinders were cast using bamboo reinforcements both full culm sizes and half culm sizes. The cylinders of 300 mm height and 150 mm diameter were cured for 28 days and subjected to splitting tensile load at 7 days, 14 days and 28 days. Three samples were tested for each conditions and the average value computed. Specifications tableValue of the data•The test data allows for investigation on the use of bamboo as another possible material •The data allows for the assessment of the possibility of replacing fine aggregate with laterite •The data presented can be used to examine bamboo culm sizes in the strength of concrete.1The data presented information on splitting tensile strength of pure concrete and lateritic concrete reinforced with bamboo. Failure load of reinforced lateritic concrete cylinders with full culm and half culm bamboo reinforcement at 7, 14 and 28 days of curing were given. Splitting Tensile strength of pure concrete and lateritic concrete cylinders with full culm bamboo reinforcement at 7, 14 and 28 days of curing.2The aggregate materials and cement used for this research were collected from different locations in Omu-aran, Kwara State, Nigeria . The bamboos which were also gotten from Landmark University farms were cut into the length needed some in full culm and others sliced vertically into half culms. Properties of the bamboo were as presented in


L. monocytogenes, E. coli, S. enterica serovar Typhimurium, S. aureus and toxin-producing enteropathogenic E. coli. Cells that were exposed to combinations of surfactants and salts for 5 min and up to 1 h lost their culturability on standard growth media while retaining their ATP production, fermentation of sugars and membrane integrity, which suggests intact and active metabolism. Screening also revealed major differences between Gram-negative and Gram-positive bacteria; the latter being more susceptible to VBNC induction. Combinations of such detergents and salts are found in many different environments and reflect realistic conditions in industrial and domestic surroundings. VBNC cells present in industrial environments, food-processing plants and even our daily routine represent a serious health risk due to possible resuscitation, unknown spreading, production of toxins and especially their invisibility to routine detection methods, which rely on culturability of cells and fail to detect VBNC pathogens.Effective monitoring of microbial pathogens is essential for a successful preventive food safety and hygiene strategy. However, as most monitoring strategies are growth-based, these tests fail to detect pathogenic bacteria that have entered the viable but non-culturable (VBNC) state. The present study reports the induction of the VBNC state in five human pathogens by commercially available household cleaners in combination with inorganic salts. We determined that non-ionic surfactants, a common ingredient in household cleaners, can induce the VBNC state, when combined with salts. A screening study with 630 surfactant/salt combinations indicates a correlation between the hydrophobicity of the surfactant and VBNC induction in  This maximizes utilization of resources while structural and genetic integrity are maintained, and increases tolerance to harmful conditions3. One particular survival strategy in bacteria is the ability to enter a viable but non-culturable (VBNC) state that permits endurance to unfavorable environmental conditions5. In this regard, the VBNC state is similar to dormancy with the cells retaining an intact membrane, undamaged genetic material and metabolic activity. However, in contrast to dormant cells, VBNC cells are by definition not culturable on routine laboratory media, which are commonly used for hygiene monitoring purposes6. Due to their reduced metabolic activity and increased peptidoglycan cross-linking, many VBNC bacteria have higher physical and chemical resistances compared to culturable cells9. Up until now, approximately 85 species have been shown to enter the VBNC state, including 67 pathogenic bacteria10. Understanding resuscitation of these VBNC cells remains challenging to microbiologists. However, in some cases stable resuscitation protocols have been implemented whereby the cells regained their ability to grow and again become virulent13. So far, the mechanisms for induction and resuscitation of VBNC cells have remained largely unknown. While a few bacteria resuscitate on simply removal of the inducing stress, others are dependent upon specific quorum sensing signals, autoinducers or could only be shown to resuscitate in vivo16.Microorganisms are constantly exposed to changing environmental conditions and are forced to employ various survival strategies by reversibly adjusting their physiologyL. pneumophilia, enteropathogenic E. coli, uropathogenic E. coli and S. typhi, have maintained their pathogenicity even if they were not able to resuscitate, either due to toxin production or other virulence factors17.Interestingly, some VBNC pathogens, such as 10. Indeed, bacterial stress factors present in clinical- or food-processing environments, such as starvation, chlorination, temperature extremes, antibiotic pressure or chemicals such as food preservatives, induce the VBNC state in several human pathogens22. In most industrial and food-processing environments induction of the VBNC state and complete loss of culturability is a long-term process, which might take up to months to occur under stable conditions10. In contrast, chlorine treatment, exposure to heavy metals or UV light have been shown to reduce culturability and stimulate VBNC induction rapidly24.It has been recently suggested that it is VBNC cells, which are not detected by routine growth-based methods, that account for the fact that only 20% of food-borne diseases can be linked to known pathogens and 80% remain unidentified or unspecified2, which seemed to be highly effective against the foodborne pathogen L. monocytogenes25. As non-ionic surfactants are now widely used in industrial and household cleaning agents, this novel combinational effect prompted further investigation.Recently our group discovered a surprising combinational antimicrobial effect of a non-toxic, non-ionic surfactant and MgCl26.In order to investigate possible VBNC induction and to distinguish VBNC cells from viable and dead cells, we compared the culturability of bacteria with growth-independent viability assays. We combined enzymatic tests for evaluation of metabolic activity and used fluorescence microscopy to analyze cell membrane integrity. Furthermore, we analyzed for culturability and ATP generation in five different pathogens to determine if they retain metabolic activity. Extended testing of metabolic activity was performed for one individual combination of each of the five selected pathogens by use of API 20E test strips that test for carbohydrate fermentation and catabolism of proteins and amino acids, which are essential for viabilityWe report VBNC state induction in the five pathogens by exposure to a combination of non-ionic surfactants with salts commonly used in food-processing environments.As growth-dependent methods are still used overwhelmingly for routine diagnostics, and they fail to detect VBNC pathogens, negative findings may be leading to a false sense of security. The results of this study suggest that adaptation of routine diagnostics to include more complex viability detection techniques is essential in order to correctly evaluate the risk of VBNC pathogens in our daily routine.L. monocytogenes with a few random combinations.The previously described antimicrobial effect of non-toxic non-ionic surfactants in combination with salts, was further investigated in respect of possible VBNC induction. Initially we investigated possible VBNC induction in Therefore, bacteria were centrifuged and exposed to a combination of surfactant with a salt for 1 h. After removing the combinational stress by washing and subsequent resuspension in fresh brain heart infusion broth (BHI) growth medium, tests to determine culturability, membrane integrity and cell metabolism were performed at 0 h, 1 h and 24 h in BHI medium Fig. .Figure 1L. monocytogenes after exposure to the non-ionic surfactant Lutensol XP30 in combination with MgCl2 for one hour in comparison to cells exposed to the alcohol-based disinfectant Bacillol® AF (disinfection control).Figure TMBacLightTM viability assay, indicating damaged cell membranes  and Gram-negative bacteria   Fig. .Eighteen different non-toxic, non-ionic surfactants in combination with seven different salts were tested. Gram-negative cells were classified as dead when the luminescence remained below 100,000 relative light units after 24 h. Above this threshold, cells were regarded as VBNC. This test was performed in triplicate. Combinations were marked as VBNC, if at least one out of three tests led to induction of the VBNC state . For combinations that led to VBNC induction three out of three times, the reproducibility was further tested Table .For Gram-negative bacteria, culturability was impaired almost exclusively using combinations of surfactants and carbonates, and some of the combinations induced the VBNC state after 1 h incubation. Prolonged exposure of the VBNC bacteria for 24 h increased the number of surfactant/salt combinations that led to cell death, while combinations that did not induce the VBNC state after 1 h did not show this effect.All combinations leading to VBNC induction three out of three times in initial screening were further analyzed in order to determine reproducibility Table . While sIn contrast to results obtained with Gram-negative bacteria, many more combinations affected culturability and induced the VBNC state in Gram-positive bacteria Fig. .Figure 3Eighteen different non-toxic, non-ionic surfactants in combination with seven different salts were tested. Gram-positive cells were classified as dead when the luminescence remained below 100,000 relative light units after 24 h. Above this threshold, cells were regarded as VBNC. This test was performed in triplicate. Combinations were marked as VBNC, if at least one out of three tests led to induction of the VBNC state . For combinations that led to VBNC induction three out of three times, the reproducibility was further tested Table .Although the effect for Gram-negatives was almost exclusively found in combinations with carbonates, there was no salt preference for Gram-positive bacteria. Interestingly, there was almost no combinational effect observable for surfactants with HLB values ≥15.For the Gram-negative bacteria, prolonged exposure to surfactant/salt combinations for 24 h led more often to cell death. Again, this effect was observed only with bacteria, that entered the VBNC state after 1 h, while culturable bacteria were not affected Fig. . FurtherAs non-ionic surfactants are widely used in commercial products, we investigated if common household products could induce the VBNC state. In this respect we tested six different commercial products containing unspecified non-ionic surfactants. After determination of minimum inhibitory concentrations, two products, a dish-washing liquid and a laundry detergent, were further investigated regarding their potential to induce the VBNC state in combination with seven salts, according to our initial screening protocol Table .L. monocytogenes with the laundry detergent in combination with (NH4)2CO3, the dish-washing liquid induced the VBNC state in both L. monocytogenes and S. enterica in combination with either KH2PO4 or MgCl2.While the VBNC state could only be induced in L. monocytogenes and S. enterica is comparable to those induced by pure Lutensols as shown previously  was part of the collection of bacterial strains at the Institute of Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, Austria. Escherichia coli ATCC 25922, Salmonella enterica serovar Typhimurium ATCC 14028 and Staphylococcus aureus ATCC 6538 were part of the collection of bacterial strains at Merck KGaA, Darmstadt, Germany, and the eae-positive enteropathogenic E. coli O8:H14 strain (EPEC) was obtained from AGES, Vienna, Austria.All bacterial strains were grown overnight in brain heart infusion broth (BHI) at 37 °C. Fresh BHI broth was inoculated with 1 ml of the overnight culture and cells were grown to an optical density of 0.6 at 600 nm, making most of the cells be in logarithmic growth phase.600: 0.6) were centrifuged for 5 min at 8,000 × g and the pellet resuspended in 1 ml of the respective treatment combination and incubated for 1 h at room temperature  plate and incubating overnight at 37 °C.In order to investigate possible VBNC induction, 1 ml of early log phase cells  was used. Correspondingly, 1 ml of the bacterial sample was centrifuged at 8,000 × g for 5 min, washed with PBS twice and diluted 1:1,000. Subsequently, 1.5 µl of SYTO 9 dye and 1.5 µl of propidium iodide were added to the sample, followed by mixing and 15 min incubation in the dark. After incubation, cells were captured with a 0.2 µm polycarbonate membrane filter . The filters were trapped between a slide and a coverslip and used for fluorescence microscopy. This test is based on analysis of cell membrane integrity by fluorescence microscopy. Viable cells with intact membranes are stained green by fluorescent SYTO 9, which can pass the intact or damaged plasma membrane, while propidium iodide can only pass through damaged and disrupted plasma membranes and competes with SYTO 9 for nucleic acid binding sites. Therefore, cells with intact membranes exhibit green fluorescence and cells with damaged membranes exhibit red fluorescence. The latter were considered to be dead. Photographs were taken with the Zeiss Observer Z1 inverted widefield microscope and ZEN 2012 (blue edition) software .In order to determine if cell membranes of non-culturable bacteria remained intact after exposure to the respective treatment conditions, the LIVE/DEADTM Microbial Cell Viability Assay . Correspondingly, 100 µl of bacterial culture was mixed with 100 µl luciferase reagent and incubated in the dark for 10 min at room temperature. Luminescence was recorded in relative light units (RLU) using a Tecan F100 microplate reader . Sterile BHI medium was used as negative control.To investigate if cells that had lost their culturability were still metabolically active, ATP was quantified using the BacTiter-GloBioMérieux, Marcy l′Étoile, France) was applied. This system can be used to monitor 20 different enzymatic or metabolic activities from a complex sample.To investigate the enzymatic activities of VBNC cells and their ability to metabolize sugar, the API 20 E Kit  was added to 900 µl of the surfactant/salt mixture to reach a final desired concentration (Table A screening program with all of the 18 different non-ionic surfactants and seven commonly used inorganic salts was performed three times on three separate days to elucidate the mechanisms underlying the combinational effect (Table Additionally, we tested the stability of the combinational effect, together with VBNC induction, by repeating the procedure on combinations, which led to VBNC induction in the initial screening three out of three times. These combinations were tested again three times on three different days in triplicate Table .L. monocytogenes cells during exposure to 1% Lutensol XP30 combined with 0.5 M MgCl2. While we used the same procedure as described in “Screening of the combinational effect,” we took samples during induction after 5, 10, 20, 30, 40, 50 and 60 min, washed the samples and tested culturability and VBNC induction as described above.In order to analyze the induction time of cells exposed to combinations of surfactants and salts, we tracked the development of culturability in After induction and confirmation of the VBNC state for the respective bacteria, cells were exposed to different conditions which were shown to facilitate resuscitation previously in different contexts. Therefore VBNC cells, recovering for 24 h in BHI medium, were centrifuged for 5 min at 8,000 × g and resuspended in a distinct condition solution Table . CulturaSupplementary Information 1


The results of experiments involving broiler chickens and turkeys indicate that increased dietary methionine (Met) levels may improve the antioxidant protection of tissues in fast-growing birds. This is an important consideration since viral infections induce oxidative stress. The aim of this study was to verify the hypothesis that turkey diets with increased Met content can suppress oxidation processes induced by infection caused by the haemorrhagic enteritis virus (HEV), and that the noted effect is determined by the chemical form of this amino acid: DL-methionine (DLM) or DL-hydroxy analogue of Met (MHA).Dietary Met content above 40% higher than the level recommended by the NRC (1994) intensified lipid peroxidation in the small intestine, leading to an increase in malondialdehyde (MDA) and lipid peroxide (LOOH) levels, but it also stimulated antioxidant mechanisms in the blood and liver of turkeys infected with HEV. In comparison with DLM, MHA contributed to more severe symptoms of oxidative stress, such as elevated MDA levels in the intestines, and a decrease in glutathione peroxidase (GPx) activity and ferric-reducing ability of plasma (FRAP).In HEV-infected turkeys, diets with increased Met content did not exert a clear antioxidant effect, which was noted in uninfected birds. The prooxidant activity of Met observed in the small intestinal wall was suppressed in the blood and liver of turkeys, most likely due to intensified synthesis of uric acid and glutathione. In comparison with MHA, DLM had a more beneficial influence on the analysed parameters of the redox status in the small intestine, blood and liver of turkeys. Due to the low level of methionine (Met) in natural feed ingredients, commercial poultry diets have been supplemented with feed-grade Met, mainly DL-methionine (DLM) or Met hydroxy analogue (MHA), as DL-2-hydroxy-4-(methyl) butanoic acid . ExperimLess severe symptoms of oxidative stress in the intestines in response to dietary Met supplementation, observed by Ruth and Field , may be In view of the above, the results of recent studies seem interesting because they suggest that increasing dietary Met levels can improve immune and antioxidant protection in young clinically healthy turkeys , 15, 34.The objective of this study was to verify the hypothesis postulating that turkey diets with increased Met content can suppress oxidation processes induced by infection caused by the haemorrhagic enteritis virus (HEV), and that the noted effect is determined by the chemical form of this amino acid: DL-methionine (DLM) or DL-hydroxy analogue of Met (MHA).The study was conducted on 120-d-old female Hybrid Converter turkey poults  with permission 45/2013 of the Local Ethical Committee for Animal Experiments located at University of Warmia and Mazury . The birds were kept in separate isolated experimental boxes in the Pavilion of Avian Experimental Infections of safety class PCL 3, at the Department of Avian Diseases of the University of Warmia and Mazury in Olsztyn. A three-stage negative pressure cascade was maintained in the experimental boxes and passageways in the Pavilion. Air entering and leaving the building was passed through HEPA H13 filters to prevent uncontrolled cross-infections between groups and infections caused by environmental agents. All turkeys were vaccinated against turkey rhinotracheitis (TRT) at 1 day of age , against ND at 10 days of age  and against Ornithobacterium rhinotracheale (ORT) at 28 and 49 days of age . Vaccination program was identical to that applied on most commercial turkey farms in Poland.The birds were randomly assigned to four dietary treatments, with 30 birds per group, treated with different levels and sources of supplemented Met. The total number of birds in one treatment was adapted to the size of pens, according to the density used in turkey rearing. For the physiological studies, 8 birds were selected from each treatment, commonly accepted as the minimum number of animals (turkeys) with a unified genotype that ensures reliable, reproducible and statistically significant results without repeating the procedure due to high intra-group variability.4,3 EID50, administered into the crop with a probe [Two sources of supplemental Met were used, DL-Met  or MHA  butanoic acid, Novus International, Inc., St. Lois, MO). The levels of dietary Met that were tailored to meet the NRC recommendations 1994) were regarded as “low” (L), and the treatments with increased Met dietary, by above 40% as proposed by some breeding companies was indicated as “high” (H). The nutritional value of basal diets  was used for determination of plasma glucose (GLU), triacylglycerols (TAG), total cholesterol (TC), uric acid (UA), total protein (TP) and albumin (ALB) and the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatine kinase (CK) and lactate dehydrogenase (LDH). As described previously [Biochemical and antioxidant parameters were determined in blood, homogenates of the small intestinal wall and liver. Blood samples (5 ml) were collected into test tubes containing heparin (at a concentration of 20 IU/ml) and tissue samples were taken from 8 birds per group. Heparinised blood samples were centrifuged for 10 min × 3000 eviously , the foln = 8) with pooled SEM are shown, and the statistical significance was considered at p < 0.05.Statistical calculations were done with the aid of two-way ANOVA (Statistica 10.0 software) considering two main factors (dietary Met level effect and dietary Met source effect) as well as the interaction between those factors (level × source interaction). In tables, mean values (P = 0.008), lower SOD activity (P < 0.001), and higher levels of LOOH and MDA in the small intestinal wall . Higher CAT activity (P = 0.009) and lower MDA concentrations (P = 0.043) were noted in the small intestinal wall of turkeys receiving DLM-supplemented diets compared with those fed MHA-supplemented diets. A Met dosage × source interaction (P = 0.004) was observed for SOD activity because the higher dietary DLM level decreased SOD activity, whereas the higher MDA level had no influence on SOD activity. A significant Met dosage × source interaction (P = 0.006) observed for MDA levels in the small intestinal wall of turkeys resulted from different effects exerted by dietary Met sources: DLM did not induce changes in MDA concentrations whereas the higher MHA level increased MDA concentrations.The applied dietary treatments affected selected parameters of the redox status in the small intestinal wall Table . TurkeysP = 0.019). A significant Met dosage × source interaction (P = 0.013) was also observed for plasma UA levels: the higher DLM level had no effect on the above parameter whereas the higher MHA level increased UA concentrations in the blood plasma of turkeys.No significant differences were found in the analysed blood biochemical parameters of turkeys fed diets with different Met content and sources, except for plasma UA levels Table , which iP = 0.012) and LOOH (P = 0.035) concentrations, and tended to decrease MDA levels in the blood plasma of turkeys (P = 0.062). In comparison with DLM, MHA decreased vitamin C concentrations (P < 0.027) and GPx activity (P < 0.001), increased the activities of SOD and CAT (both P < 0.001), and decreased FRAP values and LOOH concentrations (P = 0.001). A significant Met dosage × source interaction was noted for some redox parameters: (1) the higher DLM level increased and the higher MHA level decreased GPx activity (P < 0.001), (2) the higher DLM level did not affect and the higher MHA level increased GSH + GSSG concentrations (P < 0.001), (3) the higher DLM level decreased and the higher MHA level increased FRAP values (P < 0.001).Multiple changes in the blood redox status were noted in response to different dietary Met levels and sources Table . DietaryP = 0.009 and P < 0.001, respectively) and LOOH concentrations (P = 0.042), and a decrease in MDA levels (P = 0.002). In comparison with DLM, MHA decreased vitamin C concentrations and increased SOD activity (both P < 0.001) in the liver of turkeys. A significant Met dosage × source interaction (P = 0.039) was observed for CAT activity which did not change in response to the higher DLM level but increased in response to the higher MHA level. The higher MHA level had no influence on hepatic MDA concentrations, whereas the higher DLM level decreased MDA concentrations .Both experimental factors, i.e. the level and source of dietary Met, affected parameters of the redox status of the liver in turkeys Table . IncreasIn previous studies, increased dietary Met levels improved the antioxidant protection of intestinal mucosa. Increased glutathione production, higher levels of total antioxidant capacity and reduced protein oxidation were observed in the intestinal mucosa of chickens fed DLM-supplemented diets . Lower MIn the present experiment, undesirable changes were noted in some redox parameters of the small intestinal wall in turkeys, including a decrease in SOD activity and an increase in LOOH and MDA concentrations and CAT activity, in response to diets with higher inclusion levels of dietary Met. Our results, which do not corroborate the findings of Park et al.  and ShenThe results of earlier studies indicate that increased dietary Met levels exerted antioxidant effects in poultry, manifested by elevated plasma UA levels , 28 and In the present experiment, an increase in UA levels and considerable changes in the analysed redox status parameters were noted in the blood plasma of turkeys, possibly due to the antioxidant activity of Met (higher in the case of DLM) and oxidative stress caused by HEV infection. The presence of oxidative stress in cells and tissues, induced by bacterial and viral infections in poultry, has also been reported by other authors. The symptoms of oxidative stress included an increase in MDA levels, and a decrease in GSH concentrations and the activities of antioxidant enzymes , an incrIn another experiment with uninfected turkeys  fed idenThe results of this study indicate that dietary Met content approximately 40% higher than the level recommended by the NRC (1994) intensified lipid peroxidation in the small intestine, leading to an increase in MDA and LOOH levels, but it also stimulated antioxidant mechanisms in the blood and liver of turkeys infected with HEV. In comparison with DLM, MHA increased the activities of SOD and CAT, and decreased GPx activity and FRAP values.In HEV-infected turkeys, diets with increased Met content did not exert a clear antioxidant effect, which was noted in uninfected birds. The prooxidant activity of Met observed in the small intestinal wall was suppressed in the blood and liver of turkeys, most likely due to intensified synthesis of uric acid and glutathione. In comparison with MHA, DLM had a more beneficial influence on the analysed parameters of the redox status in the small intestine, blood and liver of turkeys.


Switching on chondroitinase helps spinal-injured rats to recover reaching and grasping movements. Chondroitinase is a promising preclinical therapy for restoring function after spinal cord injury. Burnside  Chondroitinase ABC is a promising preclinical therapy that promotes functional neuroplasticity after CNS injury by degrading extracellular matrix inhibitors. Efficient delivery of chondroitinase ABC to the injured mammalian spinal cord can be achieved by viral vector transgene delivery. This approach dramatically modulates injury pathology and restores sensorimotor functions. However, clinical development of this therapy is limited by a lack of ability to exert control over chondroitinase gene expression. Prior experimental gene regulation platforms are likely to be incompatible with the non-resolving adaptive immune response known to occur following spinal cord injury. Therefore, here we apply a novel immune-evasive dual vector system, in which the chondroitinase gene is under a doxycycline inducible regulatory switch, utilizing a chimeric transactivator designed to evade T cell recognition. Using this novel vector system, we demonstrate tight temporal control of chondroitinase ABC gene expression, effectively removing treatment upon removal of doxycycline. This enables a comparison of short and long-term gene therapy paradigms in the treatment of clinically-relevant cervical level contusion injuries in adult rats. We reveal that transient treatment (2.5 weeks) is sufficient to promote improvement in sensory axon conduction and ladder walking performance. However, in tasks requiring skilled reaching and grasping, only long term treatment (8 weeks) leads to significantly improved function, with rats able to accurately grasp and retrieve sugar pellets. The late emergence of skilled hand function indicates enhanced neuroplasticity and connectivity and correlates with increased density of vGlut1+ innervation in spinal cord grey matter, particularly in lamina III–IV above and below the injury. Thus, our novel gene therapy system provides an experimental tool to study temporal effects of extracellular matrix digestion as well as an encouraging step towards generating a safer chondroitinase gene therapy strategy, longer term administration of which increases neuroplasticity and recovery of descending motor control. This preclinical study could have a significant impact for tetraplegic individuals, for whom recovery of hand function is an important determinant of independence, and supports the ongoing development of chondroitinase gene therapy towards clinical application for the treatment of spinal cord injury. Since the majority of spinal injured individuals are injured at the cervical level  vector system. In this system al level  and a toal level , we inveChABC gene has been described previously .The mammalian-compatible ChABC gene  cells grown in SensiCell™ medium supplemented with 10% foetal calf serum (FCS) were plated into 6-well plates and left overnight to achieve 70% confluency. Cells were then washed twice with Dulbecco’s modified Eagle medium and transfected using Xfect™ (Clontech) in serum free medium for 4 h, with a total of 3 µg DNA consisting of the TRE.ChABC and PGK.GARrtTA plasmids in a ratio of 1:4 and 0.3 µg pAdVAntage™ plasmid (Promega). pAdVAntage™ was added to enhance expression of the 10 GC/ml for each vector. Other lentiviral vectors used in this study were: LV.PGK.ChABC  were generated as described previously  Act 1986, approved by the Animal Welfare and Ethical Review Body (AWERB) of King’s College London and conducted under Home Office Project License 70/8032. Methods and results are written in accordance with the ARRIVE guidelines for publishing in vivo research.Eighty-five adult female Lister Hooded rats  were used in these studies. Rats were housed under a 12-h light/dark cycle with ChABC gene therapy system, negative control rats received a contusion injury and either no treatment , or dox-i-ChABC vector injection without doxycycline administration . Positive control rats received LV.PGK.ChABC vector injection . Dox-i-ChABC treated rats received dox-i-ChABC dual vector injections and were administered doxycycline for 2.5 weeks . All tissue was processed at 2.5 weeks for evidence of ChABC gene expression at the mRNA level and for histological evidence of digested CS-GAGs, see below. Sample sizes for biochemical quantitation are consistent with prior data , dox-i-ChABC dual vector injection and doxycycline administration for 2.5 weeks (short-term dox-i-ChABC), dox-i-ChABC dual vector injection without doxycycline administration (dox−) and GFP vector injection and doxycycline administration for 8 weeks (GFP). ChABC gene expression was assessed by qPCR at 2.5 weeks, 5 and 8 weeks (n = 3 per group per time point). Animals were assessed weekly in behavioural tasks . The present study had no a priori expectation as to effect size, as dox-i-ChABC gene therapy has not been used before in this context. To select sample size, G*Power 3.17 was used to suggest a sample size of n = 7 per group would be sufficient to detect an effect size less than that observed in a previous cervical contusion study using LV.PGK.ChABC in the horizontal ladder task  were given perioperative analgesia  and anaesthetized . Skin was shaved and cleansed with sequential chlorohexidine and iodine swabs. Overlying muscle was retracted and a dorsal laminectomy of C6 vertebral process and half of C5 vertebral process was performed. Periosteum was removed. Rats then received a midline 225 kdyn spinal cord contusion injury at level C5/6 using an Infinite Horizon impactor and 3-mm diameter rounded impact tip (Precision Systems Instrumentation). Immediately following injury, rats underwent midline intraspinal injection of viral vectors at two sites, 1 mm rostral and 1 mm caudal to the injury, using a fine glass pulled-pipette and microdrive pump . The pipette was lowered 1.5 mm in the dorsoventral axis then retracted 0.5 mm and vectors were injected at a rate of 200 nl/min. The pipette was left in place for a further 2 min to ensure vector diffusion. Viral vectors were injected at a volume of 1 µl per site  or 0.5 µl . Following intraspinal injection, overlying musculature was sutured in layers, skin was closed and anaesthesia was reversed . Body temperature was maintained at 37°C using a self-regulating heating mat throughout and animals recovered for at least 1 h in an incubator  and were then returned to home cages placed partially on heated mats overnight (∼35°C). For animals receiving doxycycline, this was administered at a dose of ∼80 mg/day via custom chow (Envigo). In the acute postoperative period following surgery, extensive welfare checks were carried out, including provision of accessible chow and hydration gel in all cages. During this period animals were housed in soft bedding and absorbent-lined cages with less sawdust, to maximize ability to locomote, before returning to standard husbandry conditions.Adult female Lister Hooded rats (n = 12) was prepared for comparisons of T cell-related gene expression in animals receiving the dox-i-ChABC dual vector system compared to animals with the same dual vector system without the immune-evasive (GAR) component. Rats received a 225 kdyn contusion injury at the C5/6 level, as described above, followed by intraspinal injections 1 mm rostral and caudal to the lesion with 1 µl of LV.PGK.rtTA and LV.TRE.ChABC  (n = 3) or LV.PGK.GARrtTA and LV.TRE.ChABC  (n = 3), or received no intraspinal injection . Animals injected with inducible vectors received daily doxycycline (80 mg) for 2.5 weeks and were then deeply anaesthetized with sodium pentobarbital  and transcardially perfused with 50 ml PBS supplemented with EDTA (12.5 mM). Two sections of spinal cord rostral and caudal to the 8 mm injury epicentre were rapidly dissected and frozen on dry ice and stored at −80°C until processing. Tissue was thawed on ice and homogenized in 600 μl TRIzol® reagent (Thermo Fisher Scientific). An aqueous (RNA-containing) phase was generated using 1:10 bromo-chloro-propane, mixed 1:1 with 70% isopropanol and centrifuged at 12 000 rpm to precipitate RNA. RNA samples (3000 ng) were pooled in a 1:1 ratio from the rostral and caudal samples and converted into cDNA using the high capacity RNA-to-cDNA™ kit (Applied Biosystems). Thirty nanograms of RNA per reaction was quantified using prevalidated Taq Assays for interferon gamma (IFNγ) and CD8b (Thermo Fisher). GAPDH served as the housekeeping gene, using prevalidated GAPDH primers (Roche). RT-qPCR (TaqMan™ assay) was performed using an automated cycler (Roche LightCycler® 480 II) and mRNA expressed as ΔΔCt relative to control tissue from uninjured rats (n = 3).To assess the immune-evasive properties of dox-i-ChABC, an additional cohort of animals  or were non-weight bearing. This was expressed as a percentage of total steps. Following injury, animals were assessed weekly on this task. They were assessed in chronological numerical order, which was independent and unrelated to treatment group, during the morning.Prior to injury, animals were trained 15 min daily for 2 weeks to reach for sucrose pellets , with their dominant paw, through a window in a Plexiglas® box. Pellets were placed in an indented well, retrieval from which requires grasping and paw supination rather than dragging of the pellet towards the mouth. During baseline and testing, rats were offered 10 practice pellets and then 20 ‘trial’ pellets which were scored from the dominant paw. ‘Hits’  were recorded and expressed as a percentage of ‘trials’ . Videos from the 8-week time point were split into single frames and the middle proximal phalange position recorded in sequential frames using a Manual Tracker plugin (Fiji). Traces were smoothed via a single interpolation for graphical representation. The y-coordinate, representing window opening, was used to select x–y information as to paw location during ‘grasp phase’. This is defined as having been initiated when the paw passes through the window. Heat maps of relative paw position during this time were generated using the NumPy and Matplotlib/Pyplot histogram2d function in Python 3.6. From these traces and known frame length, the average length of time the rat took to successfully grasp the pellet was also calculated from onset of first passage of the paw through the window opening to successful retrieval of that pellet.A representative sample of rats were taken from the short and long-term dox-i-ChABC treatment groups (ChABC gene at a concentration of 100 nM (Forward: 5′-AGAGCCGTAGGCGTCTCTCT-3′. Reverse: 5′-AGCGTTGAGGGTCATCTCTC-3′). GAPDH served as the housekeeping gene, using prevalidated GAPDH primers (Roche). RT-qPCR (TaqMan™ assay) was performed using an automated cycler (Roche LightCycler® 480 II). The log fold change in mRNA expression of ChABC was calculated from ΔΔCt values.Animals were deeply anaesthetized with sodium pentobarbital  and transcardially perfused with 50 ml PBS supplemented with EDTA (12.5 mM). A section of spinal cord spanning injury epicentre and injection site (∼8 mm) was rapidly dissected and frozen on dry ice and stored at −80°C until processing. Tissue was thawed on ice and homogenized in 700 μl TRIzol® reagent (Thermo Fisher Scientific). An aqueous (RNA-containing) phase was generated using 1:10 bromo-chloro-propane, mixed 1:1 with 70% isopropanol and centrifuged at 12 000 rpm to precipitate RNA. RNA was converted to cDNA using the high capacity RNA-to-cDNA™ kit (Applied Biosystems). Thirty nanograms of RNA per reaction was quantified using primers generated against the modified Animals were deeply anaesthetized with sodium pentobarbital  and transcardially perfused with PBS followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Tissue spanning the lesion site (C5/6), plus C3/4, C7/C8, T10 and lumbar spinal cord was dissected and post-fixed overnight, cryoprotected in 30% sucrose, embedded and frozen in O.C.T. compound and then cut into serial 20 µm transverse or sagittal sections using a cryostat. To detect ChABC enzyme activity, immunohistochemistry for the terminal non-reducing C-4-S product, indicative of ChABC digestion of CS-GAGs, was performed . A pixel value threshold was assigned as representative of positive immunostaining and maintained across all analysis for C4/5 analysis and again for C7/8 analysis. Pixels above this threshold were summed and averaged for laminas I–II, III–V and VI–X, across three sections per animal in four to five animals per treatment group alongside three uninjured controls. Averaged threshold grey matter data per group were then converted to heat maps to display density of vGlut1+ staining using NumPy and Matplotlib/Pyplot histogram2d function in Python 3.6.n = 3) in each experimental group.Three transverse sections per animal were imaged at ×20 on a Zeiss LSM 710 confocal microscope and regions of interest applied over an area comprising the intermediolateral columns and ventral horns. These regions were assigned a pixel threshold representative of positive immunostaining that was maintained across all analysis, whereby pixels above this threshold were summed and averaged across each animal  is the rat. Parametric statistical analyses were performed using IBM SPSS Statistics 24 or GraphPad Prism 7. Behavioural data were analysed by two-way repeated measures analysis of covariance (RM-ANCOVA), where baseline performance was a covariate. Assumptions of ANCOVA, including normality, homogeneity of error variances and sphericity were tested and satisfied. Histological and electrophysiological data were analysed by one-way ANOVA  or t-test. All statistics and post hoc tests are stated in the text and correction for multiple comparisons performed where appropriate. Error bars are SEM unless stated.Numerical values are reported as mean ± standard error of the mean (SEM). The experimental unit  = 13.17, P = 0.0018, one-way ANOVA, Tukey’s post hoc; F = 18.18, P = 0.006, one-way ANOVA, Tukey’s post hoc; in vivo indication that our GAR peptide confers immune-evasive properties to the inducible vector system. In all subsequent studies, we used the GARrtTA stealth system (termed dox-i-ChABC).Expression of IFNγ (known to be predominantly produced by T cells) and CD8b (a CD8+ T cell marker) was examined in tissue immediately rostral and caudal to the epicentre  in animals receiving intraspinal injections of dox-inducible vectors, which were designed with either a classical transactivator (rtTA) to activate gene expression upon doxycycline administration A, or an in vitro. HEK cells transfected with both plasmids were treated with a range of concentrations of doxycycline for 48 h. Culture medium was then isolated and assessed for ChABC activity via colorimetric (spectrophotometric) assay. Without doxycycline administration, medium from co-transfected cells had some low basal ChABC activity (14% ± 4.5 U/ml), which was not increased by 1 ng/ml doxycycline administration. In contrast, application of 10 ng/ml doxycycline initiated the induction of ChABC gene expression (43% ± 4.4 U/ml), relative to maximal activity achieved by application of 100 ng/ml doxycycline (87% ± 7.6 U/ml) (in vitro co-transfection of dox-i-ChABC plasmids confer dox-sensitive expression of active ChABC enzyme.We next sought to assess whether the PGK.GARrtTA and TRE.ChABC constructs could be used to induce synthesis of active ChABC enzyme in the presence of doxycycline .6 U/ml) B. Thus, in vivo, to the injured spinal cord. LV.PGK.GARrtTA and LV.TRE.ChABC  LV.PGK.ChABC. LV.PGK.GARrtTA and LV.TRE.ChABC co-injection without doxycycline administration resulted in very low gene expression  . QPCR was used to measure ChABC mRNA expression in spinal cord tissue at different post-injury time points after different doxycycline regimes  = 11.23, P < 0.001, one-way ANOVA, Tukey’s post hoc]. Relative to rats injected with LV.PGK.GFP , at 2.5 weeks following dox-i-ChABC vector injection with doxycycline administration ChABC expression was induced . Without doxycycline administration (dox−) there was a low level of ‘leaky’ transgene expression, and this low level transgene expression without doxycycline was apparent at all time points  = 5.204, P = 0.0489, one-way ANOVA, Tukey’s post hoc], whereas removal of doxycycline at 2.5 weeks resulted in lower transgene expression . By 8 weeks ChABC expression decreased to baseline levels following removal of doxycycline at 2.5 weeks, in contrast to sustained expression of ChABC mRNA with maintained doxycycline administration   = 6.875, P = 0.02, two-way RM-ANCOVA, Bonferroni post hoc]. A trend for improvement with dox-i-ChABC treatment was observed for hindlimb performance, although this was not statistically significant   = 9.643, P = 0.001, two-way RM-ANCOVA, Bonferroni post hoc; P = 0.002, P = 0.001, respectively), but also significantly greater improvement than the short-term dox-i-ChABC treatment group (P = 0.033). Scores at Week 8 were 43.4% ± 5.8, 19.2% ± 5.7, 7.5% ± 5.3 and 11.9% ± 5.4 for long-term dox-i-ChABC, short-term dox-i-ChABC, dox− and GFP, respectively  (ectively –4.t-test P = 0.028) (ChABC gene therapy (with doxycycline administered for the full 8 weeks) promotes recovery of skilled paw use and accurate grasping, indicating that long term CSPG digestion may be required for significant neuroplasticity of descending systems involved in skilled reaching.To investigate this effect further, a representative sample of animals from the two dox-i-ChABC groups were analysed at the final 8-week time point using manual kinematic tracing. Reaches that led to successful pellet retrieval were traced based on the position of the middle proximal phalange of the paw. = 0.028) F. Thus, F = 3.961 one-way ANOVA P = 0.036, Fisher’s LSD]  = 1.452, P = 0.2701, one-way ANOVA; F = 9.762, P = 0.001 one-way ANOVA, Tukey’s post hoc and F = 10.47, P = 0.0007 one-way ANOVA, Tukey’s post hoc, respectively] with a distribution pattern within grey matter similar to that found in the uninjured spinal cord. Further examination of vGlut1 expression in sections co-stained with NeuN revealed intense vGlut1 expression distributed throughout lamina III–X in the long-term dox-i-ChABC treated group, compared to markedly lower levels and distribution of vGlut1 in short-term dox-i-ChABC group  = 1.629, P = 0.2347, one-way ANOVA; F = 4.715, P = 0.0213, one-way ANOVA, Tukey’s post hoc; F = 1.629, P = 0.2347, one-way ANOVA; F = 7.807, P = 0.0092 for intermediolateral columns and F = 7.169, P = 0.0118 for ventral horn, one way ANOVA, Tukey’s post hoc; Following spinal cord injury, descending excitatory input is severely disrupted. Since ChABC treatment has previously been shown to promote neuroplasticity A. RegionBC group . VGlut1 BC group  and agaiBC group A. RegionChABC gene and enables secretion of the active enzyme and extensive matrix digestion. Removal of doxycycline returns ChABC gene expression to baseline levels, with some low ‘leaky’ transgene expression. We use this to reveal that short-term ChABC gene therapy is sufficient to replicate prior reported functional improvements and find that long term administration elicits additional recovery of skilled forelimb reaching and grasping behaviours and this is associated with vGlut1+ fibre remodelling in the spinal cord.Here we demonstrate a novel immune-evasive gene switch that enables regulated delivery of ChABC in the injured mammalian spinal cord. This provides an experimental tool to control delivery (by effectively switching the gene on and off) and understand the role of timing in ChABC treatment, as well as a step towards creating a clinically applicable viral vector system. Dox-i-ChABC effectively mediates temporal control over CS-GAG removal, where administration of doxycycline, a safe and widely used pharmacological agent, induces high expression of the in vivo. One previous attempt to generate Tet-On chondroitinase, using an adenoviral vector encoding chondroitinase AC has been reported and used in a human astrocytoma cell line . A side by side comparison of rtTA and GARrtTA inducible vectors driving luciferase gene expression (enabling long term bioluminescence imaging) showed that rtTA-mediated gene expression undergoes shutdown upon repeated induction in rats, whereas GARrtTA-mediated gene expression is maintained; rtTA shutdown was associated with cell and tissue damage, presumably due to immune cell activation . These data, together with the current findings, support previous in vitro findings, which used a bioassay for human antigen presentation to demonstrate that GAR protects rtTA from human cytotoxic T cell mediated recognition, confirming that the chimeric transactivator GARrtTA had an immune-evasive advantage over classical rtTA  indicates that recovery of skilled hand function was not attributable to neuroprotection, but more likely to be due to neuroplasticity as a result of long term CS-GAG digestion. Accordingly, these animals also had a greater density of vGlut1+ innervation in the spinal cord rostral and caudal to the injury. VGlut1+ populations of neurons in the spinal cord constitute proprioceptive afferents synapsing on motor neurons , the International Spinal Research Trust and EndParalysis  and Wings for Life Spinal Cord Research Foundation (WFL-NL-17/16 to J.V.).Brain online.Supplementary DataClick here for additional data file.


AIDS is a worrying public health issue in China and lacks timely and effective surveillance. With the diffusion and adoption of the Internet, the ‘big data’ aggregated from Internet search engines, which contain users’ information on the concern or reality of their health status, provide a new opportunity for AIDS surveillance. This paper uses search engine data to monitor and forecast AIDS in China.A machine learning method, artificial neural networks (ANNs), is used to forecast AIDS incidences and deaths. Search trend data related to AIDS from the largest Chinese search engine, Baidu.com, are collected and selected as the input variables of ANNs, and officially reported actual AIDS incidences and deaths are used as the output variable. Three criteria, the mean absolute percentage error, the root mean squared percentage error, and the index of agreement, are used to test the forecasting performance of the ANN method.Based on the monthly time series data from January 2011 to June 2017, this article finds that, under the three criteria, the ANN method can lead to satisfactory forecasting of AIDS incidences and deaths, regardless of the change in the number of search queries.Despite the inability to self-detect HIV/AIDS through online searching, Internet-based data should be adopted as a timely, cost-effective complement to a traditional AIDS surveillance system. China’s first AIDS case was reported in 1985, and the HIV epidemic in China started to spread among intravenous drug users in the late 1980s. Since 1995, China has experienced a rapid increase in the number of cases of HIV/AIDS caused by paid plasma donors in central China. In recent years, especially since 2007, sexually transmitted AIDS cases have steadily increased and consist of over half the reported HIV/AIDS infections -1, where x denotes either any input or the output.The relationship between Internet query searching and actual HIV/AIDS epidemics may be influenced by many factors, such as the regional Internet penetration rate, the health care-seeking behaviour of Internet users, or the media’s influence power. In other words, the relationship may be nonlinear, and the linear regression model may not fit it well. Since no specific assumption needs to be made, an MLP model of ANNs is also used in this paper. In terms of hidden layers and nodes, a one-hidden-layer BP network type is used, and the number of hidden nodes is determined by trial and error . Moreoven is the total period, yi is the actual value, and To evaluate the forecasting performance of ANNs with AIDS search trend data, we employ three criteria: the mean absolute percentage error (MAPE), the root mean squared percentage error (RMSPE), and the index of agreement (IA). They are given as follows:formance –26.The forecasting performances of the MLP model of ANNs for AIDS are calculated on the basis of the test set, which accounts for 10% of all the observations, and the full set, that is, all the observations. In this section, the 80% training set is used to train the networks, and the 10% test set and the full set are used to evaluate the forecasting performance of the networks trained with the training set. After training the ANNs with the training set to realize the best forecasting of AIDS in the test set, we further use the optimal networks to predict AIDS in the full set to determine whether the forecasting performance remains unchanged. Meanwhile, we also report the optimal prediction horizons of AIDS incidences and deaths resulting from the MLP model of the ANN method. First, the optimal net is obtained based on the evaluation of the test set, then the values of the MAPE/RMSPE/IA and the topology structures are determined from recursive forecasts for the next one to six months. The forecasting performances under different horizons are reported in The Internet provides almost limitless real-time, cheap data on consumers’ searching behaviours, while ANNs provide solid novel methods for accurate forecasting. However, there might be several limitations to our study. First, the Baidu search trend data regarding AIDS might lack representation, since they are restricted to users of the Baidu search engine. Internet access is uneven in China and between rural and urban areas. Search query data represent only part of AIDS victims’ searching behaviours and thus are just a complementary but important method for monitoring real-time AIDS incidences and deaths. On the other hand, due to social discrimination against AIDS victims, HIV-infected people are usually unwilling to report their disease or they resort to informal treatments. Besides, social ignorance regarding HIV and AIDS makes HIV carriers fail to realize their disease. As a result, the incidences and deaths may be under-reported in the official statistics.Second, we did not compare the forecasting performance between the ANN method and the traditional time series models, such as the autoregressive and moving average, vector autoregressive, and vector error correction models, since the data contain only 78 observations and the variables are not necessarily stationary, which may reduce the credibility of the estimations produced by these time series models. However, a recent study has suggested that the ANN method can be a promising alternative to traditional linear methods . The ordThird, some search behaviours may be generated by news reports, special events, or a ‘celebrity effect’ rather than actual disease activity. For example, the search volumes of some search queries, such as ‘AIDS workers are advised to rest’ and ‘NBA famous player Johnson is infected with AIDS’, are due to news reports that an AIDS victim had been fired by his employer and a famous basketballer named Johnson had been infected by HIV, respectively. However, these queries generally have a low correlation coefficient with actual AIDS incidences and deaths and were removed from the list of inputs of the MLP model. Another concern is that the sharp increase in the search trend of AIDS queries on particular days, say, 1 December 1, that is, World AIDS Day, also does not reflect the actual AIDS incidences or deaths but rather indicates people’s attention to AIDS. However, it is apparent from Finally, because of the lack of provincial or city-level data, we only conducted a study at the national level. China is a large country with diversified geographies, cultures, ethnicities, and population distribution. Thus, our results may have limited policy implications for how local governments can use search trends data to monitor local AIDS incidences and deaths. Moreover, we only use monthly data rather than weekly data or daily data, which are also unavailable. We admit that a study with weekly or daily data will lead to a more accurate forecasting performance on AIDS because of their merits of real-time and high-frequency observations. As an exploratory study, our framework using Baidu search trend data to forecast AIDS incidences and deaths can easily be extended to the provincial or city level.However, despite the limitations of online search data, the ANN method, as an information-processing system inspired by biological neural networks , does noANNs perform well in forecasting China’s monthly AIDS incidences and deaths with Baidu trend data. Three criteria of forecasting performance, the MAPE, the RMSPE, and the IA, all indicate that the MLP model of ANNs can result in accurate forecasting of concurrent AIDS incidences and deaths with Baidu search trend data. Moreover, the forecasting performance is relatively robust to a change in the number of search queries and forecasting horizons.Our results have strong policy implications for both AIDS surveillance and the forecasting of other diseases. As people increasingly engage in their work and lives online, their traces on the Internet can be used to monitor their behaviours. Although HIV/AIDS can only be detected by professional laboratory instruments, potentially infected victims’ searches through search engines with keywords relating to AIDS reveal the future risk of an AIDS/HIV outbreak. Thus, AIDS/HIV surveillance based on online search queries is becoming increasingly urgent. It costs less and is more immediate than traditional surveillance based on hospital data, which are lagged by days, weeks, or even longer . In our S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Inappropriate over-the-counter supply of antibiotics in pharmacies for common infections is recognised as a source of antibiotic misuse that can worsen the global burden of antibiotic resistance.To assess responses of community pharmacy staff to pseudo-patients presenting with symptoms of common infections and factors associated with such behaviour.A cross-sectional pseudo-patient study was conducted from Jan-Sept 2017 among 242 community pharmacies in Sri Lanka. Each pharmacy was visited by one trained pseudo-patient who pretended to have a relative with clinical symptoms of one of four randomly selected clinical scenarios of common infections  and a bacterial uncomplicated urinary tract infection. Pseudo-patients requested an unspecified medicine for their condition. Interactions between the attending pharmacy staff and the pseudo-patients were audio recorded (with prior permission). Interaction data were also entered into a data collection form immediately after each visit.n = 65) were inappropriately given for the viral infections. Antibiotics were provided for 55% of the urinary tract infections, 50% of the acute diarrhoea, 42% of the sore throat and 15% of the common cold cases. Patient history was obtained in less than a quarter of the interactions. In 18% (44/242) of the interactions staff recommended the pseudo-patient to visit a physician, however, in 25% (11/44) of these interactions an antibiotic was still dispensed. Pharmacy staff advised the pseudo-patient on how to take (in 60% of the interactions where an antibiotic was supplied), when to take (47%) and when to stop (22%) the antibiotics supplied. Availability of a pharmacist reduced the likelihood of unlawful antibiotic supply  but not appropriate practice.In 41% (99/242) of the interactions, an antibiotic was supplied illegally without a prescription. Of these, 66% (Illegal and inappropriate dispensing of antibiotics was evident in the participating community pharmacies. This may be a public health threat to Sri Lanka and beyond. Strategies to improve the appropriate dispensing practice of antibiotics among community pharmacies should be considered seriously. Medicines use is appropriate  when patients receive medicines appropriate to their clinical needs, in doses that meet their individual requirements, for an adequate period of time, and at affordable prices . If any Inappropriate use of antibiotics is a global problem, particularly in the Asian region , 5. It iSelf-medication with antibiotics is also a major contributory factor to inappropriate use of antibiotics in the community . The emeA systematic review of nine surveys conducted in the Asian region, found that self-medication with non-prescription antimicrobials among the general public was 58%  . StudiesTherefore, community pharmacists, being the first healthcare professional most people in LMICs approach for medical advice, such as common viral infections, are in the best position to help people with appropriate use of medicines. Pharmacists have the antibiotics knowledge necessary to ensure rational use of antibiotics  and can However in many LMICs, community pharmacists are selling antibiotics inappropriately for self-limiting viral URTIs –35, acutThis pseudo-patient study was part of a larger study conducted among Sri Lankan CPs from January to September 2017. There were two arms to this study; one of which involved pseudo-patients’ direct antibiotic product requests (DPR) from 242 CPs throughout Sri Lanka . The curThe pseudo-patient approach can be considered as a robust methodological tool for pharmacy practice research, especially as the knowledge of being observed can lead to behavioural change , 41. Desn = 369) was calculated based on the results of a previous pilot study  and the sample size calculation has been detailed in a previous publication [The sample size for this study was derived from a previous phase: a self-reported cross-sectional country survey conducted among CP staff in Sri Lanka. The survey sample size , and a bacterial uncomplicated UTI). Three levels of requests were made by the pseudo-patient to obtain an antibiotic. The first level of request consisted of requesting an unspecified medicine to alleviate the reported symptoms of the common infection. If an antibiotic was not given, the pseudo-patient used the second level of the request; “Can’t you give me something stronger?” If the pharmacy staff did not provide an antibiotic, the pseudo-patient openly stated, “I would like an antibiotic,” which was considered as the third level of request. If the pharmacy staff asked any questions related to reported symptoms, pseudo-patients were trained to answer according to the pre-determined scenarios.In addition, advice provided by pharmacy staff and the availability of a pharmacist during the visit were noted. The availability of a pharmacist was confirmed as follows, a research assistant observed the pharmacy licence displayed in the pharmacy with a photograph of the pharmacist. If the photo displayed did not match the attending pharmacy staff or there was no photo displayed, then the pseudo-patient asked “Can I talk to your pharmacist, please?” The availability of the pharmacist was then based on the response to this question. In Sri Lanka, the licence issued by the National Medicine Regulatory Authority to run a community pharmacy should be displayed in the pharmacy with the photo of a pharmacist who owns the pharmacy or is employed .Although as part of the visit the pseudo-patient did not ask why an antibiotic was not provided, any reason stated spontaneously by the pharmacy staff was captured from the audio-recording and reported accordingly.Immediately after each visit, the pseudo-patient and research assistant completed the data collection sheet Table  togetherP value of < 0.05 was considered as statistically significant. SPSS version 24 was used for all the analyses.Descriptive statistics such as frequencies (%) were used to describe the data. Pearson’s chi-square test and binary logistic regression analysis were performed using independent predictors  to evaluate the possible factors associated with antibiotic supply without a prescription for reported common infections. The A total of 242 pharmacies were visited by the pseudo-patients. The types of pharmacies which agreed to the pseudo-patient visits included, private chain pharmacies , private single pharmacies , semi-government pharmacies  and pharmacies in private hospitals . The clinical scenario of uncomplicated UTI of adult female was presented to 62 CPs and the other three scenarios - acute sore throat (adult), common cold (four-year-old child) and acute diarrhoea (adult), were presented equally among 180 pharmacies.2  = 22.15, P < 0.001). In two-thirds of the instances antibiotics were sold inappropriately for underlying viral infections (65/99) including acute sore throat, common cold and acute diarrhoea. In the majority of instances an antibiotic was sold upon the 1st or 2nd level of request  without the pseudo-patient requesting an antibiotic by name, and the rest were supplied on the 3rd level of request , that was not impacted on antibiotic supply between viral and bacterial infections .Overall, in 41% (99/242) of instances, antibiotics were sold illegally without a prescription in response to the pseudo-patients reported clinical symptoms Table . The aduOverall, only a few pharmacy staff asked the pseudo-patients about concurrent medical conditions 10%; 25/242), any action that has already been taken  and concurrent medicines used . In 18% (44/242) of the instances, pseudo-patients were recommended to see a physician. However, in about a quarter of them  an antibiotic was still provided. Only a quarter of the pseudo-patients with UTI  were advised to see a physician  where an antibiotic was sold, the pseudo-patients were further questioned about their symptoms or concurrent medical conditions. The questions related to action that has already been taken , drug allergies , and concurrent medicines used . About half of the pseudo-patients were advised on how and how often to take the provided antibiotic, and about a quarter of them were advised on when to stop taking the antibiotic. Availability of a pharmacist in the pharmacy had no impact on patient counselling. In none of the pharmacies did staff inquire about the pregnancy status of the pseudo-patients when selling an antibiotic for a childbearing-aged female pseudo-patient presenting with symptoms of UTI Table . None ofThe most common antibiotic sold to pseudo-patients with sore throat was erythromycin , amoxicillin for common cold , metronidazole for acute diarrhoea 79%; 23/29) and ciprofloxacin for female UTI cases  . None of the pharmacy staff discussed the reported symptoms with the pseudo-patient. They did not discuss issues such as severity of the current health condition, possible aetiology of the infection, risk of emergence of ABR if an antibiotic is inappropriately supplied for a viral infection and risk of supplying an antibiotic for a possible bacterial infection (UTI) without being diagnosed by a physician. Instead, they simply denied giving an antibiotic with or without stating a reason. The median interaction time between the pseudo-patient who did not receive an antibiotic and pharmacy staff was also 2 min (IQR = 1–3 min).To the best of our knowledge, this is one of the first two pseudo-patient studies conducted in Sri Lanka, including all different types of community pharmacies throughout the country, to evaluate pharmacy staff’s behaviour when presented with symptoms of common infections.Despite Sri Lankan laws explicitly prohibiting the supply of any antibiotic without a prescription, regardless of the patient’s medical condition or symptoms, this study found that antibiotics were not only commonly provided without a prescription  for common infections, but also inappropriately for viral infections. The antibiotics were supplied without even being specifically requested by the pseudo-patients. In addition, pharmacy staff failed to adequately inquire about the presenting symptoms, give correct advice or offer alternative OTC products. However, the overall supply was lower when the presenting common infection was that of a child’s, and when a pharmacist was present.The current results showed that unlawful antibiotic supply was high (41%) and this finding was supported by DPR pseudo-patient visit findings from the same pharmacies, where antibiotics were provided without a prescription to pseudo-patients in 61% of the interactions . The majat el., found that community pharmacy staff in Northwest Ethiopia dispensed antibiotics inappropriately for self-limiting acute diarrhoea and URTIs [This study also revealed inappropriate supply of antibiotics in response to reported symptoms of common infections of viral aetiology. As the pseudo-patient clinical scenarios were representing possible viral infections  and a probable bacterial infection (uncomplicated UTI), the expected behaviour of the staff for the reported scenarios was to effectively obtain relevant medical and lifestyle-related history, advise the pseudo-patient appropriately, provide an OTC medicine or non-pharmacological treatment  or refer them to a physician (in the case of UTI). Despite this fact about two thirds of staff who gave out an antibiotic, had supplied them inappropriately for viral infections. The potential reasons for such behaviour of community pharmacy staff have been discussed in a recent self-reported national survey conducted among community pharmacy staff in Sri Lanka which mainly highlighted staff’s inadequate clinical experience and knowledge about antibiotics . Similar behaviour has also been observed in many other LMICs , 35, 49.When comparing the findings of the DPR arm of the study  to the cThe observed low proportion of antibiotic supply (15%) for the reported paediatric scenario is a positive sign. This could be due to either pharmacy staff’s concern about vulnerable paediatric patients or their lack of clinical competency in dealing with such patients, or perhaps both. As none of the staff determined the cause of the infection and failed to educate the pseudo-patient appropriately whether an antibiotic was needed or not, also supports the argument above about limited clinical training and therefore knowledge of staff.Further, only about half of the pseudo-patients who obtained antibiotics, received any form of counselling or advice. Counselling patients on “when to stop taking antibiotics” does not appear to be part of the current process of providing antibiotics in Sri Lankan pharmacies. A similar inadequate patient history taking and lack of patient counselling was also observed in the DPR arm of the pseudo-patient study . This isAlthough it was revealed that the presence of a pharmacist in the pharmacy may have been associated with a lower likelihood of antibiotic supply without a prescription, the presence of the pharmacists did not impact the counselling received by the pseudo-patient nor result in an appropriate response to the reported symptoms of common infections. Therefore, this supports the argument above about limited clinical training and the knowledge of staff. It is also evident from the literature that un-qualified pharmacy staff or pharmacists with poor clinical knowledge may be contributing to inappropriate antibiotic supply , 51.The inappropriate provision of antibiotics and inadequate counselling provided to pseudo-patients observed in this study challenges the goal of appropriate use of antibiotics in communities, and can contribute to global antibiotic misuse . In turnAlthough repeated training and rehearsals were made to ensure consistency between pseudo-patients and to increase the internal validity of the data collected, it is still possible that some interpersonal differences among pseudo-patients may have impacted the behaviour of the pharmacy staff. This approach may also have limited external validity, since in normal circumstances the pharmacist would probably have much more information about the client. A real patient has the tendency to communicate freely about his/her pathology, therefore, the outcomes measured by this method may vary from real situations . FurtherIt is evident from this study that antibiotics are given out from pharmacies illegally without a prescription and clinically inappropriately. Presence of a pharmacist in the pharmacy may have reduced the illegal supply but it did not appear to impact appropriate practice.Immediate action is sought from all stakeholders including healthcare professionals, local policy makers as well as global agencies such as WHO, and the public, to curb this public health issue. In addition to strict implementation of policies, awareness and educational interventions must be implemented to improve appropriate antibiotic dispensing practice among pharmacists and their staff.