Xa21 and Xa33 through marker-assisted backcross breeding (MABB). Through two sets of backcrosses, the two BB resistance genes were transferred separately to DRR17B. In this process, at each stage of backcrossing, foreground selection was carried out for the target resistance genes and for non-fertility restorer alleles concerning the major fertility restorer genes Rf3 and Rf4, using gene-specific PCR-based markers, while background selection was done using a set of 61 and 64 parental polymorphic SSR markers respectively. Backcross derived lines possessing either Xa21 or Xa33 along with maximum genome recovery of DRR17B were identified at BC3F1 generation and selfed to develop BC3F2s. Plants harboring Xa21 or Xa33 in homozygous condition were identified among BC3F2s and were intercrossed with each other to combine both the genes. The intercross F1 plants (ICF1) were selfed and the intercross F2(ICF2) plants possessing both Xa21 and Xa33 in homozygous condition were identified with the help of markers. They were then advanced further by selfing until ICF4 generation. Selected ICF4 lines were evaluated for their resistance against BB with eight virulent isolates and for key agro-morphological traits. Six promising two-gene pyramiding lines of DRR17B with high level of BB resistance and agro-morphological attributes similar or superior to DRR17B with complete maintenance ability have been identified. These lines with elevated level of durable resistance may be handy tool for BB resistance breeding.Bacterial blight (BB) disease reduces the yield of rice varieties and hybrids considerably in many tropical rice growing countries like India. The present study highlights the development of durable BB resistance into the background of an elite maintainer of rice, DRR17B, by incorporating two major dominant genes,  Rice production needed to be increased 42% by 2050to feed the demands of an ever-increasing human population globally , 2. ExplXanthomonas oryzae pv. Oryzae (Xoo). It is one of the most devastating diseases in rice [viz., Xa4, xa5, xa13, Xa21 have been extensively used for development of BB resistant rice varieties [Table 1). However, large scale and long-term cultivation of varieties and hybrids with a single gene results in the breakdown of resistance due to a high degree of pathogenic variation [Xa genes can enhance the durability and spectrum of resistance against BB [BB disease is caused by a gram-negative bacterium called  in rice . The bac in rice . Applica in rice , 9. Bree in rice . To datearieties , 15  is a recently released high-yielding and fine grain rice variety possessing BB genes, xa5, xa13, and Xa21 [Xa21 [Xa33 [‘Improved Samba Mahsuri’ . The F1s obtained from the two crosses were analysed by extracting DNA through the method described by [Xa21; [Xa33; [1s were backcrossed with the recurrent parent DRR17B to generate BC1F1s, which were then screened for the presence of the target resistance genes using the gene-specific markers. The positive plants for Xa21 and Xa33 were selected and further screened for the non-presence of major fertility restorer genes, Rf4 and Rf3 using tightly linked markers, viz., DRCG-RF4-14 and DRRM-RF3-10, respectively [1F1 plants possessing BB genes and a non-restoring allele concerning Rf4 and Rf3 in homozygous condition were selected following the procedure described by [ISM and 64 markers specific for the cross DRR17B/FBR1-15EM) through background selection to identify a single BC1F1 plant from each cross possessing maximum recovery of the recurrent parent genome. The selected plant was backcrossed once again with DRR17B.Marker-assisted backcross breeding strategy was adapted for targeted introgression of ribed by  and usin; [Xa33; ) to idenectively . BC1F1 pribed by . These p3 generation, and BC3F1 plants of DRR17B possessing either Xa21 or Xa33 and maximum recovery of recurrent parent genome were then selfed to obtain BC3F2s. Plants homozygous for either Xa21 or Xa33 were identified among the BC3F2 plants and the best plants from the two crosses were intercrossed to obtain intercross F1s . ‘True’ ICF1 plants were identified by screening with molecular markers specific for Xa21 and Xa33 and were then selfed to generate intercross F2s . Plants homozygous for both Xa21 and Xa33 were identified among the ICF2 plants using the gene-specific markers. The identified plants were advanced through the pedigree method of selection  up to ICF4generation. Marker-assisted selection procedures were followed as recommended by [Xa21 and Xa33 genes, while the protocol described by [Rf4 and Rf3.The process of marker-assisted backcrossing was repeated until BCended by  for deteribed by  was adopXanthomonas oryzae pv. oryzae (Xoo) collected from BB disease endemic across major rice cultivation agro climatic locations in India, viz., IX-020 , IX-133 , IX-027 , IX-200 , IX-002 , IX-403 , IX-090  and IX-281  were used to screen the ICF4 lines of DRR17B  along with the donor parents/resistant check, ‘ISM’ (possessing xa5+xa13+Xa21), FBR1-15 (possessing Xa33) and BB recurrent parent and susceptible check (DRR17B and TN1) were screened under glasshouse conditions for their resistance/susceptibility against BB. The Xoo strains were cultured and stored as described by [8−9 CFU/ml at maximum tillering stage (45 to 55 days after transplanting) through the methodology of [Eight virulent isolates of the BB pathogen, ribed by . The ricology of . Approxi4) were field evaluated in randomized complete block design in Kharif 2014 (i.e. July-October/Wet season 2014) for the following agro-morphological traits involving days to 50% flowering (days), plant height (cm), number of productive tillers (No.), panicle length (cm), grains per panicle (No.) and spikelet fertility. Each entry was planted in 20 rows of 4m length with a spacing of 15 x 20 cm between rows and within rows. Days to 50 percent flowering was recorded based on number of days from sowing to 50% population flowering on a whole plot basis. Plant height (cm), number of productive tillers (No.) and panicle length (cm) were recorded from 5 competitive plants from each plot chosen at random and the mean values computed for different lines. Five individual panicles harvested separately from five plants were collected to compute for the average grain number per panicle (No.). The ILs were crossed with IR58025A line and evaluated for spikelet fertility based on seed setting of each cross. The percentage was calculated based on seed setting per panicle as described in [Improved lines (ILs) of DRR17B , Analysis of variance (ANOVA) and Least Significance Difference (LSD) . Analysi1s derived by crossing DRR17B with ‘ISM’  and FBR1-15  were backcrossed with DRR17B to obtain BC1F1s, which were then screened with the gene-specific markers. A total of 61 and 65 BC1F1 plants were observed to be positive for the target genes in Cross I and Cross II, respectively. The positive plants were screened with markers specific for Rf3 and Rf4,and a total of 15 and 11 plants were identified to be devoid of both the fertility restorer genes concerning Cross I and Cross II, respectively. These plants were then subjected to background selection using a set of polymorphic SSR markers (61 markers for Cross I and 64 for Cross II). Plant # IIRRGP3 from Cross I, with a recurrent parent genome (RPG) recovery of 73.7% and Plant # IIRRGP22 from Cross II, with a RPG recovery of 75% were identified to be the best ones (i.e. having a maximum recovery of DRR17B genome) and were used for further backcrossing. The process of marker-assisted backcrossing was carried out until BC3F1 generation (details given in Table 2). At BC3F1, plant # IIRRGP3-87-64 from Cross I with RPG recovery of 93.4% and plant # IIRGP22-73-10 with RPG recovery of 93.7% were identified to be superior and were selfed to generate BC3F2s. With regards to the BC3F2s produced from Cross I and Cross II, 39 and 52 plants were identified to be homozygous for Xa21 and Xa33, respectively. Among these, a solitary plant, which was morphologically similar to DRR17B, was identified from Cross I  and intercrossed with each other to generate intercross F1s . Out of 68 ICF1s, 63 were identified to be heterozygous for both Xa21 and Xa33 (i.e. true intercross F1s), and they were selfed to obtain ICF2 generation. At ICF2, a total of 309 plants were screened with markers specific for Xa21 and Xa33 and 18 were identified to be double homozygotes . A total of nine plants out of the 18, which were identified to be phenotypically similar to DRR17B, were further advanced until ICF4 generation through phenotype-based pedigree selection. At ICF4 generation, six promising lines which were similar to the recurrent parent were identified  and analysed for their resistance to BB, sterility maintenance ability and also characterized for important agro-morphological traits. Among the six ILs, line # IIRRIC102-26-7 exhibited the highest recurrent parent genome recovery with more than 95% along with minimal linkage drag on carrier chromosomes .The true FXoo . The resistant check and the donor for Xa21 gene ‘ISM’ (9)  showed a score of 3 against all the isolates (with an average lesion length ranging from 1.6 to 3.6 cm). FBR1-15 (10), the donor for Xa33 gene and improved DRR17B lines possessing Xa33 (# IIRRGP22-73-10-15-13-2 (2)) showed a resistance score of 3 with most of the isolates (with lesion lengths ranging from 1.7 to 4.8 cm), with two isolates, IX-002 and IX-281 recorded moderate resistance reaction with a score of 5 (average lesion lengths ranging from 7.3 to 9.7 cm and 7.5 to 9.2 cm).The ILs of DRR17B containing only Xa21 (# IIRRGP3-87-64-22-4-50 (1)) showed a resistance reaction against two isolates viz., IX-002 and IX-090 with a score of 3 , while with three isolates,viz., IX-020, IX-027 and IX-281, the line with only Xa21 exhibited moderately susceptibility with a score of 7 . Further, the line showed highly susceptible reaction with a score of 9 with three other three isolates viz., IX-133, IX-200 and IX-409, respectively. The ILs of DRR17B containing both Xa21 + Xa33 (3–8) exhibited a significantly higher level of resistance, showing a score of 1 against all eight isolates with lesion lengths ranging from 0.1 to 1 cm .The recurrent parent, DRR17B (11) (with lesion lengths ranging from 18.8 to 33.1 cm) and susceptible check TN1 (12) (with lesion lengths ranging from 20.9 to 33.8 cm) showed a disease score of 9 against all the eight isolates of the Table 4). Comparison of five agro-morphological parameters  revealed thatall the six ILs are isophenic in their panicle length and number of productive tillers to DRR17B, while significant differences were observed with respect to the number of grains per panicle. The ILs viz., IIRRIC10-8-94, IIRRIC102-26-7, IIRRIC123-58-3 and IIRRIC172-77-12 possessed more number of grains per panicle with respective to DRR17B viz., 301.6, 360.4, 308 and 317 respectively . However, all selected six lines showed comparatively shorter plant height than recurrent parent. While panicle length of, line # IIRRIC102-26-7 was observed longest among all six panicle (24.16 cm), the remaining five ILs exhibited equal or less than the recurrent parent DRR17B (average length of 23.24 cm: Table 4). Line # IIRRIC102-26-7 exhibit highest numbers of productive tillers per plant (average of 12), all remaining five ILs were similar to thir recurrent parent (10–11.2). The to 50% flowering, of all the six ILs flowered earlier (92–102 days), as compared to DRR17B (105 days).The current study screened the six ILs for their maintenance ability. Out of six, three lines showed partial spikelet fertility, while the remaining three lines  showed complete spikelet sterility when crossed with the WA-CMS line, IR58025A  using eight virulent isolates of Xoo. All the ILs possessing Xa21+Xa33 were observed to show significantly higher level of resistance against BB when compared to the donor parents, ‘ISM’ and FBR1-15. Single gene containing lines of DRR17B (i.e. possessing either Xa21 or Xa33), the recurrent parent DRR17B and the BB susceptible check TN1 .It is earlier known that Xa21 confers broad spectrum resistance against many of the virulent pathotypes of Xoo in India [Xa21 in BB resistance gene pyramiding programmes [Xa21 containing lines of DRR17B indicating that Xoo isolates, which are capable of overcoming Xa21 conferred resistance are fast-developing [Xa33were observed to show a better level of resistance as compared to the lines having Xa21. Furthermore, DRR17B lines possessing both Xa21 and Xa33 were observed to be highly resistant against all the eight virulent isolates of Xoo, thus, indicating the suitability of deployment of Xa33 either singly or in combination with Xa21. Earlier, two elite restorer lines, KMR3R, and RPHR1005 were improved for BB resistance by introducing Xa21 [Xa33 has been deployed in different genetic backgrounds by different research groups [Xa21 has been combined with Xa33 in the genetic background of an elite maintainer line, i.e., DRR17B and the gene-pyramid lines demonstrated a higher level of resistance as compared to lines possessing a single resistance gene .Phenotypic screening for BB resistance was carried out in this study among selected single gene containing BCin India  and seveogrammes , 41, 42.veloping , 44. Inting Xa21 , 38. Simh groups , 39, 40.ISM’ (with Xa21 + xa13 + xa5) has displayed satisfactory level of resistance with a score of 3 against all eight isolates. Interestingly, when Xa21 gene was combined with another major dominant gene- Xa33, such breeding lines exhibited the highest level of resistance with a score of 1. This indicates that the gene combination Xa21 + Xa33 displayed a broad spectrum of resistance and hence can be recommended for deployment in hybrid rice improvement programs as both Xa21 and Xa33 are both dominant and will express in the F1 hybrid.It is a known fact that long term cultivation of rice varieties possessing single resistance gene can result in the breakdown of resistance by faster development of virulent pathogens , 44, 45.Rf3 and Rf4, coupled with phenotype-based selection for certain key agronomic characters helped in near-complete recovery of good features of DRR17B along with identification of few ILs with complete maintenance ability (Table 4). In addition to improving BB resistance of DRR17B, The current study also focused on the identification of ILs of DRR17B possessing plant height which is significantly shorter than DRR17B, as shorter plant is preferred for deployment as good maintainers [viz., RMSIC 10-8-94, RMSIC 10-19-138, RMSIC 102-26-7, RMSIC 123-34-84, RMSIC 123-58-3 and RMSIC 172-77-12 and these lines could serve as better maintainers as compared to DRR17B. Interestingly, when compared to DRR17B, some of the ILs exhibited advantage concerning grain number per panicle. These lines include RMSIC 10-8-94, RMSIC 102-26-7, RMSIC 123-58-3 and RMSIC 172-77-12  were observed concerning number of days to 50% flowering in some of the backcross derived plants (Table 4). No significant differences were observed between improved versions of DRR17B and recurrent parent DRR17B concerning other agro-morphological characters or grain type and the lines mostly resembled the original recurrent parent. The approach of coupling of MABB with phenotypic selection adopted in this study helped to regain most of the key agro-morphological traits of DRR17B, while simultaneously helping in the selection of some superior backcross derived segregants of DRR17B possessing BB resistance.Similar to the approach adopted in the current study, several earlier studies also resorted to phenotype-based selection for key agro-morphological traits along with marker-assisted selection while improving elite varieties and parental lines for one or more target traits through MABB , 38, 48.ntainers . Signifiviz., IIRRIC102-26-7, IIRRIC123-34-84, and IIRRIC172-77-12) displayed complete maintainer ability highlighting the necessity of phenotypic confirmation for maintenance ability, despite a rigorous marker-assisted selection for rf3 and rf4 alleles in this study. This could be attributed to the existence of minor fertility restorer genes/QTLs as reported earlier [Xa21 + Xa33 are being converted as CMS lines by crossing with DRR17A through MABB.The ILs of DRR17B were test crossed with IR58025A (WA-CMS line) to check their maintainer ability. Three lines . The ILs Viz., IIRRIC102-26-7, IIRRIC123-34-84, and IIRRIC172-77-12 were exhibited complete maintainer ability as like DRR17B and remaining lines were partial maintainers.The six ILs of DRR17B exhibited high level of BB resistance against the BB isolates, when compared with the recurrent parent DRR17B. Whereas in agro-morphological characters like plant height, day to 50% flowering and number of grains per panicle etc, variations were observed. All the improved lines were shorter than the recurrent parent. With regards to Days to 50% flowering all the improved lines were little early (92–103 days) than DRR17B (105 days). Some the improved lines Xa21 and Xa33 by marker-assisted backcross breeding (MABB) strategy. The double gene pyramided lines of DRR17B expressed high level of resistance against eight different virulent isolates of Xoo and their resistance levels was comparable with triple resistance gene pyramided rice variety, ‘ISM’ (possessing Xa21 + xa13 + xa5) and were also significantly better than the single gene containing lines (possessing Xa21 or Xa33). Three promising double-gene pyramided lines of DRR17B with high level of BB resistance, agro-morphological attributes similar to or superior to the DRR17B with complete maintainer ability would be helpful in development of superior rice hybrids with durable, broad-spectrum resistance.The present study has resulted in development of improved versions of an elite maintainer of rice, DRR17B possessing durable resistance against BB through incorporation of two major dominant genes conferring broad-spectrum resistance,


This assay reacted with the hepatopancreas (HP) of EHP-infected shrimps, and the highest copy numbers were found in HP and feces samples from Southeast Asian countries (106–108 copies mg−1), while HP samples from Latin America, Artemia, and EHP-contaminated water showed lower amounts (101–103 copies mg−1 or mL−1 of water). No false positive was found with the normal shrimp genome, live feeds, or other parasitic diseases. This tool will facilitate the management of EHP infection in shrimp farms.We developed a qPCR assay based on the  Enterocytozoon hepatopenaei (EHP), an intracellular spore-forming parasite, is a major threat to the commercial shrimp industry. The infection is specific to the tubular epithelial cells of the hepatopancreas (HP) of shrimps. Shrimps in an advanced stage of EHP infection show signs of lethargy, reduced feed intake, soft shells, and an empty midgut [Penaeus monodon [Enterocytozoon, in the family Enterocytozoonidae [P. vannamei cultured in various Asian countries, including China, Thailand, Indonesia, Malaysia, Vietnam, and India [The microsporidian y midgut . This pa monodon . It was zoonidae . Subseqund India ,5,6,7. Rnd India .Severe EHP infections have become common in ponds, and result in substantial economic losses to shrimp producers, since they are associated with inhibited growth in cultured shrimps . EHP infβ-tubulin gene sequence has sufficient variability for molecular diagnosis at the species level [β-tubulin gene.Several EHP diagnostic methods based on SSU rRNA are available, including PCR, in situ hybridization, and loop-mediated isothermal amplification assays ,2,3,4,9.es level . For morβ-tubulin gene sequence for EHP-specific quantification. This assay was then applied to examine shrimp and feces samples collected from various countries to quantify the level of EHP present. The method was also applicable for screening live shrimp feeds and water samples.In this study, we developed a qPCR assay based on the β-tubulin gene of the EHP genome (GenBank no. KY593130), using Primer Express v. 3.0 software (Life Technologies). The DNA fragment (84-bp) was amplified using the forward primer (EHP-bTub-F2: 5′-GATTTGAGAAAATTGGCGGTTAA-3′) and the reverse primer (EHP-bTub-R2: 5′-TTCTGAACACAGAGGCGCATA-3′), while the TaqMan probe (5′-TGATTCCATTTCCACGACTGCACTTTTTC-3′) was synthesized and labeled with 6-carboxyfluorescein (FAM) on the 5′ end and N,N,N′,N′-tetramethyl-6-carboxyrhododamine (TAMRA) on the 3′ end. The qPCR assay was performed by combining 1 µL extracted DNA and a qPCR mixture containing 2.5 μl TaqMan Faster Universal PCR Master Mix (Life Technologies), 0.3 μM of each primer, and 0.1 μM TaqMan probe for a final volume of 10 μl. Amplification was performed by thermal cycling for 20 s at 95 °C followed by 40 cycles of 3 s at 95 °C and 30 s at 60 °C. Amplification and data analysis were performed using a StepOnePlus PCR system (Life Technologies).The primers and TaqMan probe were designed based on the β-tubulin gene from the EHP isolate 16-505/HP (β-tubulin gene is a single copy gene in the EHP genome. The plasmid was designated pEHP-tubB-2 (3885-bp) and the concentration was determined by measuring OD260nm (2.38 × 108 copies per 1 ng of DNA). For a standard curve, the purified pEHP-tubB-2 plasmid was used, and the sensitivity was determined by preparing 10-fold dilutions. A standard curve presenting concentrations from 102–108 copies µL−1 is shown in Perezia sp. microsporidium [Paramoeba sp. amoeba [β-tubulin gene sequence are specific to EHP, showing no reaction to another shrimp parasites. In addition, DNA from different shrimp species and live shrimp feed were tested to ensure that the primers did not cause false positives  using a Microsep Advance Centrifugal Device (PALL Corporation). Approximately 30 mg of HP, feces, and artemia samples and 300 μL of water samples were used for DNA extraction using QIAamp tissue kit (Qiagen). After DNA extraction, the concentration of DNA was measured by a spectrophotometer . The EHP copy number was highest in the HP samples from Indonesia, with a range between 8.0 × 103–1.4 × 108 copies mg−1, followed by those from Thailand (2.5 × 102–9.1 × 107 copies mg−1) and Vietnam (1.9 × 103–4.8 × 107 copies mg−1). In contrast to the SE Asian samples, HP samples from Latin America presented a relatively low EHP quantity, with copy numbers between 2.1 × 101–1.3 × 102 copies mg−1. In the feces sample, 106–1.7 × 107 copies mg−1 were found, while Artemia samples contained EHP DNA at 4.3 × 103–2.8 × 105 copies mg−1. Finally, EHP was detected in the water samples at 1.7 × 103 copies per mL−1.To evaluate practical use, a total of 86 samples were collected from a population of EHP-affected vannamei . These sArtemia. This indicates good applicability of the qPCR assay regardless of the source of EHP and their host species. For the Artemia samples, substantial amounts of EHP (as DNA copies) were detected. Live shrimp feeds, including polychaetes, squids, and Artemia biomass, have been suspected to be carriers for EHP, so EHP quantification by qPCR in live shrimp feeds can be used as a tool to aid in disease prevention in broodstock populations. Using qPCR, we also confirmed that feces contained high numbers of EHP copies, indicating that this may act as an important source of infection in ponds. In penaeid shrimps, EHP multiplies rapidly in the HP tubule epithelium cells, and EHP spores are released into the digestive system, causing white feces in ponds [All samples tested in this study came from shrimps exhibiting slow growth in farms from SE Asia and Latin America, and from contaminated in ponds .β-tubulin gene, applicable in EHP management in shrimp farming.In conclusion, EHP infection is notorious in farmed shrimp ponds, and such EHP infections are likely to continue to be prevalent. The situation requires careful monitoring of EHP levels in shrimps and their feed using a highly specific diagnostic method. In addition, quantification of EHP in live feeds, pond environments, or feces becomes more important for EHP management. This study describes a new quantification method for EHP based on the


Background: Antimicrobial resistance (AMR) is an increasing global threat, and hospital-based antimicrobial stewardship programs (ASPs) are one of the effective approaches to tackle AMR globally. This study was intended to determine the attitude of key healthcare professionals (HCPs), including physicians, nurses, and hospital pharmacists, towards AMR and hospital ASPs.Methods: A cross-sectional study design was used to collect data from HCPs employed in public teaching hospitals of Punjab, Pakistan, from January 2019 to March 2019. A cluster-stratified sampling method was applied. Descriptive statistics, Mann Whitney and Kruskal Wallis tests were used for analysis.Results: A response rate of 81.3% (881/1083) for the surveys was obtained. The majority of the physicians  perceived AMR to be a serious problem in Pakistani hospitals (p < 0.001). Most of the HCPs considered improving antimicrobial prescribing  accompanied by the introduction of prospective audit with feedback , formulary restriction  and regular educational activities  as effective ASP methods to implement hospital ASPs in Pakistan. A significant association was found between median AMR and ASP scores with age, years of experience, and types of HCPs (p < 0.05).Conclusions: The attitude of most of the HCPs was observed to be positive towards hospital-based ASPs regardless of their poor awareness about ASPs. The important strategies, including prospective audit with feedback and regular educational sessions proposed by HCPs, will support the initiation and development of local ASPs for Pakistani hospitals. Antimicrobial resistance (AMR) is regarded as one of the most daunting challenges that the world is currently facing . It has K. pneumonia was found to be involved in 81% of the deaths , Ivory Coast (16.8%), Kenya (27.7%), Nigeria (29.6%), Ethiopia (42.8%), South Africa (52%), and Cameroon (72%)  .Acinetobacter and more than 70% of P. aeruginosa isolates to be multidrug-resistant (MDR). Moreover, the resistance against third-generation cephalosporins was also noted to be higher . The emergence of resistance against colistin is further worsening the situation, which is why Pakistan recently launched its National Action Plan (NAP) to help reduce AMR  is regarded as a globally accepted tool to address the challenge of AMR . The focIn Pakistan, the efforts to develop regional hospital-based ASPs were started in 2014 by Medical Microbiology and Infectious Diseases Society . HoweverPakistan is one of the renowned countries of Asia, comprised of four provinces, including Punjab, Khyber Pakhtunkhwa, Balochistan, and Sindh . Among aA literature review was undertaken to construct and conceptualize the questionnaire . An effoThe final version of the questionnaire comprised of 34-items and had six sections. The demographic information about HCPs, including the type of profession, age, gender, experience, and specialty, was asked in the first section of the questionnaire. The second and third sections of the questionnaire contained four questions related to awareness of AMR and five questions related to the triggering factors of AMR rated on a 7-point Likert scale (1 = not a problem to 7 = very serious problem and 1 = does not contribute to 7 = strongly contribute). Ten Questions related to patient care and ways to eradicate AMR were asked in the fourth section of the questionnaire, with options 1 = strongly disagree to 7 = strongly agree. The fifth section of the questionnaire focused on ASP strategies comprised of five questions. Here again, a 7-point Likert scale was opted (1 = very unhelpful to 7 = very helpful). The last section of the questionnaire has four questions about the previous involvement of HCPs in AMR and ASPs with three options “yes,” “no,” and “do not know.”A pilot study was conducted on thirty HCPs to ensure the internal consistency of the questionnaire . The value of Cronbach alpha measured for sections two to six was more than seven indicating an acceptable level of internal consistency.A multistage cluster-stratified sampling method was used to recruit HCPs, including physicians, nurses, and hospital pharmacists. In the first stage, six administrative divisions  of the Punjab province were selected out of nine by cluster sampling, with names of divisions drawn from a hat Figure 1Trained data collectors with a pharmacy background collected the data from HCPs. The principal investigator (PI) informed all the data collectors about the aims and objectives of the study. To approach different HCPs, data collectors visited six teaching hospitals located in six administrative divisions of Punjab. The in-charge physician of the ward or department was briefed about the study purpose, and later facilitated the distribution of the questionnaire among HCPs. The data collectors addressed all the queries that arose from the HCPs. The PI also inspected the working of data collectors by conducting unscheduled and random visits to the hospitals to ensure the quality of the data collection. All the participating HCPs provided verbal consent before completing the questionnaire.p-value <0.05 was set to be statistically significant. Post-hoc analysis (Bonferroni correction) was also carried out to determine the difference in intergroup variables. In order to prevent the inflation of type one error, the p-value was based on the total number of comparisons, such as for three group p-value < 0.017 was considered statistically significant . Based on the results of the Kruskal–Wallis test, a follow-up analysis (the Mann–Whitney) was also conducted to determine where exactly the statistically significant difference occurs among the different HCPs . Moreover, logistic regression was also applied to comparative estimates.To summarize the data, and calculate frequencies, and percentages the Statistical Package for the Social Sciences  was used. Kolmogorov–Smirnov and Shapiro–Wilk tests were employed to detect the data normality. Since the data did not follow the normal distribution, median, and interquartile ranges (IQRs) were measured. The categorical data were presented in proportions that agreed with a “6” and “7” Likert scale response. Mann-Whitney and Kruskal-Wallis tests were used on continuous data. Median AMR scores (measuring awareness about AMR and its contributing factors), AMR eradication scores (measuring AMR eradication approaches), and ASP scores (measuring ASP strategies) were also calculated, which were compared using demographic variables. A The Biomedical Ethics Committee of Xi’an Jiaotong University approved the study (no. 2018-528). Additionally, permission to conduct this study was also taken from the University of Veterinary and Animal Sciences. Respondents were informed of the voluntary nature of their participation, and confidentiality was guaranteed.There was a total of 881 HCPs completed the survey, of whom 46.5% (410) were physicians, 45.1% (397) nurses, and 8.4% (74) pharmacists. The overall response rate was 81.4% . Many of the participating HCPs  were female as the nursing staff in Pakistan is mostly comprised of female nurses. Most of the HCPs  were aged between 25-30 years. A large proportion of HCPs were working in the medicine , pediatric , and surgery  departments. The majority of the HCPs had 1–5 years of clinical experience Table 1.p < 0.001). However, physicians  were less likely to consider AMR as a serious threat in their practicing hospitals compared to pharmacists  and nurses . The greatest number of HCPs  deemed AMR as a serious issue in the Pakistani community outside of the hospital (p < 0.001)  than pharmacists  and nurses  viewed AMR to be a serious problem in Pakistani hospitals (< 0.001) Table 2.p < 0.001).A large number of HCPs perceived that use of antimicrobials in the Pakistani community  and hospitals  as being a triggering factor for AMR however, they were in less agreement  that use of antimicrobial in their hospitals was a contributing factor (p < 0.008). Most of the HCPs  did not agree that the use of antimicrobials in Pakistani animals and the agriculture sector lead to AMR (p < 0.001)  compared to nurses  and pharmacists  agreed that use of antibiotics without a prescription is an important factor contributing to AMR (< 0.001) Table 2.p = 0.014); however, a large number of HCPs  agreed that better use of antibiotics would reduce the momentum of resistant infections (p = 0.034). Similarly, 67.9% (599/881) HCPs believed that the irrational use of antibiotics is not ethical (p < 0.012).Only a handful of the HCPs  deemed that AMR affects patients under their supervision (p < 0.001). More than 50% of the HCPs agreed that a formal policy for the use of antimicrobial  accompanied by local antimicrobial guidelines and protocols  should be introduced in their hospitals  were more likely than pharmacists  and nurses  to agree that improving prescribing of antimicrobials could decrease AMR . Nevertheless, nurses  were more in agreement to establish an ASP team. Furthermore, most of the HCPs  showed a positive attitude to become part of an ASP team (p < 0.004).Nurses  were less in support than physicians  and pharmacists  of introducing a computer application to rationalize the use of antimicrobials (p < 0.001). Many HCPs  were not in favor of restricting the prescribing of all antibiotics (p = 0.001); however, they  showed a positive attitude to limit the prescribing of certain antibiotics (p = 0.004). The other imperative approaches to which HCPs agreed were readily accessible microbiological data  and regular education sessions   than physicians  and pharmacists  considered regular hospital-wide audit and feedback on antibiotics as being a helpful ASP strategy  Table 4.A larger number of physicians  and nurses  than pharmacists  were involved in taking care of patients with resistant infections nevertheless, more than half of the HCPs  agreed that the rate of resistance is escalating over the last 10 years. Only a fraction of HCPs  had heard of an “antimicrobial stewardship program.” Moreover, only 3% (26/881) HCPs said that they have worked in a healthcare setting with a functional ASP Figure 2p < 0.05). The median ASP attitude score of nurses was significantly higher than pharmacists .A significant association of types of HCPs was found with median attitude scores on AMR and ASP. The physicians were more positive in attitude than nurses towards AMR  and ASP strategies .The age of the HCPs also had a significant association with median scores as HCPs between the ages of 36–40 years had more awareness than younger HCPs about AMR   Table 5.p = 0.012] and physicians  were significantly more likely to involve in dealing patients with resistant infections than other HCPs  OR: 3.824; 95% CI (1.351–10.826);This multicenter and multidisciplinary study provides the latest insight about the perspective of all key HCPs, including physicians, pharmacists, and nurses from a low- and middle-income country (LMIC).The majority of each of the HCP respondents were of the view that AMR is a global threat, and it is a severe issue of Pakistani hospitals, including their practicing hospital. It has already reported in several studies that AMR is a global issue . A recenA significant difference in perception about AMR was observed between HCPs as nurses were less likely to consider AMR as a serious problem in the community possibly due to their diminished role in community healthcare settings as they are mostly engaged in hospital settings, as also highlighted by a Thai study . MoreoveImproper and excessive use of antimicrobials in agriculture and animal sector is widespread across the world , but in More than 50% of HCPs agreed that patients could buy antibiotics over the counter without any valid prescription  and this could contribute to AMR. These results comply with studies conducted in other developing countries, including China, Ethiopia, and Thailand . Nurses In accordance with the previous studies , only 36More than half of the HCPs considered all AMR eradication strategies, such as policies and systems to improve antimicrobial prescribing accompanied by regional antimicrobial guidelines and protocols, helpful to limit the progression of AMR in Pakistan. The systems comprised of optimal antibiotic use have already shown effectiveness in terms of clinical benefits . TherefoFormulary restriction and hospital-wide audits with feedback are two imperative hospital ASP strategies . All parIn our study, many HCPs  were involved in taking care of patients with resistant infections, which further strengthens their perception of AMR as of grave concern. However, this is not always true as some studies have shown an inconsistent relationship in physicians who have taken care of patients with resistant infections, and the seriousness with which they take the AMR problem in their hospital . For exaA poor understanding by all HCPs of ASP was found, as only 15.2% of HCPs were familiar with this term. This may be because hospital-based ASPs have not been implemented in all Pakistani hospitals, and HCPs lack awareness of and training towards this type of program. Furthermore, the lack of conferences and workshops about ASPs could be another reason. Training with short-term courses outlining core elements of ASPs should be offered to all HCPs prior to implement ASPs in Pakistan successfully . This poThere are some limitations of this study that should be noted before policymaking based on our findings. First, this study was conducted in six of the nine administrative divisions of Punjab province. While more than half of the Pakistani population lives in Punjab, and the six divisions were selected by cluster sampling to minimize the bias, the findings may not apply to other parts of Pakistan. Second, the nature of participation of HCPs was voluntary, which could cause selection bias, and views of non-participating HCPs are unknown. Third, this study was carried out in tertiary care public hospitals of Punjab, and the attitudes of HCPs working in the non-public hospitals may differ. However, HCPs working in the public sector hospitals in Pakistan also work in the private sector during off-hours. Irrespective of the above limitations, this multidisciplinary and multicenter study highlights the latest views of all major stakeholders of ASPs towards AMR and ASPs from an LMIC. Furthermore, the Government of Pakistan may consider the findings of this study for the development of local guidelines to implement hospital ASPs across all healthcare settings.AMR was viewed as a serious issue by most of the HCPs. Formulary restriction and hospital-wide audit with feedback on antibiotic use accompanied with regular educational sessions are key strategies proposed by HCPs to implement hospital ASPs in Pakistan. The significant differences in perceptions and attitudes of different professionals towards AMR and ASPs found in this study will help to develop and tailor regional ASPs. Regardless of inadequate ASP familiarity, the attitude of most of the HCPs was positive about hospital-based ASPs.The datasets generated and/or analyzed during the current study are not publicly available due to privacy restrictions but are available from the corresponding author on reasonable request.The Medical Ethics Committee of Xi’an Jiaotong University (2018-528) approved the study. Additionally, permission to conduct this study was granted by the University of Veterinary and Animal Sciences. Respondents were informed of the voluntary nature of their participation, and confidentiality was guaranteed. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.Conceptualization: KH and YF. Data curation: KH, AG, and MJ. Formal analysis: KH and JC. Investigation: KH. Methodology: KH and YF. Resources: KH and YF. Software: WJ, MZ and CY. Supervision: YF and MR. Writing—original draft: KH. Writing—review and editing: YF and MR. All authors read and approved the final manuscript.This work was funded by the National Natural Science Fund (71974156), “Young Talent Support Plan,” “High Achiever Plan” of the Health Science Center, Xi’an Jiaotong University, and the Central University Basic Research Fund (2015qngz05).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The molecular weight fractions of <1 kDa accounted for almost half of KS-DON. Hydrophilic compounds accounted for more than 75% of KS-DON. Three fluorescence peaks were identified: soluble microbial byproducts (A); protein-like substances (B); and humic acid-like substances (C). It is concluded that the KS-DON in Taihu Lake sources has higher bioavailability and higher risk of endogenous release. Ecological dredging and establishment of constructed wetlands are possible measures to reduce the release of endogenous nitrogen.KCl-extractable sediment dissolved organic nitrogen (KS-DON) extracted from sediments near drinking water intakes of six drinking water sources in Taihu Lake in China was partitioned into hydrophobic and hydrophilic fractions and high/low molecular weight fractions. The results showed that the total dissolved nitrogen (TDN) contents of the extracts ranged from 67.78 to 128.27 mg/kg. KS-DON was the main TDN species, accounting for more than 50%, with NH Nitrogen is an essential element to living organisms, particularly in primary productivity in aquatic ecosystems . DissolvMany studies have confirmed the bioavailability of DON in sediments ,13,14, iAlthough DON content is usually low in surface water, it has become a critical issue in terms of drinking water treatment, because of reactions with chlorine that generate disinfection byproducts (DBPs) during the disinfection/oxidation processes ,20. ChloTaihu Lake is an important source of drinking water in eastern China, with an important role in regional economic and social development, in addition to impacting human quality of life and wellbeing ,25. The 2 and a mean depth of 1.90 m, is the third largest shallow freshwater lake in China, located in the southern part of the Yangtze River Delta, China [Taihu Lake , with an area of 2425 kma, China  , Xukou Bay (Y2), Gonghu Bay , Western shore-line (Y6). Y1–Y6 are important drinking water sources across the Taihu lake, serving 10 million people in the Taihu Lake Basin. Y1 , Y2  and Y3  are located in grass-type zones with better water quality, surrounded by outflow rivers ; Y4 , Y5  and Y6  are located in algal-type zones, especially in Y6, surrounded by main inflow rivers.Surface sediment samples (0–10 cm) were collected near the drinking water intakes with a grab sampler in April 2015. Three adjacent surface sediment samples were collected from each site and mixed evenly in the field. These sediment samples were placed into sealed polyethylene tubes and temporarily stored in iceboxes at 4 °C. After immediate transfer to the laboratory, the samples were stored below −20 °C and then freeze-dried at −50 °C using FD-1D-50 freeze-dryers . The dried samples were homogenized using an agate mortar and passed through a 100-mesh sieve before analysis.W/V) for 1 h in a horizontal shaker at room temperature, then centrifuged at 5000 r/min for 15 min at 4 °C and filtered through 0.45-μm Millipore filters (mixed cellulose ester membrane) to remove suspended solids and residual bacteria. Extracts were stored in the dark, kept at 4 °C prior to the experiments, and analyzed within 1 weeks of collectionThe sediment samples were extracted using a 1 mol/L KCl solution  spectrophotometry, whereas NH4+-N was measured using the salicylate hypochlorite method. TDN was measured via the alkaline potassium persulfate digestion-UV spectrophotometric method. All the determinations were done according to the Chinese National Standard Methods  [3−-N and NH4+-N) [NO., 2002) . The con NH4+-N)   were used: (i) 1-kDa nominal molecular weight limit (NMWL); (ii) 3-kDa NMWL; (iii) 5-kDa NMWL; and (iv) 10-kDa NMWL. Extract samples at a volume of 200 mL were filtered through each membrane in series. Fifty milliliters of raw water and of each filtrate was retained for further analysis. The percentage of KS-DON in the samples collected using each membrane size was calculated as follows (Equations (2)–(6)):Based on Yeh et al.’s  study, wExtract was fractionated by using different resins  ,42. The Three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy  was used to characterize KCl-extractable sediment DOM. The excitation (Ex) wavelength was set from 200 to 450 nm at 5-nm intervals, corresponding to emission (Em) wavelengths from 250 to 550 nm at the same intervals. The scanning speed was set at 1200 nm/min. The spectrum of deionized water was recorded as the blank.Fluorescence regional integration (FRI), a quantitative technique proposed by Chen et al. , integrap < 0.05 for all tests.The water quality parameters of extracts were expressed as the mean ± standard deviation of the three replicate samples. One-way analysis of variance (ANOVA) was used to analyze the differences of nutrient in the sediment between six sites. Statistical analysis was performed using the SPSS 20.0 statistical package , and the level of significance used was 3−-N showed no significant differences (p > 0.05) between six sites, while the concentrations of TDN, NH4+-N and DON differed (p < 0.05), and the TDN, NH4+-N and DON content of sites in the Algae-type zones were higher (p < 0.05) than the Grass-type zone. The content of TDN showed as follows: Y6 > Y5 > Y4 > Y3 > Y2 > Y1. The concentrations of TDN in the six sediment samples from Taihu Lake ranged from 67.78 mg/kg to 128.27 mg/kg. The sediment NH4+-N content ranged from 17.64 mg/kg to 34.78 mg/kg, and accounted for 24–31% of sediment TDN content. Its distribution pattern across the six sample sites was the same as that of sediment TDN. The sediment NO3−-N content ranged from 9.87 mg/kg to 25.70 mg/kg, and accounted for the lowest percentage fraction of sediment TDN compared with other nitrogen species. It is likely that this result is because the sediment is an anaerobic environment, which is unfavorable to the formation of NO3−-N sediment NO3−-N. The KS-DON content ranged from 39.23 mg/kg to 76.63 mg/kg, contributed to 68.0–82.5% of sediment TDN, consistent with previous studies [The nitrogen species and concentrations in each extracts are shown in  studies .The sediment DON content of the Taihu Lake is closely related to the level of pollution, and research has shown that the more serious the pollution, the higher the concentration of DON in the sediment . SedimenThe KS-DON composition of each molecular weight fraction is shown in The results showed that KS-DON of the Taihu Lake mainly comprised small molecular weight fractions, which is similar to the results of Tao et al.  and showAccording to the published literature, humic and fulvic acids are the main components of SH, and hydrophilic (non-humic) organic matter (CH and NH) are attributed to proteins, amino acids, and carbohydrates ,46. As sThe KS-DON fractionation results demonstrated that KS-DON from Taihu Lake mainly comprised hydrophilic groups. Research has shown that plant and animal debris can degrade to proteins, polypeptides, and amino acids, increasing the content of hydrophilic DON . The hydi,n) for the KS-DON are presented in I+II,n for region of simple aromatic proteins(region I and II), ranging from 50.13% to 61.76%. This quantitative result was consistent with visual analysis of the location of peaks within regions I and II of the EEM spectra  to PI+II+IV,n for the protein-like regions  were calculated  in the short term after ecological dredging, because of the higher NH4+-N content in the porewater, so it was suggested that ecological dredging in winter with lower water temperature could reduce the internal loading in order to achieve positive efficiency.The purpose of ecological dredging is to remove surface sediments containing high-impact salts, including suspended, semi-suspended flocculent colloids of nutrients deposited on the surface of silt, or dormant algae or biological debris of animals and plants. Liu et al.  have repWetlands and higher aquatic plants can play the role of wave dissipation and physical retardation, thus promoting particulate deposition and reducing sediment resuspension . BesidesThe characteristics of KS-DON in the six drinking water sources from Taihu Lake were investigated in the current study. KS-DON was the main species of TDN and accounted for >50% of TDN. Molecular weight fractions of <1 kDa, 1–3 kDa, 3–5 kDa, 5–10 kDa, and >10 kDa accounted for 43.78%, 20.22%, 7.71%, 7.23% and 21.06% of TDN, respectively. NH was the main component of KS-DON and averagely contributed 43.84% of KSDON in the sediment samples. CH, SH, and WH contributed 33.78%, 14.29%, and 8.09% of KS-DON, respectively. There were three fluorescence peaks: (i) soluble microbial byproducts; (ii) protein-like substances; (iii) and humic acid-like substances. It is concluded that KS-DON in Taihu Lake is mainly hydrophilic and low molecules with high bioavailability, low humification and high risk of KS-DON endogenous release. Therefore, measures should be taken not only to control the input of exogenous pollution, but also to reduce the risk of endogenous N-pollution release, such as using ecological dredging and establishment of constructed wetlands.


Tires are an important vehicle component, as car handling, safety and fuel economy depend for a major part on the tire composition and construction. As a consequence, tires are improved continuously. The most prominent improvement in the recent past was the use of a silica-silane filler system in passenger car tread compounds, instead of traditionally used carbon black. For recycling and re-use of end-of-life car tire rubber one of the most promising recycling methods is devulcanization: re-plasticizing the vulcanized rubber by selectively breaking the sulfur bridges between the polymer molecules. In the present paper, the influence of silica, which is present in the passenger car tires granulate, on both devulcanization and subsequent revulcanization, is investigated. In a step-wise approach it is shown that the presence of silica influences both devulcanization and revulcanization. The best tensile strength of the revulcanizate, using a carbon-black-based revulcanization formulation, was 5 MPa. This could be improved to 6.5 MPa by using 2.8 phr of 1,3-DiPhenylGuanidine (DPG) in the revulcanization formulation. After addition of a silanization step during revulcanization by adding 3.2 phr bis[3-(TriEthoxySilyl)Propyl] Tetrasulfide (TESPT), a silane, to the formulation, the tensile strength of the revulcanizate was further improved to 8 MPa. With these results it is shown that the silica in the granulate can be used to improve the revulcanization properties. To check the benefits of using pure tire tread material for the devulcanization and subsequent revulcanization, of both a carbon black and a silica-based virgin tread compound, it is shown that a tensile strength of the revulcanizate of 13 MPa can be reached. This shows the potential of devulcanized rubber when the various tire components are separated before whole car tire material is granulated as the beginning of the recycling. The performance of passenger car tires is continuously improved, supported by governmentally imposed labeling systems on relative performance in terms of safety and environmental aspects, noise and fuel economy. Due to the sheer amount of 10Finetuning of the process conditions leads to a shift towards devulcanization. It was shown that damage of the polymer chains can be limited considerably by tailoring the process conditions ,2. For EAn additional challenge is the production of a clean car tire granulate. During grinding, the rubber is separated from remaining steel, fibres and contaminations. The preferred particle size of the Ground passenger car Tire Rubber (GTR) is determined by the degree of purity required for the final application, and the costs. Besides, the granulate is a mixture of different rubber compounds from all tire components. An important, relatively recent, change in passenger car tire tread compositions is the use of silica as reinforcing agent rather than the more common carbon black. This is because of the significant improvement of wet grip and rolling resistance of the tires, leading to improved safety and better fuel economy of vehicles. However, as earlier work has shown, devulcanization of silica-based rubber is more difficult than of its carbon black counterpart, as indicated by a Horikx-Verbruggen analysis ,5 to detThe devulcanization process applied in this study is a thermo-chemical-mechanical twin-screw extruder based, continuous process, upscaled from a small scale batch process, as described by Saiwari . In the In previous studies, the devulcanization agent DiPhenyl DiSulfide (DPDS) was used, but due to the smell of the final product and a reported scission of the polymer chains , an alteBecause of a relatively low tensile strength of the first batch of revulcanized material, the devulcanization process was thoroughly improved. As the feedstock contained a considerable amount of silica, the revulcanization process and compound formulations were optimized for this.To study the influence of a mixture of all kinds of compounds as can be found in passenger car tire granulate, both a silica-based and a carbon black-based tread compound were prepared and stress strain properties before and after devulcanization (and subsequent revulcanization) were measured. By microscopy of the fracture surfaces of the revulcanizates, the influence of silica on devulcanization are shown.The introduction of silica in passenger car tire treads started more than 20 years ago ; therefoWith additional analysis it was shown that the silica content varies around an average of 23 phr. By Fourier Transform Infrared Spectroscopy (FTIR) analysis it was shown that the remaining GTR-ash after TGA clearly contains a certain amount of silica: curve B in By Scanning Electron Microscopy-Energy Dispersive X-ray analysis (SEM-EDX), the presence of silica particles up to 250 Devulcanization of GTR was done in a continuous process with the twin-screw extruder. The formulations as given in Verbruggen  has showBecause of the powdery consistency of the devulcanization agents, swelling before devulcanization was not necessary. Just before use, the GTR was manually mixed with TDAE, TDTBP and DBD until the mixture was homogeneous. Mixing time was 5–10 min at room temperature, depending on the amount of GTR. The functions of TDAE are multiple: it acts as a processing aid during devulcanization, as an additive to facilitate the diffusion of devulcanization agents into the GTR particles, and to prevent dusting while dosing powdery devulcanization aids.The screw design of the extruder was based on a minimal shear concept: minimize the application of shear during the processing at high temperatures and perform shear at lower temperatures after the chemical devulcanization, see Initially, the revulcanizates were prepared with formulation 1 of In a previous, unpublished, study the influence of a silica reinforced vulcanization system on the performance of the devulcanization process was investigated by S.Saiwari  using thThe physical bonds between carbon black and the polymers as opposed to the chemical bonds between silica, silane and the polymers, which result in a different kind of network;The nature of the chemical bonds  present between the silane coupling agents and the polymers;A combination of these two.This indicated that the amount of polymer that could be released from the polymer network of a silica reinforced compound by devulcanization is substantially lower than for a carbon black reinforced rubber. Possible explanations are, see As the applied devulcanization process does not break mono-sulfidic crosslinks, and the chemical bonds between silane and polymers are presumed to be primarily monosulfidic, a higher percentage of these type of links are supposed to be the main reason. In addition, it was found during the experiments of the present study, that the degree of devulcanization of tire granulate was not in relation to the tensile properties found after revulcanization of this material.This must be due to the complex composition of the devulcanized granulate based on the mix of all tire components. Also, it is mentioned that unsaturated elastomers in general and SBR in particular are sensitive to recombination during devulcanization, especially for temperatures above 200  studies ,17. For For silica it is known that its surface is covered with silanol moieties, as depicted schematically in However, as the amount of silica in the GTR is considerable, as described before, the question may be raised if there are free silanol moieties left or regenerated after devulcanization. Therefore, DPG was added to the formulation to compensate for the acidity of the silica. The amount of DPG was set at 2 phr, similar to the concentration used in the silica-based model compound, As the improved results due to the addition of DPG show, a certain amount of free silanol moieties is still present on the silica surface. To make use of these for building an additional silica network, a silanization step before the revulcanization process with a coupling agent  is again necessary to further improve the tensile properties. By this secondary silanization, as shown in ress see .These results demonstrate that the first improvement by the addition of DPG alone remedied a certain acidity due to the silica in the GTR and hence in the devulcanizate. The second improvement by the silanization with additional TESPT indicates that the silica still has free silanol moieties left for a second silanization, enabling a further improvement of the properties of the material.To emphasize the benefits of separating the different tire components and using pure tread compounds, an additional study into these was done. As was discussed before, silica reinforced compounds are more difficult to be devulcanized than carbon black reinforced ones. In the earlier study  the mateA part of both vulcanizates were ground at room temperature, sieved with the laboratory set of sieves to obtain a fraction of 2–3.5 mm and subsequently devulcanized. Devulcanization of these small batches was performed in the Brabender mixer with a chamber volume of 50 mL. The mixer was preheated to a temperature of 220 Subsequently, the devulcanizate was dumped into liquid nitrogen to cool down and to prevent contact with oxygen. Afterwards, it was post-treated on a mill in a similar way as the DGTR, as described before. The devulcanized compounds were revulcanized again, using the same formulations as were used before for the prior vulcanization with respect to the zinc-oxide content, stearic acid, sulfur, TBBS, DPG and TESPT. These components were assumed to have been fully consumed during the prior vulcanization which turned out to be a practical approximation. For preparing the revulcanizates the procedures as described in Dumbbells were cut from all samples and tested for their stress strain properties according to ISO 37. Pictures were taken of the fracture-surfaces of the dumbbells after the tensile tests by microscopy.Tensile properties for both compounds, prior to devulcanization, were a tensile strength of 20 MPa for the silica-based compound at 380% strain and 15 MPa for the carbon black-based at 400% strain. After de- and revulcanization, the compounds showed a tensile strength of 13 MPa and a strain of 280% for the silica-based and 13 MPa and 330% for the carbon black-based compound, as shown in Hence, after revulcanization of the carbon black-based compound about 95% of the tensile strength could be recovered, but for the revulcanized silica compound this was only about 70%. Although this is significantly less compared to the carbon black-based compound, it is much better than could have been expected from the decrease in crosslink density of similar compounds as shown by the Horikx-Verbruggen diagrams in The latter implies a lower amount of devulcanized rubber containing larger parts of non-devulcanized rubber. This must be due to the fact that the silica-silane-polymer bonds mainly consist of mono-sulfidic crosslinks which cannot be broken by the devulcanization process, as shown in The Ground Tire Rubber (GTR) used in this investigation was obtained from Genan, Dorsten, Germany. It is a commercial ground passenger car tire, medium grade with a normal-like size distribution of dimensions 1 to 3.5 mm, see Continuous devulcanization was performed in a KrausMaffei ZE 25 UTX co-rotating twin-screw extruder , length 42D with D = 25 mm, with 3 de-aeration positions between the supply funnel and outlet and an elongated die, with dimensions of a rounded rectangular slit of 20 mm × 40 mm and length of 100 mm, as can be seen in The extruder was operated at 10–30 rpm with a total residence time in the extruder and the elongated die of approx. 10 min at 20 rpm. To minimize oxidative degradation during the devulcanization, the extruder was equipped with nitrogen supply in the supply funnel, at the position of elements 36–37 and at the entrance to the die. The barrel temperature was set at 130 Modulus, tensile strength and elongation at break were determined with a Zwick tensile tester, ZwickRoell GmbH & Co, 89079 Ulm, Germany using dumbbell shaped samples according to ISO 37 type II. A Thermo Gravimetric Analysis (TGA) was performed with a TGA550 from TA Instruments, New Castle, DE19720, USA. For FTIR spectrometric analysis, a PerkinElmer Spectrum 100,PerkinElmer Inc, North Billerica, MA 01862, USA was used and SEM-EDX analysis were performed with a Jeol JSM 6400 SEM, Jeol SemAforce digital Image acquisition software and a Noran Voyager energy Dispersive X-ray analyzer, all from JEOL USA, Inc., Peabody, MA 01960, USA. For microscopy, a Keyence 3D, Keyence corporation, Itasca, IL 60143, USA was used.GTR contains an increasing amount of silica over recent times, as result of the progressive use of this filler in modern tread compounds to improve tire performance. It was shown that devulcanization of such silica containing GTR is more difficult than of a carbon black-based GTR. The presence of silica in GTR has a double effect: It makes the rubber more difficult to devulcanize than GTR without silica, which leads to a coarser structure after revulcanization and to lower values of the tensile strength: a value of 3.5 MPa could only be obtained. By using additional DPG as secondary accelerator and adding new coupling agent TESPT, resp. an additional silanization mixing sequence in the revulcanization formulation, the tensile strength could be restored to 8 MPa. In To emphasize the benefits of separating tire treads from the rest of the tires before processing, it was shown with tread compound studies with 80 phr carbon black or 90 phr silica as fillers that it must be possible to keep the tensile properties of a revulcanizate of a devulcanized silica-based compound at approximately 70%, from 20 MPa to 13 MPa, while for a carbon black-based compound at around 95%. The decrease in the tensile properties between revulcanized silica-based compounds and the original material, compared to those of the carbon black-based compounds, indicates that separation of the different tire parts before devulcanization to separate silica-containing and non-containing parts, may significantly improve the overall quality of the devulcanizates.


M. bovis shedding has been previously reported in cattle and wildlife hosts (badgers and wild boars), but the observed differences were attributed to dose and route of infection, rather than host genetics. This article addresses the extent to which cattle infectivity may play a role in bTB transmission, and discusses the feasibility, and potential benefits from incorporating infectivity into breeding programmes. The underlying hypothesis is that bTB infectivity, like resistance, is partly controlled by genetics. Identifying and reducing the number of cattle with high genetic infectivity, could reduce further a major risk factor for herds exposed to bTB. We outline evidence in support of this hypothesis and describe methodologies for detecting and estimating genetic parameters for infectivity. Using genetic-epidemiological prediction models we discuss the potential benefits of selection for reduced infectivity and increased resistance in terms of practical field measures of epidemic risk and severity. Simulations predict that adding infectivity to the breeding programme could enhance and accelerate the reduction in breakdown risk compared to selection on resistance alone. Therefore, given the recent launch of genetic evaluations for bTB resistance and the UK government's goal to eradicate bTB, it is timely to consider the potential of integrating infectivity into breeding schemes.Host resistance and infectivity are genetic traits affecting infectious disease transmission. This Perspective discusses the potential exploitation of genetic variation in cattle infectivity, in addition to resistance, to reduce the risk, and prevalence of bovine tuberculosis (bTB). In bTB, variability in  Mycobacterium bovis, is responsible for an estimated 10–15% of human TB cases (Bovine tuberculosis (bTB) is a zoonotic disease, which can compromise both human health and international livestock trade. Zoonotic TB caused by TB cases , 3. AddrTB cases –4.In the UK, bTB has been the most pressing animal health problem, with financial losses amounting to over £175 m per annum . HoweverSelective breeding can complement classic disease control strategies, reducing the requirement for biosecurity measures and movement restrictions which have a major economic impact for herds undergoing a bTB breakdown , 10. WitVeterinarians and epidemiologists have long considered reducing host infectiousness as an effective means to decrease disease transmission , 22. InfEmerging evidence suggests that infectivity can be, at least to some extent, under host genetic control , 29–33. In this Perspectives article, we (a) review existing evidence that cattle may genetically differ in their bTB infectivity, (b) outline data and methodology requirements for estimating genetic infectivity for bTB, (c) discuss the benefits from considering infectivity in genetic evaluations, and (d) identify key challenges and future research opportunities for incorporating infectivity, in addition to resistance, in cattle breeding programmes aiming to reduce bTB prevalence.M. bovis have been reported in various studies, but those have been mostly attributed to phenotypic variation rather than host genetics. For example, the number and frequency of episodes of shedding of M. bovis in cattle, were found to be dose- and infection route-dependent  genetically related individuals are spread over different epidemics (herds/breakdowns), (ii) individual epidemics occur in “closed” groups with minimum between-group transmission, and (iii) individual infection times differ, and are known or can be inferred. These requirements appear to be satisfied by bTB. The UK national bTB eradication scheme has generated systematic repeated records of SICCT test results for a large number of herds containing related animals. In addition, due to movement restrictions imposed on herds undergoing a breakdown, herds can be considered as closed groups during the breakdown period, and data collected can be used to infer infectivity. Although the exact time of cattle infection with  methods , 58.It remains to be tested with field bTB data, how various sources of uncertainty affect genetic infectivity estimates. For example, these methods assume knowledge of the true infection status of an individual, which raises the question whether SICCT and other monitoring records are appropriate for this purpose. Of these, SICCT is the most commonly available measurement but its relatively poor sensitivity is well documented, i.e., its ability to correctly identify infected individuals; published sensitivity estimates range from 26 to 91% –63. WhetM. bovis and hence reduce a major source of infection for the environment .bTB has been a seemingly intractable problem in the UK in recent decades and understanding how cattle genetics influences bTB spread is important for eradication. Under the hypothesis that some cattle infected with bTB are genetically more infectious than others, reducing the occurrence of cattle with higher genetic infectivity through selective breeding would (i) reduce bTB transmission between cattle by removing highly infectious individuals comprising a major risk factor for herds, (ii) reduce shedding of ironment  and wild0 , but also less likely to transmit bTB infection, if infected. Selection on breeding values for resistance and infectivity is expected to reduce the population R0 , 39, i.e0 , hence co−cattle , 68, whegligible , 69. InvA genetic-epidemiological simulation model can be used to assess the relative benefits of using a selection index that includes both resistance and infectivity compared to selecting on resistance alone. For this purpose we extended a stochastic epidemiological Susceptible-Latent-Infectious-Test sensitive model for bTB that originally assumed genetic variation only in resistance  with parThe epidemiological benefits and additional gain expected from adding infectivity to the breeding goal depends on its genetic correlation with other traits of economic interest. A classic example of the impact of adverse correlated responses, is the reduction of cattle fertility following selection on milk yield, due to adverse genetic correlation with milk yield . Based oIn principle, bTB surveillance schemes such as those implemented in the UK, RoI, and NZ, would permit researchers to pioneer the estimation of infectivity genetic effects without the need to collect new data. Current genetic evaluations for bTB resistance  use phenM. bovis, and obtaining more reliable disease phenotypes. Separating the effects of the infectious dose from host response is extremely challenging in field situations where exposure may not be uniform (M. bovis genome sequence information, can help to infer transmission routes and obtain information on the networks of who-infects-whom (Challenges arising in the analysis of bTB data to uncover genetic infectivity include accounting for multiple and poorly understood transmission routes of  uniform . Howevercts-whom , 75–78, cts-whom . More rects-whom , and by Host infectivity is an important trait for disease transmission and emerging evidence suggests that it may be under genetic control to some extent; however, the role of genetic infectivity of cattle in bTB spread remains to be explored. Infectivity might be difficult to capture from noisy field data; however, the UK bTB surveillance database and newly developed statistical methods provide the opportunity to estimate genetic effects for infectivity. Exploiting genetic variation in infectivity as a complementary bTB control method is a low-investment high-return approach, as it can be developed at minimal cost using data already available. Simulation studies suggest that breeding for both disease resistance and infectivity can complement and substantially enhance current disease control approaches toward bTB eradication. Using UK data to determine genetic regulation of disease transmission can create a platform for controlling bTB in other countries and for controlling other infectious diseases.ST, AA, RS, GB, JW, and AD-W conceived the perspectives. ST drafted the manuscript, designed and carried out the simulations and participated in the interpretation of findings. JW and AD-W contributed to the initial draft and simulation designs. All authors contributed to later versions of the manuscript, read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The transition from recreational drug use to compulsive drug‐seeking habits, the hallmark of addiction, has been shown to depend on a shift in the locus of control over behaviour from the ventral to the dorsolateral striatum. This process has hitherto been considered to depend on the aberrant engagement of dopamine‐dependent plasticity processes within neuronal networks. However, exposure to drugs of abuse also triggers cellular and molecular adaptations in astrocytes within the striatum which could potentially contribute to the intrastriatal transitions observed during the development of drug addiction. Pharmacological interventions aiming to restore the astrocytic mechanisms responsible for maintaining homeostatic glutamate concentrations in the nucleus accumbens, that are altered by chronic exposure to addictive drugs, abolish the propensity to relapse in both preclinical and, to a lesser extent, clinical studies. Exposure to drugs of abuse also alters the function of astrocytes in the dorsolateral striatum, wherein dopaminergic mechanisms control drug‐seeking habits, associated compulsivity and relapse. This suggests that drug‐induced alterations in the glutamatergic homeostasis maintained by astrocytes throughout the entire striatum may interact with dopaminergic mechanisms to promote aberrant plasticity processes that contribute to the maintenance of maladaptive drug‐seeking habits. Capitalising on growing evidence that astrocytes play a fundamental regulatory role in glutamate and dopamine transmission in the striatum, we present an innovative model of a quadripartite synaptic microenvironment within which astrocytes channel functional interactions between the dopaminergic and glutamatergic systems that may represent the primary striatal functional unit that undergoes drug‐induced adaptations eventually leading to addiction. Astrocytes may channel functional interactions between the dopaminergic and glutamatergic systems within a quadripartite synaptic microenvironment which may represent the primary striatal functional unit that undergoes drug‐induced adaptations that eventually lead to addiction. A1adenosine 1 receptorA2Aadenosine 2A receptorAcbnucleus accumbensAcbCnucleus accumbens coreAcbSnucleus accumbens shellAdenoadenosineaDLSanterior dorsolateral striatumA‐Oaction‐outcomeAstroastrocyteATPadenosine triphosphateB‐endbeta‐endorphin2+CacalciumCB1cannabinoid receptor 1CeNcentral nucleus amygdalaCNScentral nervous systemCOMTcatechol‐o‐methyltransferaseCSconditioned stimulusD1dopamine receptor D1D2dopamine receptor D2DAdopamineDATdopamine active transporterDLSdorsolateral striatumDORδ‐opioid receptorDRdopamine receptorDyndynorphineCBsendocannabinoidsEnkenkephalinGABAgamma‐aminobutyric acidGLASTglutamate aspartate transporterGLT‐1glutamate transporter 1GlutglutamateKORκ‐opioid receptorLgAlong accessLTDlong‐term depressionLTPlong‐term potentiationMAOmonoamine oxydasemGluRmetabotropic glutamate receptorMORμ‐opioid receptorMSN(s)medium spiny neuron(s)NACN‐acetylcysteinepDMSposterior dorsomedial striatumPFCprefrontal cortexPLprelimbic cortexShAshort accessVTAventral tegmental areaxCTcystine‐glutamate exchanger1Drugs of abuse induce a broad spectrum of central and systemic effects that vary depending on their mechanisms of action Nestler, . DespiteThe striatum is the main entry point of the basal ganglia, receiving glutamatergic inputs from the cortex following a topographical organisation in parallel loops Haber, . BroadlyThis cytoarchitectural landscape is highly contrasted between the ventral and dorsal territories of the striatum, which have different cortical and limbic inputs and functionally distinct roles , the most ventral territory of the striatum and long considered a “limbic‐motor interface”  that reliably predicts the outcome, the firing of these neurons shifts to the CS. Consequently, the activation of mesolimbic dopamine neurons has been suggested to convey a prediction error signal about the value of future outcomes Schultz, . Within These complementary roles of the striatum have been formalised into a putative model, wherein the Acb is considered the “Critic,” whereas the dorsal striatum is considered the “Actor,” mapping relationships between states and action propensities  dopamine‐dependent control over behaviour  via antecedent glutamatergic mechanisms in the AcbC  homeostasis  and reinstatement Kalivas,  which haThus, at the level of the synapse, upon an action potential reaching the presynaptic terminal, a sudden increase in calcium concentration triggers the release of glutamate into the synaptic cleft wherein glutamate binds to its postsynaptic ionotropic or metabotropic (mGluR1/5/6) receptors, thereby triggering depolarisation of the postsynaptic neuron and regulation of the function of that glutamatergic synapse respectively , the activation of which suppresses glutamate release by presynaptic terminals, thereby contributing to downregulate glutamatergic transmission   under continuous reinforcement and are subsequently subjected to several consecutive daily instrumental extinction sessions (often 12 days). Instrumental responding is then reinstated either by an injection of the drug (drug‐induced reinstatement) , NAC was shown to be mostly ineffective, except in a subset of individuals who had volitionally initiated abstinence at the onset of the trial  and orbitofrontal cortex  environment, including dopamine  expressed on astrocytes by adenosine released by neurons triggers astrocytic glutamate release (Li, Nomura, Aihara, & Nishizaki, in vitro and in vivo studies that astrocytes contribute to the function of dopaminergic synapses (Jennings & Rusakov, The pivotal role of astrocytes in glutamate homeostasis has been relatively well described. However, the contribution of these cells to dopamine homeostasis and its impact on glutamatergic transmission remains poorly understood, despite increasing evidence from both Apart from their role in dopamine clearance, cultured astrocytes have been shown to respond to dopamine, in that direct application of dopamine modulates cytosolic calcium signalling in astrocytes. These effects occur both in a receptor dependent manner, through D1/D2 receptors (Jennings et al., Collectively, these data suggest an important role of the DAT, the gateway for dopamine into astrocytes, in the coupling between dopaminergic synaptic activity and calcium signalling.Thus, investigation of the role of astrocytes in the regulation of striatal synaptic physiology should go beyond the prototypic tripartite glutamatergic synapse and should consider the striatal dopaminergic synapse in a quadripartite synaptic environment. A more complete understanding of this quadripartite system is paramount for our understanding of drug addiction. The contribution of dopamine signalling to the homeostasis and function of the striatal quadripartite synaptic microenvironment remains to be elucidated but could represent the cellular mechanism by which drugs of abuse, which aberrantly increase dopamine levels in the striatum, trigger long‐lasting alterations of glutamate homeostasis that span the entire striatum.Furthermore, endogenous systems, namely endocannabinoids, endogenous opiates and the adenosinergic system, have been shown to influence glutamatergic and dopaminergic homeostasis as well as the dynamic function of astrocytes in the striatum (for review see Fouyssac, Everitt, & Belin, 4The purpose of this review was to summarise experimental evidence in support of the involvement of astrocytes in the dysregulation of the glutamate homeostasis induced by exposure to drugs of abuse across the functional domains of the striatum and offer a broader mechanistic view of the striatal quadripartite synaptic microenvironment. The disruption of the homeostasis and function of this striatal microenvironment may contribute to the emergence of maladaptive drug‐seeking habits and ultimately compulsive behaviour, the hallmark of addiction. A better understanding of the neural, behavioural and psychological consequences of the restoration of glutamate levels by NAC both in preclinical and clinical models has helped shed a new light on the cellular mechanisms in the striatum that contribute to several facets of addiction. However, the drug‐induced alteration of glutamate homeostasis characterised by the downregulation of astrocytic xCT and GLT‐1 may only be the tip of the iceberg of the many adaptations these cells may undergo in response to drug exposure, including those associated with dopaminergic transmission. Further research is warranted better to understand the physiology of the quadripartite synaptic microenvironment which, we hypothesise, represents the functional unit of the striatum, and the extent to which interactions between dopaminergic and glutamatergic systems are channelled by astrocytes. The molecular landscape of the quadripartite synaptic microenvironment and its associated physiology might differ between individuals and such difference may represent molecular and cellular vulnerability factors which could account for the inter‐individual propensity to develop maladaptive drug seeking habits and eventually drug addiction (Anthony, Warner, & Kessler, The authors have no financial or potential conflicts of interest to declare.MF and DB have contributed equally to the writing of the manuscript. Click here for additional data file.


L-DOPA, L-3,4 dihydroxyphenylalanine), dopamine agonists  and MAO-B inhibitors , which can be used alone or in combination with each other. Potential risk factors include environmental toxins, drugs, pesticides, brain microtrauma, focal cerebrovascular damage, and genomic defects. This review covers molecules that might act as the biomarkers of PD. Then, PD risk factors (including genetics and non-genetic factors) and PD treatment options are discussed.Parkinson’s disease (PD) is a progressive neurodegenerative disorder caused mainly by lack of dopamine in the brain. Dopamine is a neurotransmitter involved in movement, motivation, memory, and other functions; its level is decreased in PD brain as a result of dopaminergic cell death. Dopamine loss in PD brain is a cause of motor deficiency and, possibly, a reason of the cognitive deficit observed in some PD patients. PD is mostly not recognized in its early stage because of a long latency between the first damage to dopaminergic cells and the onset of clinical symptoms. Therefore, it is very important to find reliable molecular biomarkers that can distinguish PD from other conditions, monitor its progression, or give an indication of a positive response to a therapeutic intervention. PD biomarkers can be subdivided into four main types: clinical, imaging, biochemical, and genetic. For a long time protein biomarkers, dopamine metabolites, amino acids, etc. in blood, serum, cerebrospinal liquid (CSF) were considered the most promising. Among the candidate biomarkers that have been tested, various forms of α-synuclein (α-syn), i.e., soluble, aggregated, post-translationally modified, etc. were considered potentially the most efficient. However, the encouraging recent results suggest that microRNA-based analysis may bring considerable progress, especially if it is combined with α-syn data. Another promising analysis is the advanced metabolite profiling of body fluids, called “metabolomics” which may uncover metabolic fingerprints specific for various stages of PD. Conventional pharmacological treatment of PD is based on the replacement of dopamine using dopamine precursors (levodopa,  Parkinson’s disease is the second most common neurodegenerative disease characterized by a progressive loss of dopaminergic neurons in the SN pars compacta. In PD, there is a long latency between the first damage to cells in at-risk nuclei of the nervous system, and the onset of clinical symptoms. The symptoms and signs of PD usually do not develop until 70–80% of dopaminergic neurons have already been lost . Thus, iOrexin, also known as hypocretin, is a neuropeptide hormone expressed by a small number of neurons of the dorsolateral hypothalamus. Orexin is secreted by the lateral and posterior neurons of the hypothalamus. The hormone regulates many physiological functions, such as the sleep-wake cycle , cardiov2-, H2O2, and⋅OH) can damage biological molecules basically through irreversible reactions causing degenerative processes associated with aging. One of the DNA lesions caused by ROS is an oxidized form of 8-hydroxyguanine (8-OHG) known as 8-OHdG which can be used as a biomarker of DNA damage (Reactive oxygen species (ROS) species  that is converted to dopamine by L-aromatic amino acid decarboxylase (AADC). In the brain, dopamine is used as the precursor of noradrenaline (norepinephrine) and adrenaline (epinephrine). Loss of dopaminergic neurons in the midbrain and SN of PD brains leads to the reduction of dopamine levels  as a valuable biomarker to distinguish several forms of neurodegenerative diseases. This biogenic amine and norepinephrine’s metabolite passes the BBB, and analysis of its level in serum and CSF may be helpful to determine cognitive staging in PD, distinguish PD from non-PD controls, DLB versus AD, etc. . This meIn another recent article a preclinical phase of PD is identified by analysis of dopamine metabolites in CSF. Low CSF concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and DOPA identify pre-clinical PD in at-risk healthy individuals .Catecholamine neurons are not abundant in the nervous system, and their vulnerability in PD and related diseases  is not eAccording to the “catecholaldehyde hypothesis” for the pathogenesis of PD, long-term increased build-up of DOPAL, the catecholaldehyde metabolite of dopamine, causes or contributes to the eventual death of dopaminergic neurons. LBs, a neuropathological hallmark of PD, contain precipitated α-syn. Bases for the tendency of α-syn to precipitate in the cytoplasm of catecholaminergic neurons have also been mysterious. Since DOPAL potently oligomerizes and aggregates α-syn, the catecholaldehyde hypothesis provides a link between synucleinopathy and catecholamine neuron loss in Lewy body diseases. The concept developed here is that DOPAL and α-syn are nodes in a complex nexus of interacting homeostatic systems.α-Synuclein, which is found in an aggregated and fibrillar form, has attracted considerable attention as a potential molecular biomarker of PD. . Human αApoA1 is a 28 AA apolipoprotein with ∼28 kDa molecular weight which is the main constituent of HDL particles . This aphttps://clinicaltrials.gov/ct2/results?cond=Parkinson$+$Disease&term=biomarker&cntry=&state=&city=&dist=.Recent investigation of microRNAs (miRNAs) in PD point to their emerging role as potential PD biomarkers, especially due to their presence in CSF and peripheral circulation both free and in exosomes. miRNAs are small 21–24 nucleotide non-coding RNAs that regulate gene expression post-transcriptionally. Due to their ability to cross the BBB miRNAs have a high potential as convenient PD biomarkers. In a recent study N-acetylaspartate/creatine ratios in advanced PD  and phosphorus (31P) magnetic resonance spectroscopy (MRS) are non-invasive imaging techniques that have been used to the study metabolites involved in energy metabolism, including ATP, lactate, creatine and other low molecular weight metabolite . Signifianced PD . Alteratanced PD .Today’s technology is able to detect brain’s abnormalities in PD patients using imaging techniques, such as transcranial B-mode sonography (TCS), susceptibility-weighted imaging (SWI), diffusion weighted imaging (DWI) , positroTranscranial B-mode sonography monitors the blood flow velocity of brain’s vessels by measuring the frequency of ultrasounds waves and their echoes. This inexpensive and reliable method shows the higher echogenicity of the SN in PD brains compared to normal group that possibly occurs due to increased iron and gliosis levels in SN of PD patients . The incDiffusion weighted imaging is a form of MRI that measures the rate of water diffusion through a tissue to determine the structural details of that tissue. The higher measured diffusivity means the greater mobility of water molecules that can be due to the death of cells and the reduction of the region volume. This technique can differentiate PD from multiple system atrophy (MSA) in the early stage, while the clinical symptoms of these disorders are similar. In particular, the higher diffusivity of water in middle cerebellar peduncles in MSA patients in comparison to PD patients has been reported using DWI . DWI canBoth PET and SPECT scans can detect the early onset of PD and loss of dopaminergic neurons using radiotracers and computer techniques to generate 3D images. The majority of radiotracers are non-invasive radiopharmaceuticals with a short lifetime that usually decay soon after the imaging is complete. Moreover, the 3D images of PET and SPECT scans reveal function of an organ, whereas MRI can only monitor the anatomy and structure .123iodine (123I) and 99mtechnetium (99mTc) that emit gamma rays. The DAT-SPECT imaging can monitor degeneration of presynaptic terminals in dopaminergic neurons by visualizing DAT quantity. This method is a good way to diagnose reduction of DAT in the brain, and importantly it can’t distinguish PD and other Parkinsonian Syndromes. The DAT gamma-emitting ligands, such as 123I-iometopane (123I-β-CIT), 123I-ioflupane (123I-FP-CIT), 123I-altropane (123I-IPT) are the most common DAT-density SPECT tracers. These ligands are derivatives of tropane and dopamine reuptake inhibitors that target DAT (The SPECT scan radiotracers have a longer life-time in comparison to PET radiotracers. They mostly are rget DAT .123I-iodobenzamide (123I-IBZM) (123I-IBF (123I-epidepride (123I-2′-iodospiperone (2′-ISP) is also used in some studies to monitor D2 dopamine receptors. Although this radiotracer can distinguish between PD and other form of parkinsonism due to the pattern of its uptake in basal ganglia, it produces a high imaging background and needs to be modified to improve its performance  , 123I-IB123I-IBF , and 123idepride . 123I-2′formance .123I-iodobenzovesamicol (123I-IBVM) binds to VAChT and reveals the density of acetylcholine containing vesicles. Reduction of VAChT in parietal and occipital lobes in PD patients without dementia and reduced VAChT in all lobes of the cerebral cortex in PD patients with dementia has been established by this SPECT radiotracer (The number of vesicular acetylcholine transporter (VAChT) can be monitored by SPECT radiotracers as an approach for PD early diagnosis. Acetylcholine (ACh) is a neurotransmitter that is in balance with dopamine in healthy people. The death of dopaminergic neurons and reduction of dopamine levels in PD are associated with increased Ach. iotracer .123I-metaiodobenzylguanidine (123I-MIBG) is another radiotracer that can distinguish between PD and MSA   (11C-MP4A is another PET radiotracer that monitors the level of acetylcholinesterase (AChE) activity. AChE hydrolyses deactivates Ach and terminates the signal. Impairment of cholinergic system and reduction of cortical AChE has been assessed by 11C-MP4A-PET scan. AChE activity reduces more in PDD than in PD, indicating that cholinergic dysfunction is correlated with dementia in PD (The PET scan radiotracers emit electron anti-particles (positrons) that are positively charged with the same mass as an electron. The presence of presynaptic DAT in dopaminergic neurons of striatum and SN can be assessed with 8F-dopa) , 18F-FE--FE-PE2I , 18F-β-C8F-β-CFT , 18F-LBTF-LBT999 , and11C-e (DTBZ) . Becauseia in PD .The development of neuroprotective drugs for PD is an important unmet medical need, since this disease progressively impair the patients’ quality of life and functionality in activities of daily living. The identification of new therapeutic targets is therefore of great importance. Although different medications and therapies for controlling PD symptoms are currently available, no cure for PD exists. The development of treatments for PD, based on patients’ symptoms and needs, vary from different medications to rehabilitation or even surgery. PD includes different clinical entities observed in several studies investigating the existence of PD subtypes. A cluster analysis permits to identify distinct PD subtypes according to the relevance of both motor and non-motor symptoms and select therapeutic approach according to cluster symptoms presentation .L-dopa, L-3,4-dihydroxyphenylalanine) is an effective drug. Levodopa is the immediate metabolic precursor of dopamine which is produced by TH from L-tyrosine. In the dopaminergic neurons, dopa-decarboxylase converts levodopa into dopamine. The orally taken levodopa can be decarboxylated in peripheral sites before reaching the CNS. Therefore, levodopa is available in combination with carbidopa or benserazide that are peripheral inhibitors of Dopa decarboxylase, but do not pass through the BBB. Unchanged levodopa in the presence of decarboxylase peripheral inhibitors can penetrate into the CNS and is used as a precursor of dopamine  . Formatidopamine .L-DOPA therapy and reduces its efficiency as a drug  are available as alternative PD medications. These two medications block the conversion of levodopa into methylated levodopa. Therefore, inhibition of COMT activity by these medications can extend the existence of functional levodopa preventing its degradation  . The MAOradation .Additionally, some other chemicals can be considered as potential PD medicines. Rapamycin can be a useful treatment for PD as an up-regulator of autophagy. Rapamycin induces autophagy in cells by inhibition of a specific kinase activity called mTOR. Autophagy is a potential target for PD treatment, since it initiates the clearance of protein aggregates and inhibits apoptosis . AdenosiFormation of protein aggregates that leads to neuronal cell death is a promising target for PD treatment. In January 2015, Neuropore Therapies has announced phase I clinical trial of a new drug, NPT200-11 (UCB-1332) that inhibits oligomerization of α-syn . AnotherDeep brain stimulation therapy is rarely used for certain types of brain-related disorders including PD, dystonia, obsessive-compulsive disorder and treatment resistant depression . When PDL-DOPA to dopamine. After successful preclinical studies, adeno-associated viral vectors carrying human AADC gene are recently delivered into putaminal neurons and subthalamic nucleus of PD patients. In this method, sufficient amount of dopamine production can be controlled by taking adequate levodopa dose. Orally taken levodopa can be converted into dopamine by AADC and sooth PD symptoms (The development of gene therapy of PD has made a major progress in a recent decade. Advanced PD does not give a good response to levodopa therapy. Broaden loss of dopaminergic neurons is accompanied by reduction in aromatic amino acid decarboxylase (AADC) levels that converts symptoms . Safety symptoms . Anothersymptoms . Lack ofsymptoms . The humsymptoms . Therapesymptoms . Therefosymptoms . The humsymptoms . Injectesymptoms .in vitro into 3,4-dihydroxylphenyl-acetaldehyde (DOPAL). DOPAL initiates oligomerization of α-syn into non-fibrillar, SDS-resistant aggregates  – an enzyme involved in metabolizing environmental toxins, are also related to the development of PD. For example, CYP2D6 deficient metabolizers are two times higher at risk of developing PD if they are also exposed to pesticides. Therefore, sufficient levels of CYP2D6 activity are required for the metabolism of pesticides , which are linked to the pathogenesis of PD .Reactive oxygen species and thus oxidative stress is also known as a pathological factor in PD. NADPH oxidase-2 enzyme (NOX2) is a membrane-bound oxidase present primarily in phagocytes generating ROS in phagosomes to kill bacteria. NOX2-derived ROS also damages dopaminergic neurons. Dopaminergic neurons in NOX2-knockout mice start to degenerate faster than similar cells in wild-type controls after administration of MPTP .In 1956 two neurologists, Poskanzer and Schwab hypothesized that PD is related to influenza infection . They stTables 2). The mutations in several genes, including α-syn, LRRK2, PINK1, Parkin, DJ-1, VPS35 and GBA1 are linked to PD (Table 3).Although most cases of PD are idiopathic forms of the disease, about 15% of PD patients are recognized as having a first-degree family member with this disease. Recently, the genetic factors and gene loci involving in autosomal dominant and autosomal recessive forms of PD have been discovered due to advanced molecular genetics  , PARK16, transmembrane glycoprotein NMB (GPNMB), and syntaxin-1B (STX1B) genes. Hypomethylation of the TNF-α promoter, hypermethylation of a CpG dinucleotide in synphilin-1, STX1B and hypermethylation of multiple CpG sites proximal to GPNMB are all described in PD . MoreovePathology of PD is complex and include a combination of genetics, epigenetics and environmental factors. Current medical, pathological, and experimental data support the Braak hypothesis  of spatiFE designed the article contents and wrote the original manuscript. AS revised and edited the manuscript, searched for additional related literature and discussed the writing with FE.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.