Meloidogyne javanica, were recovered. Morphological and morphometric studies were carried on the characters of females, males, J2s and perineal patterns. Species-specific Sequence Characterized Amplified Region (SCAR) primers confirmed morphological studies, and all these populations produced specific band in 670 bp using Fjav and Rjav primers. Genetic diversity of different populations was studied by Inter Simple Sequence Repeats (ISSR) and Random Amplified Polymorphic DNA (RAPD) markers implementing 10 primers for each approach. Results revealed a relatively low genetic diversity . The analyses of molecular variance indicated that the variation resulted from genotypic variations within region and variances among regions are 81% and 19% for RAPD, and 86% and 14% for ISSR, respectively. On the other hand, FST and Nm values are 0.140 and 1.535 for ISSR while these values are 0.188 and 1.079 for RAPD. So it can be concluded that there is a great deal of gene flow between populations due to the movement of plant material contaminated with nematodes, which results in high mixing between populations. ISSR and RAPD datasets failed to group populations according to their geographic region. There were no pathotypes or other intraspecific biological entities observed in the species.In order to investigate different species of root-knot nematodes associated with vegetable production in southern regions of Iran, 37 populations of the most predominant species,  Meloidogyne , which their population increase to damaging levels within a few seasons under susceptible crops, are so common in subtropical and tropical vegetable production that frequently they are taken to represent “nematodes” in general . The populations of M. incognita  Chitwood, 1949 and M. javanica  Chitwood, 1949 were the most recovered populations (more than 80%) from vegetable fields worldwide . Five species including M. arenaria  Chitwood, 1949, M. cruciani Garcia-Martinez, 1982, M. hapla Chitwood, 1949, M. incognita and M. javanica are the recovered RKN in vegetable production in Iran  of the genus  in Iran .Considering that morphological and morphometrical methods for the identification species of RKNs are considerably time consuming and there is a lot of ambiguity, using species-specific primers could be a rapid method for detection  , the sma species .M. enterolobii  with a random sequence and markers are considered to be uniformly distributed along the genome, whereas ISSR primers is based on the amplification of regions flanked by repeating sequences which found only between microsatellite loci. In the other hands, annealing temperature in RAPD is lower than ISSR .M. javanica collected from major vegetable growing areas in southern regions of Iran, using RAPD and ISSR markers.The aim of the present study is to infer the genetic intraspecific diversity among populations of M. javanica were collected from vegetables  and weeds of vegetable fields in several regions of southern Iran, during 2018 and 2019  and 4 μl proteinase K (600 μg/ml) were added, respectively. The samples were incubated at 65°C for 1 hr and at 95°C for 10 min. After incubation, the tubes were centrifuged for 2 min at 38,000 g and kept at –20°C for next uses.One full egg mass was put in 16 μl ddHPCR was performed containing 1 μl of DNA template, 8 μl mastermix (Amplicon Red), 1 μl of each primer (10 pmol) and 4 μl ddH2O to a final volume of 15 μl. Four set species-specific primers  were useM. javanica, 54°C for M. incognita, 61°C for M. arenaria, and 58°C for M. hapla) and 45 sec at 72°C, followed by a final step of 5 min at 72°C. All steps were repeated twice to insure the results. PCR success was visualized loading 4 μl of the PCR product into a 1.7% TBE buffered agarose gel  and visualized with ethidium bromide.The PCR amplification profile consisted of 5 min at 94°C, 35 cycles of 30 sec at 94°C, 30 sec at annealing temperature (64°C for 2O. Ten primers including (CCA)5, (GTG)6, (ATG)6, (GAG)4GC, (GACA)4, (GA)8C, (AC)8T, (CA)8G, (CTC)4GC, and (GAGA)4GG , and 8 μl ddHGAGA)4GG  were impGAGA)4GG .PCR ampliﬁcation reactions were programmed for an initial denaturation at 94°C for 3 min, followed by 35 cycles of 30 sec at 93°C, 90 sec at 48°C, 1 min at 72°C, and a ﬁnal extension of 10 min at 72°C. PCR success was visualized by loading 7 μl of the PCR product into a 1.7% TBE buffered agarose gel .2O. Ten primers , and 8 μl ddH primers  were use primers .For RAPD amplifications, the thermos-cycle profile was 5 min at 94°C; 35 cycles of 1 min at 94°C, 1 min at 35°C and 2 min at 72°C, followed by a final step of 5 min at 72°C. PCR success was visualized loading 7 μl of the PCR product into a 1.7% TBE buffered agarose gel .FST (Fixation index) and Nm (Number of migrants) used to calculate the differences of populations and amount of gene flow between populations. These two indices are inversely correlated, and by increasing fixation index, Nm is decreased  software package . The genecreased .M. javanica, M. incognita, M. arenaria, and M. hapla are 670 bp, 1200 bp, 420 bp, and 960 bp, respectively. The species-specific primers of M. javanica (Fjav and Rjav) yielded one unique fragment of approximately 670 bp in 37 populations, and no PCR product was obtained in the control template  for the four major species . Expectea region . Morphom5 primer of ISSR and OPAD10 primer of RAPD is presented in With the 10 random primers used in the RAPD analysis, a total of 180 bands were produced. All primers resulted in ampliﬁcation and different patterns were tested for population differences. The resulting gel electrophoresis for 19 populations for (CCA)M. javanica populations. The populations of Abdan and Bardkhon exhibited the highest level of genetic diversity , whereas the lowest value was shown in the populations of Jiroft . In 164 loci, 19.08% were polymorphic in all individuals of studied populations. The mean genetic variations of total indicated by H and I, were 0.080 and 0.115, respectively.In ISSR, a total of 164 loci, with average of 16.4 loci per primer, were scored. P, H, and I were used to estimate genetic diversity within the M. javanica populations with RAPD analysis were 0.043 to 0.162 (The Nei’s (1978) genetic distances (D) between pairs of to 0.162 . The lowto 0.162  and an iM. javanica populations according to their geographical region and host plants. The UPGMA trees generated using Dice coefficient for RAPD and ISSR are presented in The UPGMA trees obtained with RAPD and ISSR data showed some differences in tree topologies between calculations based on Dice and Jaccard. The positions of some populations are different by ISSR and RAPD, although the number of clades was consistent as both methods revealed four clades. None of these clades grouped FST and Nm for RAPD are 0.188 (P = 0.010) 1.079 however for ISSR are 0.140 (P = 0.010) and 1.535.In analyses of molecular variance analysis, percentages of molecular variance within and among populations were 11.36% and 2.62% for RAPD, and 8.26% and 1.33% for ISSR, respectively . FST andM. javanica was the most predominant species of RKN infesting vegetable fields and greenhouses in southern Iran. Although there was a slight genetic variation at the intraspecific level, populations of M. javanica generally showed a low level of genetic diversity. In the present study, surveyed Meloidogyne species were characterized using morphological and morphometric characters and confirmed with species-specific SCAR-PCR. The genetic diversity among populations of M. javanica was further explored by ISSR and RAPD markers.This is the first comprehensive study of RKN species in southern regions of Iran which demonstrated that M. enterolobii, M. javanica, and M. incognita was still challenging using various molecular techniques, except for SCAR-PCR , and it seems that these two markers may work independently  from each other. The PCR-based techniques using RAPD and ISSR markers were shown to be helpful for obtaining detailed information about the diversity that exists among M. javanica populations. Perhaps including more populations from distant geographic regions, and even different countries, provide better understanding of the genetic structure and gene flow of these nematodes. Further intra- and inter-specific genetic diversity of M. javanica may be explored by implementing next-generation sequencing (NGS) techniques.In this paper, the species-specific SCAR primer technique proved to be a powerful tool to distinguish the populations of the two widely distributed RKNs (


The World Health Organization (WHO) recently classified Enterobacteriaceae resistance to third-generation cephalosporin into the group of pathogens with critical criteria for future research.E. coli (CREc) from a South African wastewater treatment plant (WWTP) was conducted using standard phenotypic and molecular biology characterization methods.A study to assess the antibiogram and beta-lactamase genes among the cefotaxime resistant E. coli (TEc) concentration (log10 CFU/mL) ranged between 5.7 and 6.8 among which cefotaxime resistant E. coli were between 1.8 and 4.8 (log10 CFU/mL) for cefotaxime antibiotic concentration of 4 and 8 mg/L in the influent samples. Effluent samples, heavily influenced by the chlorination had only 0.3 log10 CFU/mL of TEc. Fifty-one cefotaxime resistant isolates were selected out of an overall of 75 isolates, and subjected to a new round of testing, with a follow up of 36 and 48 isolates for both colistin and gentamicin, respectively as guided by initial results. Selected CREc exhibited resistance to amoxicillin-clavulanic acid , colistin sulphate , ciprofloxacin , gentamicin  and intermediate-resistance to meropenem . Extended spectrum-beta-lactamase genes detected, viz.: blaCTX-M  and blaTEM  and concurrent blaCTX-M + blaTEM , but no blaSHV was detected. Carbapenem resistance genes, blaKPC-2 , blaOXA-1 , blaNDM-1  were also detected. Approximately, 10.5 - 36.8% (n = 38) co-occurrence of two or more beta-lactamase genes was detected in some isolates. Out of the selected number (n = 30), 7(23.3%) were enterotoxigenic E. coli (ETEC), 14 (46.7%) were Enteroaggregative E. coli (EAEC), but no enteropathogenic E. coli (EPEC) was detected.Approximate total Resistance to cefotaxime and the presence of a wide range of beta-lactamase genes exposed the potential risks associated with these pathogens via occupational and domestic exposure during the reuse of treated wastewater. Escherichia coli has been accepted as both a biological indicator of water contamination and a human pathogen implicated in both systemic  and superficial infections (skin or wound infection) [E. coli is part of the normal flora of the gastrointestinal tract, shed along with faecal waste also including release by infected and convalescent individuals through poorly managed wastewater [E. coli released depends partly on the antibiotic susceptibility pattern of the organism, as this determines the clinical treatment option(s) available [fection) –6. E. costewater , 8. The vailable . In specvailable  inferredvailable –13, as svailable . Koopmanvailable  reportedE. coli (and other members of the Enterobacteriaceae) to cefotaxime has been categorized to be of a critical threat to the public health by the World Health Organization, WHO [On the global level, the resistance of ion, WHO , for whiion, WHO , 16.blaCTX-M [blaCTX-M may also contain blaTEM and/or blaSHV, especially among clinical isolates [blaCTX-M gene bearing Enterobacteriaceae isolates while Korzeniewska and Harnisz [. D’Andrea et al. [blaCTX-M as the primary basis of resistance to the third-generation cephalosporins in E. coli isolated from clinical and environmental samples. In Nicaragua, blaCTX-M genes were detected among ESBL-producing E. coli from hospital wastewater samples [Resistance to cefotaxime  is often due to production of enzymes like extended-spectrum beta-lactamases, ESBLs encoded by the beta-lactamase genes, such as blaCTX-M –19. The isolates , 21. If isolates  showed t Harnisz  reported samples .E. coli with these ESBLs genes, their threat becomes more critical if they also possess carbapenem resistance genes, blaVIM, blaOXA-1, blaKPC-2, blaNDM-1, etc. [E. coli with carbapenem resistance and carbapenem resistance genes in the treated hospital effluent possesses potential epidemiological threat globally, as the bacteria may initiate difficult to treat infection among the exposed groups [E. coli and other bacteria originating from different sources interact within the wastewater matrix and may modify bacterial ecosystems therein [E. coli.For cefotaxime resistant -1, etc. . Carbaped groups –27. Trand groups , 29. Excd groups . E. coli therein , dependiE. coli in the effluent that could constitute a threat. The aim of this study, therefore, was to assess and enumerate the cefotaxime resistant E. coli in influent and effluent samples of a wastewater treatment plant in Durban, South Africa and the corresponding sensitivity pattern to other selected antibiotics, as well as the presence of ESBL genes and other selected beta-lactamase genes in them.Some reports inferred that the conditions in WWTPs give room for the interaction of antibiotic-resistant bacteria and the exchange of mobile genetic elements –32. Surv3 containing industrial, municipal and hospital inflow. The total influent Chemical Oxygen Demand (COD) was 250–1100 mg/L and the total effluent COD was 75 mg/L. Other WWTP information are tabulated in Table The sampling was done in a WWTP, treating municipal wastewater from Durban, Kwazulu Natal Province, South Africa in February (summer) and August (winter), 2017. The WWTP receives inflow in the range of 12,000–14,000 mIn five sampling rounds representing different seasons both pre-grated influent and final (post-chlorinated) effluent samples were collected. The rationale for collecting pre-grated samples was to have the samples in the exact forms it entered the WWTP. To have the samples in the form in which the effluents released to the recipient water, post-chlorinated final effluent samples were collected. The samples transported in a cold chain analyzed within 12 h of collection.Quantification of the bacteria was done using standard membrane filtration. Membrane Fecal Coliform (mFC) agar  purchased from Quantum Biotech, South Africa was used as the medium for quantification. The media was prepared with 1% rosolic acid  purchased from Hach International. Ten-fold serial dilutions representative for the influent and effluent ranges of concentration were prepared using sterilized saline solution (0.85% (w/v) NaCl). The prepared samples were filtered through 0.2 μm cellulose acetate filters  in triplicates. The filters were placed on the mFC agar plates supplemented with 4 mg/L or 8 mg/L cefotaxime antibiotic  and on agar plates without antibiotic supplements in line with UNESCO regulations for another ongoing project in our laboratory. Though 4 mg/L was optimized to be adequate for satisfactory results, continuation of both 4 mg/L and 8 mg/L throughout was to see potential variance over a while.E. coli forms blue colonies. Grey and/or pink colonies might not be coliforms. The selectivity of the media was determined by estimating the ratio of blue and total colonies.The resulting culture was incubated at 37 °C for 24 h. Enumeration of the total number of colonies depended on observation of specific colonial colorations . E. coli colonies of ≤24 h were suspended in 200 μL aliquots of distilled water. The suspension containing the isolates was mixed by vortexing until a mixture with thoroughly dissipated isolates formed. This was boiled at 95 °C for 15 min and subsequently centrifuged at 15000 rpm; at 4 °C for 15 min where after, the supernatant was removed and stored at − 20 °C for further assays.Isolation of the DNA was done by the boiling method using the protocols of Salehi et al. . The youE. coli was done using the primers  specific for a conserved region of E. coli alanine racemase  gene specified in Table PCR based species-specific identification of E. coli WG5 was used as a positive control.The cycling conditions for the PCR reaction started with an initial 6 min denaturation step at 95 °C, followed by 35 cycles containing denaturation at 95 °C for 20s, primer annealing/extension at 72 °C for 1 min 30 s, and a final extension for 5 min at 72 °C. Agarose gel electrophoresis was performed at 80 V for 45 min in 2% agarose gel in Tris Acetate-EDTA (TAE) buffer, stained with Sybr Safe DNA gel stain .E. coli was performed following standards described by Cheesebrough [Phenotypic antibiotic susceptibility pattern of the selected and identified cefotaxime-resistant sebrough  and Clinsebrough . Fifty-oMultiple antibiotic resistance index (MAR Index) was determined per isolate as the ratio of the number of antibiotics to which an isolate showed resistance (X) to the total number of antibiotics against which the isolate was tested (Y) –40.\docThis was determined on all the isolates concerning all the antibiotics used.blaCTX, blaTEM and blaSHV  buffer, stained with Sybr Safe DNA gel stain .blaVIM, blaOXA-1, blaKPC-2, blaNDM-1 using the primers listed in Table blaVIM, the cycling conditions included the initial denaturation at 94 °C for 3 min, followed by a stage of 36 cycles containing denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 1 min and a final extension at 72 °C for 5 min. PCR cycling conditions for blaOXA-1 began with initial denaturation at 95 °C for 2 min, followed by a stage of 30 cycles containing denaturation at 94 °C for 45 s, annealing at 55 °C for 30 s and extension at 72 °C at 1 min. This was followed by a final extension stage at 72 °C for 5 min.Singleplex PCR was performed for the detection of blaKPC-2 was done with cycling conditions that included an initial denaturation at 95 °C for 5 min, followed by a stage of 40 cycles containing denaturation at 95 °C for 15 s, annealing at 66.1 °C for 30 S, extension at 72 °C for the 30 S and final extension at 72 °C for 10 min [blaNDM-1, the cycling conditions began with denaturation at 95 °C for 15 min, followed by a stage of 30 cycles containing 95 °C for 1 min denaturation, 61.1 °C for 1 min annealing, and extension for the 30 S at 72 °C. This was followed by a final extension for 10 min at 72 °C [The detection of r 10 min . For blaat 72 °C .E. coli were further pathotyped to determine which strains they were, using the primers listed in Table E. coli pathotypes, the thermal cycling conditions for Enteroaggregative E. coli (EAEC), Enteropathogenic E. coli (EPEC) and Enterotoxigenic E. coli (ETEC)  were as follows: the initial denaturation/enzyme activation step at 95 °C for 15 min, followed by 35 cycle consisting of denaturing at 94 °C for 45 s, annealing at 55 °C for 45 s, extension at 68 °C for 2 min and the final elongation at 72 °C for 5 min [Isolated and identified or 5 min . The cycor 5 min  while thE. coli and carbapenemase positive E. coli were determined using the chi-square test at p value < 0.05 (significant). The test of significance for seasonal variation (p < 0.05) was done using GraphPad software, version 5.01.Statistical analysis was performed for descriptive statistics. Plating of the samples was done in triplicates. The values were plotted using Box and Whiskers, Microsoft Office Excel 2016  which automatically calculated and depicted the standard deviations (SDs) and the spread of data. Differences in antibiotic resistance between ESBL positive E. coli were resident in the wastewater. A very high mean total E. coli count of over 6 log10 CFU/mL was found in the influent sample  did not show reduction in the cefotaxime resistant E. coli counts beyond what lower concentration showed in a slight variation.Large counts of cefotaxime-resistant E. coli count (log10 CFU/mL) ranged from 5.7 to 6.8, with cefotaxime resistant E. coli count (log10 CFU/mL) ranging between 1.8 to 4.9 for cefotaxime concentration of 4 mg/L and 2.3 to 2.8 at 8 mg/L. The influent counts (log10 CFU/mL) for the total E. coli count (NA) as well as cefotaxime-resistant E. coli (A4 and A8) for all the summer samples  are presented in Fig. 10 CFU/mL) that ranged from about 6.4 to 6.6, with one wide outlier of count 0, plotted separately from the range 6.4–6.6. The cefotaxime (4 mg/L) resistant coliforms counts (log10 CFU/mL) ranged from 0 to 4.7 while those for 8 mg/L ranged from about 2.6 to 2.9, presented in Fig. An approximate total count log0 CFU/mL count log0 CFU/mL count log0 CFU/mL p < 0.05) with an approximate total E. coli count (log10 CFU/mL) in winter that ranged from 5.2 to 6.6, cefotaxime-resistant E. coli count (log10 CFU/mL) ranged from 2.5 to 4.8 at concentration of 4 mg/L and 1.0 to 5.2 at concentration of 8 mg/L. The trend was also similar for other coliforms with a total count (log10 CFU/mL) that ranged from about 4.7 to 6.3; cefotaxime (4 mg/L) resistant coliforms counts (log10 CFU/mL) ranged from 2.3 to 3.5 and cefotaxime (8 mg/L) resistant coliforms counts (log10 CFU/mL) ranged from 0 to 2.9. The winter counts of E. coli and the other coliforms are represented in Fig. Seasonal variation was not significant (count log0 CFU/mL E. coli growth and rarely very low growth (range in log10 CFU/mL = 0 to 0.3) in the effluent concentration  however showed some appreciable counts (log10 CFU/mL) that ranged from 0 to 3.6 as in Fig. E. coli and coliform growth. Almost all the cefotaxime-supplemented plates for effluent showed no growth. The count of 1 CFU/mL in very few cases was estimated as 0 log10.Meanwhile, there was consistently no total and cefotaxime-resistant E. coli to additional antibiotics is presented in Table E. coli also showed 35.3% resistance to amoxicillin-clavulanic acid, 76.5% to colistin sulphate, 47.1% to ciprofloxacin, and 87.5% to Gentamicin.The results of the antibiotic susceptibility testing from cefotaxime resistant E. coli isolates had a MAR index above 0.2. The corresponding figures were 41.2% with a MAR index of 0.5 and 29.4% with an index of 0.75  of the E. coli occurred. For extended-spectrum beta-lactamase (ESBL) genes, blaCTX-M was detected in 52.6% of the isolates and blaTEM in 84.2%, while no blaSHV was detected. Both blaCTX-M and blaTEM were concurrently detected among 36.8% of the E. coli isolate. Carbapenem resistance gene, blaKPC-2 was detected among 15.8% of the isolates, blaOXA-1 among 57.9% of the E. coli isolates, while blaNDM − 1 occurred in 15.8% of the E. coli isolates. No blaVIM was detected. Carbapenem-resistance genes, blaKPC-2 and blaOXA-1 were found in 15.8% of the isolates. blaNDM-1 and blaOXA-1 were also concurrently detected in 10.5%. Both ESBLs genes and carbapenemase genes were concurrently detected in some isolates, with the highest being blaTEM and blaOXA-1 simultaneously detected in 52.6% of the isolates. Fewer isolates showed the presence of the putative genes for carbapenemases than ESBLs. In addition, there was statistically significant difference (p < 0.05) between most carbapenem-resistance genes  and ESBL genes (blaCTX-M and blaTEM). The details of the beta-lactamase genes are displayed in Table The presence of various beta-lactamase genes among the cefotaxime-resistant n = 30) of the selected isolates were confirmed as pathogenic; 7 (23.3%) were heat-stable (ST) enterotoxigenic E. coli (ETEC), 14 (46.7%) were Enteroaggregative E. coli (EAEC), but no enteropathogenic E. coli (EPEC) and enteroinvasive E. coli (EIEC) were detected. Figure The results of the pathotyping showed that 70% n = 0 of the E. coli and determining antibiogram to other antibiotics, as well as the presence of beta-lactamase genes in the Gram-negative bacteria from a WWTP from Durban, South Africa. This aim is in line with the critical criteria placed on Enterobacteriaceae resistant to third-generation cephalosporin and carbapenem antibiotics by WHO [E. coli to the environment through wastewater effluents remains an issue of public health concern, depending on downstream human exposure. A large number of total E. coli count (log10 CFU/mL) that ranged from 5.7 to 6.8 or cefotaxime-resistant E. coli as depicted in Fig. E. coli) is expected in influent since E. coli is a normal part of the gut flora [E. coli) might also be due to wastewater from hospital inflow. These resistance attributes are expected from the clinical origin [E. coli might be a warning sign of probable lack of pretreatment for the hospital outflow.We conducted this study with the aim of quantifying the cefotaxime-resistant s by WHO . The discount log0 CFU/mL l origin , 21, 46 l origin , the preE. coli and cefotaxime-resistant E. coli (range in log10 CFU/mL = 0 to 0.3)  that ranged from 0 to 3.6. This is possible because transformation only requires a competent cell to pick up resistance genes in the environment, even if the donor is from another genus [The effluent concentration of both total  the new . These cer genus , 49.E. coli. Fluoroquinolone resistant isolates of E. coli from sewage had also been reported by Colomer-Lluch et al. [E. coli is synonymous with having a 12.5% chance of therapeutic failure or total resistance, in line with the position of WHO [Enterobacteriaceae around the world and resistance to colistin itself is directly linked with the agricultural use of human antibiotics [Though many cefotaxime-resistant Gram-negative bacteria are already recognized as critical threats , their fh et al. . The corh et al. . A figurn of WHO . The coln of WHO . Resistan of WHO . Colistiibiotics . The resibiotics .E. coli exhibited multiple antibiotic indices (MAR Index) > 0.2 which means that the isolates might have originated from high risk source (s), where antibiotics are probably abused [The discharge of the antibiotic-resistant bacteria from WWTPs to the environment is reported widely , 58–60. y abused , 40, 59.y abused , the MARy abused .E. coli strains from individuals without history of colistin usage [E. coli from a boy without any record of prior administration of colistin but rather probable zoonotic exposure from the family’s pigs. This is why animal or infected human feces like in waste and wastewater are considerable hotspots [E. coli isolated from the sampled WWTP can be difficult to treat in an individual if contracted via occupational exposure.The high resistance to colistin sulphate (76.5%) detected in this study is considered alarming. Over the past few years now, attention around the world has been especially focused on the epidemiology of resistance to colistin in bacteria. Researchers link the emergence of colistin resistance to prior exposure to the sublethal concentration by the bacteria, but studies have reported the isolation of colistin-resistant in usage . In a stin usage  isolatedhotspots , 65. Colhotspots . So, infblaTEM, blaCTX-M, blaSHV, blaVIM, blaOXA-1, blaKPC-2 and blaNDM-1, which might be responsible for some detected resistance to the cefotaxime and other beta-lactam antibiotics [blaVIM was not detected, blaOXA-1, blaKPC-2 and blaNDM-1 might be responsible for intermediate resistance to meropenem by the cefotaxime-resistant E. coli [blaCTX-M, blaSHV and blaTEM have also been reported in both hospital and municipal wastewater effluents [Several antibiotic resistance determinants were detected in this study. These include ibiotics . Though  E. coli , 68. Sevffluents , 69. Theffluents , since tffluents . ConcurrE. coli and K. pneumoniae. Most rampant putative ESBLs’ genes were blaCTX-M genes in which the similar gene types were detected in both clinical and environmental isolates [E. coli (MDR-Ec), have been isolated from a healthy human [Ec is sometimes shed in feces released into WWTP through the user interface. Intraspecific and interspecific transfer of resistance gene was expected to be high in the WWTP due to high bacterial density. This transfer may include rampant blaCTX-M .Reports in other studies , 72 showisolates , 74. Mulhy human  and the n = 30) of the selected isolates were confirmed as pathogenic due to their pathotypes (Fig. Since 70% n = 0 of the E. coli and other coliforms but the effluent contained only a very scanty concentration of total E. coli and other coliforms. Some of the cefotaxime resistant E. coli also showed resistance to amoxicillin-clavulanic acid, colistin, meropenem, ciprofloxacin, gentamicin, trimethoprim-sulphamethoxazole, nitrofurantoin and tetracycline. The presence of cefotaxime-resistant and indeed multidrug-resistant Enterobacteriaceae in wastewater influent showed that these organisms, classified with critical threats, were possibly harbored in the population. These, cefotaxime-resistant E. coli, were pathogenic as revealed by their pathotypes. These resistant isolates bore extended-spectrum beta-lactamase genes and carbapenem resistance genes singly and concurrently, which make them of potential clinical origin. It is imperative for hospitals to pretreat their wastewater effluent to avoid the critical threat associated with these kinds of bacteria to public health, in case of leakage and human exposure.The influent samples in the WWTP contained a large number of Cefotaxime-resistant


Inhibition was assessed with the sustained attention to response task (SART). This paradigm also allows to examine the effect of different degrees of response automatization, which is a known modulator for response inhibition, but does not seem to be substantially impaired during acute intoxication. As a neurophysiological correlate of response inhibition and control, we quantified EEG-derived theta band power and located its source using beamforming analyses. We found that alcohol-induced response inhibition deficits only occurred in the case of response automatization. This was reflected by decreased theta band activity in the left supplementary motor area (SMA), which may reflect modulations in the encoding of a surprise signal in response to inhibition cues. However, we did not find that differences in baseline  GABA or glutamate levels significantly modulated differences in the size of alcohol-induced inhibition deficits.Alcohol increases GABAergic signaling and decreases glutamatergic signaling in the brain. Variations in these neurotransmitter levels may modulate/predict executive functioning. Matching this, strong impairments of response inhibition are one of the most consistently reported cognitive/behavioral effects of acute alcohol intoxication. However, it has never been investigated whether baseline differences in these neurotransmitters allow to predict how much alcohol intoxication impairs response inhibition, and whether this is reflected in neurophysiological measures of cognitive control. We used MR spectroscopy to assess baseline  GABA and glutamate levels in the anterior cingulate cortex (ACC) and striatum in  The aberrant consumption of alcohol is a significant cause of physical, mental, and social problems . Acute aSevere cognitive deficits are consistently found in the domain of inhibitory control, which is highly important to control  drinking, especially in the face of negative consequences . While rRegarding underlying neurophysiological processes, theta frequency oscillations play an important role ,17,18,19However, it is important to consider that theta oscillations reflecting cognitive control are closely related to gamma-aminobutyric acid (GABA) and glutamate levels in the medial frontal cortex  and the In short, we hypothesized that the effect of acute alcohol intoxication on response inhibition depends on the degree of response automatization. As a consequence, we expected increased alcohol-induced impairments in inhibitory control with an increased degree of response automatization. We further hypothesized that these deficits might be caused by an alcohol-induced increase in GABA and decrease in glutamate levels in the striatum and ACC. If this was the case, inter-individual differences in striatal and ACC GABA and glutamate should correlate with the magnitude of the alcohol-induced inhibition deficit. Lastly, we examined whether the investigated effects were reflected in the theta band and associated activity modulations in medial frontal structures.n = 39 healthy young male subjects between 18 and 30 years of age participated in the study. The sample size was based on an earlier study by Quetscher et al. [A group of r et al. , who tesr et al. , and repr et al. . Before The study design and the administration of alcohol were identical to the protocol used in previous intoxication studies carried out by our group ,9,52. WeWe administered individual amounts of alcohol, aiming for an average peak breath alcohol concentration (BrAC) of ~1.2‰. The amount of vodka  that each participant received was individually determined based on their estimated total body water (TBW). The Excel sheet which we used for the calculation of individual beverage amounts can be found in the Each participant was given 1.98 g of alcohol per estimated liter of total body water. The alcohol was administered in the form of equal parts of vodka (40 vol. %) and orange juice. Both were measured and mixed at room temperature to minimize the risk of stomach aches. Irrespective of the individual amount that was served, all participants were asked to finish their drink within 30 min, and to wait another 30 min until the start of the experiment. In this time, the participants watched thee episodes of The Big Bang Theory in order to provide standardized entertainment that lacked extensive discourse on drinking and party-related topics and was intended to helped to prevent negative mood swings, as well as differential effects of the engagement in conversation with the experimenters ,56. AfteThe sustained attention to response (SART) task allows to investigate the effect of different degrees of response automatization on inhibitory control processes. The task was originally introduced by Robertson et al.  and had All participants were seated at distance of about 57 cm from a 17 inch thin-film transistor (TFT) monitor and were asked to respond using a QWERTZ keyboard. In this GO/NOGO type task, the digits “1” to “9” were presented in a random order, with one digit shown in each trial. They were displayed in white font with randomly varied font size . Each trial started with a 250 ms presentation of a single digit. The digit was followed by a mask (a circle with a cross inside). The inter-trial interval randomly varied between 1100 and 1600 ms. Participants were asked to rest their right index finger on the response button (space bar) and react to any digit except “3” by quickly pressing the button. Whenever the digit “3” was presented, the participants had to inhibit this response. The task was divided into three equally sized blocks.In the first block, there were eight times more GO trials than NOGO trials , so the participants likely had a strong automatic response tendency within this block. In the second block, the amount of GO trials and NOGO trials was equal , so the participants likely had a weaker response tendency than in the first block. In the third block, there were eight times more NOGO trials than GO trials , so the participants likely had no pronounced automatic response tendency within this block. Each block consisted of 260 trials. All stimuli (digits) representing GO trials were presented equally frequently. Altogether, it took about 25 min to complete the task.The concentrations of GABA+ in the striatum and the ACC were examined using 1H-MR-spectroscopy (MRS). MRI and MRS data were acquired using a 3T “Prisma” scanner  and a 32 channel (receive only) headcoil. Subsequent to the localizer, a high-resolution 3D T1-weighted sagittal Magnetization Prepared RApid Gradient Echo (MPRAGE) sequence (1 mm isovoxel) was measured and multiplanar reconstructed for exact voxel placements. Next, 30 × 30 × 30 mm voxels of interest (VOIs) were placed in the left as well as the right striatum to be able to average out laterality effects, and a 20 × 20 × 60 mms VOI was placed over the midline to cover both the left and right ACC. The positioning of the VOIs is shown and described in Mescher-Garwood point-resolved spectroscopy (CMRR-MEGAPRESS) sequence  developed by Edward J. Auerbach and Małgorzata Marjańska [To improve spectral resolution, a special shim adjustment based on Gradient Echo (GRE) Double Acquisition  was performed, followed by manual shimming, to achieve a full width at half maximum (FWHM) value below 15 Hz. MRS data were acquired using the Center for Magnetic Resonance Research arjańska ,65 and parjańska , which farjańska  and genearjańska ,70 with arjańska ). Referearjańska . This waEEG data recording followed the standard protocol applied by our group. We used an EEG cap with 60 Ag-AgCl electrodes at standard equidistant scalp positions. Electrode impedances were kept below 5 kΩ, and the data was recorded against a reference electrode at position Fpz, using a QuickAmp amplifier . The data were initially recorded at a sampling rate of 500 Hz. Brain Vision Analyzer 2.1. software  was used for offline data pre-processing and EEG data analyses. During this process, the data were downsampled to 256 Hz and a band-pass filter (infinite impulse response filter from 0.5 to 20 Hz with a slope of 48 db/oct) was applied. Technical or irregular movement artifacts were then eliminated with the help of a manual raw data inspection. An independent component analysis  was then applied to remove periodically recurring artifacts like eye movements or pulse artifacts. Lastly, a second manual raw data inspection was conducted to remove any residual artifacts that might have prevailed in the EEG data up to this point. In the next step, EEG data were segmented in a target-locked fashion. Each segment started 2000 ms before and ended 2000 ms after target stimulus onset (set to time point zero) to ensure having enough full cycles at theta frequency. Only correctly answered trials were included in the analyses. Additionally, an automated artifact rejection procedure was applied. Here, we excluded segments with amplitudes below −100 and above 100 μV. The maximally allowed value difference in a 200 ms interval was set to 200 μV and the lowest acceptable amplitude difference in a 100 ms time span was set to 0.5 μV. Furthermore, we eliminated the reference potential by conducting a current source density (CSD) transformation. In addition to serving as a spatial filter, CSD transformation may improve the identification of electrodes that best reflect ERP peaks and associated cognitive processes ,73. A baσf = width of Gaussian shape in the frequency domain; f0 = central frequency) was set to f0/σf = 5.5. To calculate the total power, TF decomposition was separately applied to the single-trial data of each experimental condition. For the power band quantification, we focused on central theta oscillations, which are known to play an important role in executive control functions [t statistics, samples were considered as candidate members of a cluster of samples if their t value exceeded 0.05. By means of the Monte Carlo method, the reference distribution of the permutation test was approximated using 2000 random draws. A cluster was considered significant if its p value was below the critical alpha level of p < 0.025. Within the theta band, we quantified the power at a frequency of 5 Hz in the time frame from 300 ms to 350 ms at electrode Cz, where the theta power reached its maximum.Time–frequency (TF) decomposition was achieved using a continuous wavelet transform (CWT) that applies Morlet wavelets to different frequencies in the time domain. For this study, the TF decomposition was performed in 0.5 Hz intervals from 0.5 to 20 Hz. The Morlet parameter, which is used to plot the time–frequency  comprising the within-subject factors “intoxication status” (sober vs. intoxicated), “automatization block” (block 1 vs. block 2 vs. block 3), and “condition” (GO vs. NOGO). All performed tests were Greenhouse–Geisser-corrected, whenever necessary. In the results section, the mean and standard errors of the mean (SEM) are reported as a measure of variance. MRS data were integrated with the behavioral and neurophysiological data by means of correlation ,63,83,84n = 39 participants were excluded for the following reasons: N = 3 participants had to discontinue the experiment before the paradigm could be run due to vomiting after alcohol administration . N = 2 participants had to be excluded due to technical issues . N = 2 participants were excluded for slow responding (mean RT of more than 600 ms during the sober appointment). N = 1 participant stopped pressing the response button after a while on one of the appointments. This was most likely due to an accidental shift on the keyboard resulting in responding with an incorrect button (which was not recorded). Lastly, n = 1 participant secretly watched movies on his smartphone during the experiment . On average, the n = 30 participants who remained in the sample were 23.43 ± 0.717 years old (range 18–30), had a height of 181.6 ± 1.3 cm (range 169–195), a weight of 76.8 ± 1.9 kg (range 56–100), and an AUDIT score of 6.8 ± 0.6 (range 3–14).After collecting the data, nine participants from the initial sample of n = 16 participants received alcohol on their second appointment. Based on the estimated total body water in liters, the participants received an average amount of 281.63 ± 4.22 mL vodka, or 90.12 ± 1.35 gr alcohol. Immediately before performing the experimental paradigm , the participants had a mean BrAC of 1.01‰ ± 0.02. After completing the paradigm, their mean BrAC was 0.93‰ ± 0.03.N = 14 participants received alcohol on the first of their two study appointments, while the remaining F = 24.246, p < 0.001, F = 111.321, p < 0.001, F = 92.065, p < 0.001, t-tests showed significant differences for all possible contrasts , with a higher number of hits in block 3 (98.12% ± 0.40) than in block 2 (89.38% ± 1.18) or in block 1 (81.71% ± 1.76). Furthermore, there were significant interactions of intoxication status × condition  = 7.848, p = 0.009, F = 7.656, p = 0.003, F = 85.180, p < 0.001, F = 6.829, p = 0.007, F = 16.183, p < 0.001, F = 93.875, p < 0.001, F = 8.308, p = 0.003, t(29) = 3.211; p = 0.003; intoxicated = 60.37% ± 4.11; sober = 71.85% ± 3.14) and in block 2 (t(29) = 4.430; p < 0.001; intoxicated = 76.77% ± 2.67; sober = 84.69% ± 1.78). However, no such detrimental alcohol effect could be observed in block 3 (t(29) < 0.001; p > 0.999). Moreover, the intoxication effect (sober minus intoxicated) was significantly smaller in block 3 (<0.01% ± 0.30) than in either block 1 (11.48% ± 3.58) (t(29) = 3.347; p = 0.002) or block 2 (7.92% ± 1.79) (t(29) = 4.816; p < 0.001). The alcohol effect size did not differ between block 1 and block 2 (t(29) = 1.102; p < 0.280). In GO trials, there was only a main effect of intoxication status  = 8.924, p = 0.006, F ≤ 1.099; p ≥ 0.332). In short, we found detrimental effects of alcohol on response inhibition to be present in case of response automatization, and to be absent when there was no response automatization. But while the intoxication effect appeared bigger in case of stronger automatization (block 1 vs. block 2), this effect did not reach significance.The analysis of accuracy (percentage of hits) revealed a main effect of intoxication status (223) see . Post-hoF = 32.164, p < 0.001, F = 105.999, p < 0.001, t-tests showed significant differences for all possible contrasts , with slower responses in block 3 (522.1 ms ± 8.1), than in block 2 (412.7 ms ± 14.1) or in block 1 (401.1 ms ± 14.0). Moreover, there was a significant interaction of intoxication status × automatization block  = 6.171, p < 0.004, t-tests showed significant differences between the intoxicated and the sober appointment for all three blocks . Most importantly, the alcohol effect (intoxicated minus sober) was significantly larger in block 3 (64.5 ms ± 8.4) than in block 1 (32.8 ms ± 9.6) (t(29) = 2.583; p = 0.015) or block 2 (25.5 ms ± 11.5) (t(29) = 3.074; p = 0.005). The alcohol effect size did not differ between block 1 and block 2 (t(29) = 0.707; p > 0.485).The repeated measures ANOVA for RTs in correct GO trials only showed a main effect of intoxication status (175) see . Post-hoWavelet plots illustrating the target-locked time–frequency decomposition of alcohol effects on the EEG signal are given in F = 6.072, p = 0.020, 2 ± 26.67) than in the intoxicated state (194.56 μV/m2 ± 16.93). Furthermore, we found a significant main effect of condition  = 29.164, p < 0.001, 2 ± 23.16) than for GO trials (194.89 μV/m2 ± 18.47). There was also a significant main effect of automatization block  = 31.631, p < 0.001, t-tests showed significant differences for all possible contrasts , with a lower theta band activity in block 3 (173.04 μV/m2 ± 16.32), than in block 2 (224.54 μV/m2 ± 22.15) or in block 1 (250.20 μV/m2 ± 24.73). Additionally, there were significant interactions of intoxication status × condition  = 6.608, p = 0.016, F = 7.711, p = 0.004, F = 27.977, p < 0.001, F = 8.544, p = 0.001, F = 6.871, p = 0.014, 2 ± 17.96; sober state = 268.96 μV/m2 ± 32.38) and automatization block  = 35.768, p < 0.001, 2 ± 34.39; block 2 = 243.85 μV/m2 ± 25.56; block 3 = 149.66 μV/m2 ± 12.98), as well as an interaction of intoxication status × automatization block  = 10.242, p = 0.002, t(29) = 2.958; p = 0.006; sober = 387.80 μV/m2 ± 54.94; intoxicated = 246.96 μV/m2 ± 21.93) and in block 2 (t(29) = 2.649; p = 0.013; sober = 274.92 μV/m2 ± 32.96; intoxicated = 212.77 μV/m2 ± 22.25), but not in block 3 (t(29) = 1.118; p = 0.273). Additionally, the intoxication effect (sober minus intoxicated) significantly differed between all automatization blocks , with the smallest effect in block 3 (-11.03 μV/m2 ± 9.86), a medium effect in block 2 (62.16 μV/m2 ± 23.46), and the largest alcohol effect in block 1 (140.84 μV/m2 ± 47.61). Beamforming analyses revealed that the alcohol-induced decrease in theta band activity in block 1 and block 2 was related to a stronger activation of the left supplementary motor area (SMA peak MNI coordinates in mm: 41/73/72) in the sober state (as compared to the intoxicated state) in both automatization blocks . We quantified and analyzed theta frequency band power at 5 Hz and at electrode Cz in a time frame from 300 to 350 ms. There was a main effect of intoxication status (228) see . To analocks see . In the p ≥ 0.333), glutamate levels , or the GABA/glutamate ratio . Given that none of the correlations between neurotransmitter levels and behavioral measures reached significance, we decided to refrain from correlating neurophysiological measures with MRS data, as potential results could only have demonstrated behaviorally irrelevant epiphenomena. In order to investigate whether any of the MRS-assessed transmitter levels correlated with the magnitude of alcohol-induced cognitive deficits, we performed correlation analyses. In order to reduce the chances of identifying false positive effects or mere epiphenomena, we only entered behavioral difference values  as behavioral measures of inhibition that had demonstrated to be significantly impaired by alcohol intoxication . Importantly, none of these behavioral measures correlated significantly with any of the MRS-assessed GABA levels , but was absent when there was no response automatization (block 3). This alcohol-induced inhibition impairment was reflected by decreased theta band activity in the left SMA, which is functionally relevant for the execution of the right hand GO responses. Importantly, we could exclude a speed/accuracy tradeoff, as the intoxicated participants did not only commit more false alarms, but also responded more slowly when intoxicated. However, neither baseline GABA levels nor baseline glutamate levels in the striatum and ACC predicted behaviorally relevant alcohol effects on inhibition, as we found no significant correlations for blocks 1 and 2.Acute alcohol intoxication is known to cause physical and mental harm. Drunk individuals commonly show reduced inhibitory control ,85, wherTo investigate this, we applied a task which allowed to examine the effect of different degrees of response automatization on inhibitory control processes. Reproducing previous publications, we found that an acute, binge-like alcohol intoxication of 1.01‰ significantly impaired (response) inhibition ,9. More n = 14 binge drinkers and did not find this in non-binge drinkers, or investigate the acute effects of alcohol. While this requires further replication, it is hence possible that even though fronto-striatal GABA and glutamate play a key role in cognitive functioning, as well as in mediating the cognitive effects of alcohol intoxication, transmitter levels assessed at a sober baseline do not allow prediction of the degree of impairment caused by a standardized binge.Matching the behavioral results of impaired inhibitory control, we found alcohol-induced reductions in theta band power on the neurophysiological level. Further matching the modulation by response automatization, detrimental effects of alcohol on theta band power were evident when responses were automatized, and were absent when there was no response automatization. Theta band activity is important for cognitive control, especially for response inhibition ,22,23,24n = 9 participants from the initial sample because of various reasons. Therefore, the current study employed a relatively small sample size. As a result, it will also be important to replicate these results with a sample that is larger in size.Most importantly, however, it should be stressed that this lack of effect does not in any ways imply that response inhibition deficits are not partly, or even mainly, caused by alcohol-induced changes in fronto-striatal GABA and glutamate signaling. It only states that it likely not possible to predict the degree of alcohol-induced reductions in response inhibition on the basis of transmitter levels at a sober baseline measurement. In this context, it also needs to be mentioned that while the ACC VOI barely covered any other brain structures apart from the region we intended to assess, the 3 × 3 × 3 cm VOI used for striatal GABA/glutamate quantification inevitably also contained structures that were not closely related to our research question. Indeed, such a large voxel size is currently required to obtain a good signal that allows for reliable quantification of GABA+ levels , and comIn summary, we investigated whether and how the degree of response automatization and inter-individual differences in sober fronto-striatal GABA/glutamate levels modulate the effect of acute alcohol intoxication on inhibitory control. We found response automatization to be a prerequisite for finding alcohol-induced inhibition impairments. This was reflected by decreased theta band activity in the left SMA, suggesting this effect to be due to modulations in the encoding of inhibition-enhancing surprise signals. However, we could not predict the degree of alcohol-induced inhibition decline on the basis of sober neurotransmitter levels. This clearly does not imply, however, that alcohol-associated cognitive deficits cannot be induced by alcohol effects on GABAergic and glutamatergic signaling.


Saccharomyces cerevisiae P29, a sparkling wine strain, during the second fermentation under two conditions (without and with endogenous CO2 overpressure) in the middle and the end of second fermentation. Consequently, a proteomic analysis with the OFFGEL fractionator and protein identification with LTQ Orbitrap XL coupled to HPLC were performed. The classification of biological processes was carried out using the tools provided by the Saccharomyces Genome Database. Results indicate that a greater number of biological processes were identified under condition without CO2 overpressure and in the middle of the fermentation versus the end of the second fermentation. The biological processes highlighted under condition without CO2 overpressure in the middle of the fermentation were involved in the carbohydrate and lipid metabolic processes and catabolic and biosynthetic processes. However, under CO2 overpressure, specific protein expression in response to stress, transport, translation, and chromosome organization and specific processes were not found. At the end of fermentation, there were higher specific processes under condition without CO2 overpressure; most were related to cell division, growth, biosynthetic process, and gene transcription resulting in increased cell viability in this condition. Under CO2 overpressure condition, the most representative processes were related to translation as tRNA metabolic process, chromosome organization, mRNA processing, ribosome biogenesis, and ribonucleoprotein complex assembly, probably in response to the stress caused by the hard fermentation conditions. Therefore, a broader knowledge of the adaptation of the yeast, and its behavior under typical conditions to produce sparkling wine, might improve and favor the wine industry and the selection of yeast for obtaining a high-quality wine.Sparkling wines elaboration has been studied by several research groups, but this is the first report on analysis of biological processes according to the Gene Ontology terms (GO terms) and related to proteins expressed by yeast cells during the second fermentation of sparkling wines. This work provides a comprehensive study of the most relevant biological processes in  Champenoise, method consists of two consecutive fermentations and a long period of aging of the wine in contact with the yeast lees. A first fermentation occurs, where the base wine is obtained from grape must, and a second fermentation of the base wine mixed with sugar and specific yeast strains of Saccharomyces occurs in a sealed bottle. After the second fermentation, the wine is subjected to a long aging period in contact with the yeast lees for at least 9 months  at low temperatures (12‒16 °C). During this last stage, the organoleptic properties of wine are influenced by the release of certain secondary metabolites, such as glycerol, acetate, succinate, pyruvate, and esters [2 overpressure during the second fermentation in the production of sparkling wines. Regarding volatilome and its relationship with the organoleptic properties of the final wine, CO2 overpressure affected the concentration of certain aromatic compounds, such as ethyl dodecanoate, ethyl tetradecanoate, hexyl acetate, ethyl butanoate, and ethyl isobutanoate [2 overpressure [2 overpressure seems to affect the yeast stress-related proteome during the second fermentation. In fact, according to these authors, proteins involved in osmotic stress, such as glycerol biosynthesis and others required for energy storage mechanisms during nutrient starvation, stand out under second fermentation conditions [The production of sparkling wine by the traditional, or eratures 2‒16 °C. d esters ,4. Numerd esters ,6,7,8,9.utanoate . Once yeutanoate ,12. Duriutanoate . On the pressure . Penachopressure  showed tpressure . Furthernditions .2 overpressure, released by Saccharomyces cerevisiae P29 strain, on the proteins related to the GO terms of sparkling wines elaborated according to the Champenoise method. This work conducts a global study about the most relevant biological processes in S. cerevisiae P29, a sparkling wine strain, under two conditions: a control condition without pressure, or P (−), and a pressure condition, or P (+), at the middle (MF) and the end (EF) of fermentation. We designed a proteomic analysis based on an OFFGEL fractionation, and later protein identification (LTQ Orbitrap XL mass spectrometer coupled to HPLC) and selection using the Gene Ontology tools from the Saccharomyces Genome Database (SGD). Finally, the GO Term Mapping tool was used to determine the most significant biological processes and pathways found under both conditions of this study. Broader knowledge about the adaptation of the yeast and its behavior under the typical conditions of the second fermentation would improve the industrial process, and favor the selection and genetic improvement of wine yeast strains capable of carrying out this process, giving rise to a wine of quality.Since there are few researches focused on analysis of biological processes according to the Gene Ontology (GO) terms under fermentation conditions, the present work aims to study the effect of endogenous COSaccharomyces cerevisiae was used. This is a typical sparkling wine strain for secondary fermentation in a closed bottle in the Cava production. It was isolated from the designation of origin (DO) of the Penedès, Barcelona (Spain) [For this work, the P29 strain (CECT 11770) of  (Spain) .v/v) was reached, yeast cells were introduced into the bottles together with the commercial base wine composed of Macabeo:Chardonnay (6:4), an ethanol content of 10.21% (v/v), 22 g/L sucrose, and 1.5 × 106 cells/mL. The second fermentation was carried out in a thermostat chamber at 14 °C in bottles with a volume of 750 mL.Yeast strain was incubated for 5 days at 21 °C, using gentle agitation of 100 rpm for growth in a pasteurized must of the Macabeo grape variety . When an ethanol content of 10.39% ). The rest of the bottles were hermetically sealed with a shutter and with a metal crown capsule, this causes the CO2 released during the second fermentation to be trapped inside the bottle, with pressure condition (P (+)). During the second fermentation in the sparkling wine process, two sample times were taken: (a) at the middle of the second fermentation (MF) (pressure of 3 bar); and (b) at the end of the second fermentation (EF) (pressure of 6.5 bar) under CO2 overpressure condition. At the same time, when a similar consumed sugar and produced ethanol content was obtained under both conditions (sugar consumption (MF: 9.07 ± 0.26 g/L and EF: 0.3 ± 0.0 g/L) and ethanol content (MF: 10.7 ± 0.03% v/v and EF: 11.56 ± 0.04% v/v)), samples were taken from the condition without CO2 pressure. The bottles were divided into two groups for the study of COv/v ethanol, 9.9 ± 0.5 g/L reducing sugars, 0.2 ± 0.0 g/L volatile acidity, 5.2 ± 0.1 g/L total acidity, 3.28 ± 0.03 pH, 1.87 ± 0.06 g/L malic acid, 0.1 ± 0.0 g/L lactic acid; (b) under EFP(−): 11.52 ± 0.03% v/v ethanol, 0.3 ± 0.0 g/L reducing sugars, 0.24 ± 0.03 g/L volatile acidity, 5.1 ± 0.02 g/L total acidity, 3.28 ± 0.01 pH, 1.90 ± 0.06 g/L malic acid, 0.1 ± 0.0 g/L lactic acid; (c) under MFP(+): 10.85 ± 0.04% v/v ethanol, 8.23 ± 0.06 g/L reducing sugars, 0.2 ± 0.0 g/L volatile acidity, 5.2 ± 0.0 g/L total acidity, 3.28 ± 0.01 pH, 1.87 ± 0.06 g/L malic acid, 0.1 ± 0.0 g/L lactic acid; (d) under EFP(+): 11.56 ± 0.04% v/v ethanol, 0.3 ± 0.0 g/L reducing sugars, 0.23 ± 0.02 g/L volatile acidity, 5.25 ± 0.02 g/L total acidity, 3.3 ± 0.02 pH, 1.89 ± 0.06 g/L malic acid, 0.1 ± 0.0 g/L lactic acid.The wine compositions at the different sampling times were: (a) under MFP (−): 10.64 ± 0.02% The cellular viability counting was carried out using appropriate dilutions with Ringer’s solution. Then, these were plated in Sabouraud agar medium for 48 h at 28 °C. All samples were carried out in triplicate. Data are recorded as means (± standard deviation) CFU (colony-forming units)/mL.g for 10 min by a centrifuge , washed the sediment twice with sterile distilled cold water and broken through a mechanical technique in Vibrogen, using the Cell Mill V6  with 500 µm diameter glass balls. Once the cells were broken, protein extraction was performed. For this purpose, a protease inhibitor cocktail and an extraction buffer composed of 100 mM Tris-HCl (pH 8), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 2 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride (PMSF) were used. A total of 500 µg of protein was loaded into the well tray of the protein fractionator. This concentration was calculated by a Bradford test [The cells were collected from all bottles by centrifugation at 4500× ord test . Then, tord test .Saccharomyces Genome Database (SGD), using the GO Slim Mapper tool.To carry out a more reliable selection of the total proteins, the following selection criteria were taken into account, according to Dasari et al. : (i) scohttps://www.yeastgenome.org/). This classification was made using the GO Term Finder tool, provided by the database. The GO Terms are descriptive terms that allow the relating of each gene product with a molecular, cellular, and biological process context, providing a statistical value . This statistical study was performed with a level of significance (α) of 0.01.The proteins involved in each biological process were classified in different GO Terms provided by the SGD database  and 594 under MFP (+); 542 of them were common under both conditions  and 52 under MFP (+)). On the other hand, 392 proteins were identified under EFP (−) and 419 under EFP (+), with 268 proteins being common between both conditions  and 150 under EFP (+)) .The biological processes obtained in each type of condition and sampling times were elucidated from the GO term of biological processes and pathways. Gene Ontology is a tool that describes how and where gene products work in biological systems. It is structured in three interrelated parts that describe what genetic products do at the biochemical level and at the cellular level, and the general biological objectives to which their actions contribute .2 overpressure condition. This difference could be because, in high-stress situations, the yeast cells only carry on those biological processes that are fundamental to viability and cell maintenance. These results agree with those from Matallana et al. [Saccharomyces yeasts.A total of 57 biological processes were obtained in the middle of the second fermentation . While 1a et al. , which a6 (±2.1 × 106) CFU/mL, (10.64 ± 0.02% v/v ethanol) and under CO2 overpressure 8.02 × 106 (±1.4 × 106) CFU/mL , no significant differences were observed [2 overpressure would not affect cell viability, despite being subjected to stress.On the other hand, there were 18 specific processes under MFP (−); those processes that showed a higher protein frequency were related to metabolism, and more specifically to the metabolism of lipids and carbohydrates . Both processes were related to a series of GO Terms , and to obtaining energy . Unlike yeasts under CO2 overpressure condition, they dedicate most of their energy to their survival. Processes that were differentially up-regulated (relative frequency above twofold) under MFP (+) versus MFP (−) samples were as follows:chromosome organization, translation, nucleocytoplasmic transport, response to stress, ribosome biogenesis, anatomical structure formation involved in morphogenesis, cell differentiation, chromosome segregation, membrane organization, DNA metabolic process, homeostatic process, protein targeting, aging, anatomical structure development, and cellular protein modification process. These processes are related to cell division , morphogenesis  and gene transcription . In view of these processes under MFP (+), yeast metabolism under CO2 overpressure conditions and its cell growth are not affected, since it counteracts not having specific processes for them by presenting a high frequency of proteins in the common processes mentioned above. However, the yeast could be stressed by CO2 pressure, since a stress response under MFP (+) higher than MFP (−) has been detected.With the aim of contrasting this hypothesis, the common biological processes at the middle of the second fermentation were identified (39 processes), and the most representative common biological processes under MFP (−) versus MFP (+) were eight processes . In view of these results, the yeast could have a more active metabolism under conditions without CO2 overpressure (EFP (−)), while under condition of CO2 overpressure (EFP (+)) 42 biological processes were identified at the end of the second fermentation : 1.14 × 106 (±0.4 × 106) CFU/mL (11.52 ± 0.03% v/v ethanol); EFP (+): 3.33 × 104 (±1.2 × 104) CFU/mL (11.56 ± 0.04% v/v ethanol). This fact may be explained since, as described above, in EFP (+), yeast autolysis may be taking place, which would help the survival of the rest of the population in this hard condition. On the other hand, the GO Terms related to the specific biological processes in EFP (−) were developmental process, intracellular copper ion transport, and oxidoreduction coenzyme metabolic process  were aging (4.03%), cell division (2.42%), cell morphogenesis (1.61%), cytoskeleton-dependent intracellular transport (0.81%), growth (2.42%), and protein maturation (2.42%). In both conditions, due to the scarcity of the carbon source (glucose), the yeast reorganizes its metabolism during the stationary phase ,30. The  process . They pror yeast ,32. Accos et al.  reported2 overpressure (EFP (−)) processes which stood out were biosynthetic process, cellular amino acid metabolic process, small molecule metabolic process, carbohydrate metabolic process, cofactor metabolic process, generation of precursor metabolites and energy, signal transduction, homeostatic process, cell death, cell wall organization or biogenesis, cellular protein modification process, and lipid metabolic process.In view of the results obtained, most of these common processes in EFP (−) are involved in maintaining the redox balance caused by oxidative stress. For this purpose, the yeast could be synthesizing amino acids, such as methionine or tryptophan. These amino acids are described in studies carried out by Peláez-Soto et al. [2 overpressure versus without CO2 overpressure, and the majority of these processes are related to gene translation . The most significant GO Terms obtained for these processes coincide with the biological processes previously mentioned. The biogenesis of ribosomes  and protein translation  stood out. Presumably, the yeast could be synthesizing stress response proteins, and is more likely to respond to stress due to CO2 overpressure, since the conditions of ethanol and glucose did not show significant differences between conditions .Regarding common biological processes, under condition without COo et al.  as cellup-value: 0.00441), DNA packaging , DNA-templated transcriptional start site selection , and transcriptional start site selection at RNA polymerase II promoter . These results suggest that yeast could be activating its transcription and translation machinery to try to cope with cell death and autolysis. Jing et al. [S. cerevisiae, this process is triggered by starvation conditions, and is considered an adaptive response, allowing the survival of younger yeast cells, due to the recycling of the compounds released into the medium by dead yeasts [The specific biological processes under EFP (+) were tRNA metabolic process and chromosome organization, which presented a frequency of 1.99% and 3.97%, respectively. Four GO Terms have been related to these two specific biological processes . These Gg et al.  reportedd yeasts . 2 overpressure was identified. However, at the end of the second fermentation, two specific processes took place under CO2 overpressure, versus six specific processes under conditions without CO2 overpressure. Most of the prominent processes were related to cell division, growth, and biosynthetic process, presumably for the maintenance of good cell viability observed without CO2 overpressure condition. At the end of fermentation, it was clear that the overpressure affects viability, since there were a smaller number of living cells in the second fermentation under this condition. However, under CO2 overpressure condition, processes were related to tRNA metabolic process, chromosome organization, and translation.This work focuses on providing a first approach to an understanding of the biological processes that take place in yeasts during the second fermentation in the production of sparkling wines through a proteomic study. This knowledge arises from the need to select yeasts capable of carrying out the second fermentation, and of tolerating various stresses to which they are subjected. We believe that stress tolerance is a good criterion for selecting enologically interesting yeasts. Despite obtaining a greater number of biological processes in the middle of the second fermentation, no specific process of the condition with COThese results represent a first approach that highlights the most relevant biological processes that take place during the second fermentation in bottle in the production of Spanish sparkling wine (Cava). Furthermore, they couldbe a starting point for future research to validate some of the possible mechanisms that take place during the second fermentation in bottle.A better understanding of yeast metabolism, its adaptation, and its behavior under the typical conditions of the second fermentation is necessary for the strain selection process and improvement of its application in the wine industry, and can be interesting for quality control and improvement of the sparkling winemaking process.


In this paper, a UPLC-MS/MS method was developed for the determination of ropivacaine and its metabolite 3-hydroxy ropivacaine in cerebrospinal fluid. The cerebrospinal fluid was processed by ethyl acetate liquid-liquid extraction. The multiple reaction monitoring (MRM) mode was used for quantitative analysis by monitoring the transitions of m/z 275.3 → 126.2 for ropivacaine, m/z 291.0 → 126.0 for 3-hydroxy ropivacaine, and m/z 290.2 → 198.2 for the internal standard. Standard curves for ropivacaine and 3-hydroxy ropivacaine in cerebrospinal fluid were conducted over the concentration range of 0.2–2000 ng/mL, demonstrating excellent linearity, and the lower limit of quantification was 0.2 ng/mL. The intraday precision of ropivacaine and 3-hydroxy ropivacaine was less than 11%, while the interday precision was less than 7%. The accuracy ranged between 87% and 107%, the average extraction efficiency was higher than 79%, and the matrix effect was between 89% and 98%. The developed method was then applied to a case of suspected poisoning of ropivacaine. S-(–)-enantiomer, is a long-acting amide local anesthetic that can provide different effects at different doses [t1/2's are 14 min and 4 h, respectively. During bilateral intercostal nerve blocks, its absorption into the blood is faster than with epidural injection. Ropivacaine is mainly metabolized in the liver, and its metabolites have a weak local anesthetic effect [Ropivacaine, which exists as the pure nt doses . At a cont doses , 3. The nt doses , 5. Aftec effect . Clinicac effect , 8. It ic effect . AdverseAt present, various methods such as HPLC-UV , HPLC-DARopivacaine, 3-hydroxy ropivacaine, and diazepam-d5 , all with purities > 98% , were puμm, Waters, USA) was used as the stationary phase, and the column temperature was set at 40°C. The mobile phase consisted acetonitrile and 10 mmol/L ammonium acetate (containing 0.1% formic acid) in water. The mobile phase was gradient eluted with a flow rate of 0.4 mL/min. From 0 to 0.2 min, acetonitrile was 15% isocratic; from 0.2 min to 1.2 min, acetonitrile was increased from 15% to 85%; from 1.2 min to 2.1 min, acetonitrile was 85% isocratic; from 2.1 min to 2.4 min, acetonitrile was decreased from 85% to 15%; from 2.4 min to 4.0 min, acetonitrile was 15% isocratic.The UPLC-MS/MS was composed of an ACQUITY H-Class UPLC and XEVO TQS-micro triple quadrupole mass spectrometer . A UPLC BEH C18 column .The desolvation gas (nitrogen) was set to 850 L/h, and the cone gas was set to 50 L/h. The capillary voltage was set to 1.5 kV, and the desolvation temperature was 450°C. The MRM mode was used for quantitative analysis. The selected MRM transitions were as follows: m/z 275.3 → 126.2 (quantification) and m/z 275.3 → 84.3 (confirmation) for ropivacaine ; m/z 291.0 → 126.0 (quantification) and 291.0 → 98.3 (confirmation) for 3-hydroxy ropivacaine ; m/z 290.2 → 198.2 (quantification) and 290.2 → 154.0 (confirmation) for diazepam-d5  .Stock solutions of ropivacaine (1.0 mg/mL), 3-hydroxy ropivacaine (0.1 mg/mL), and diazepam-d5 (0.1 mg/mL) were prepared in methanol. The stock solutions were diluted with methanol to prepare a series of standard working solutions, and all solutions were stored at 4°C until use.Blank cerebrospinal fluid was mixed with an appropriate amount of the standard working solutions to prepare a curve for ropivacaine and 3-hydroxy ropivacaine with concentrations of 0.2, 1, 5, 20, 100, 500, 1000, and 2000 ng/mL in cerebrospinal fluid. Quality control (QC) samples  were prepared in the same manner as the standard curve samples. The standard curve samples were treated by ethyl acetate liquid-liquid extraction and analyzed by UPLC-MS/MS.μL), the internal standard solution (10 μL of a 0.5 μg/mL solution), and ethyl acetate (2 mL) were added to a 5 mL centrifugation tube, which was then vortexed for 1.0 min. After centrifugation , the supernatant (1.5 mL) was removed and dried under a stream of air and then reconstituted in 100 μL of methanol. The resulting solution (80 μL) was transferred into the inner liner of the injection bottle, 2 μL of which was injected into the UPLC-MS/MS for analysis.Cerebrospinal fluid , and the intraday and interday precisions were determined by measuring QC samples at three concentration levels on three consecutive days. The intraday and interday accuracies were reported based on the average value of the QC samples at the three different concentrations and the true value for the three consecutive days. The extraction efficiency was evaluated by comparing the measured peak area of the low-, medium-, and high-concentration QC samples to the corresponding standard peak area. The matrix effect was evaluated by comparing the peak area of the low-, medium-, and high-concentration standard solution in the blank cerebrospinal fluid after sample treatment to the peak area of the corresponding standard solution.To confirm the ability to measure the diluted samples from beyond the upper limit of quantitation to within the calibration concentration range , six aliThe stability of ropivacaine and 3-hydroxy ropivacaine in cerebrospinal fluid was investigated by analyzing the QC samples of three low-, medium-, and high-concentration levels placed under three storage conditions, which include long-term stability , short-term stability (2 h at room temperature), and freeze-thaw stability (3 consecutive freezing and thawing cycles for 3 days) (-20°C to room temperature).The choice of positive and negative mode for ESI is often evaluated in methodology –26. The The liquid chromatography conditions should be chosen in order to effectively separate endogenous interfering substances –29 from The typical UPLC-MS/MS chromatograms of blank cerebrospinal fluid, fortified cerebrospinal fluid with ropivacaine, 3-hydroxy ropivacaine, and the internal standard are shown in y = 0.4069x + 0.2476, r = 0.9998, and y = 0.5768x + 0.3461, r = 0.9992, where y represents the ratio of the peak area of ropivacaine and 3-hydroxy ropivacaine to the internal standard and x represents the concentration of ropivacaine and 3-hydroxy ropivacaine in cerebrospinal fluid. The LLOQ of ropivacaine and 3-hydroxy ropivacaine in cerebrospinal fluid was determined to be 0.2 ng/mL. The detection limit of ropivacaine and 3-hydroxy ropivacaine in cerebrospinal fluid was 0.05 ng/mL, and the signal-to-noise ratio was 3.The standard curves of ropivacaine and 3-hydroxy ropivacaine in cerebrospinal fluid were acquired over the concentration range of 0.2–2000 ng/mL. The equations for the standard curve of ropivacaine and 3-hydroxy ropivacaine were It can be seen from n = 6) was identified as 98.5%, and the precision  was 5.7%. These results met the acceptance criteria, suggesting that samples exceeding the calibration curve concentrations can be diluted 20-fold to achieve concentrations within the range of the assay.After the 20-fold dilution of the six aliquots of ropivacaine  in cerebrospinal fluid, the accuracy (After analysis of the three stability conditions, which consisted of room temperature for 2 hours, –20° C for 30 days, and 3 freezing and thawing cycles, the accuracy of ropivacaine and 3-hydroxy ropivacaine in cerebrospinal fluid was between 85% and 109%, and the RSD was within 12%. These results indicate that ropivacaine and 3-hydroxy ropivacaine were both stable.μg/mL and 0.57 ng/mL by the developed UPLC-MS/MS, respectively. According to the literature, a concentration of 2.2 μg/mL of ropivacaine in plasma was reported to be toxic [A woman with pain in the right elbow was diagnosed with tennis elbow and cervical spondylosis after a physical examination, cervical X-ray, and cervical magnetic resonance imaging. The patient was then given local injections, which consisted 20 mg of ropivacaine mesylate, 20 mg of sodium hyaluronate, 3.0 g of adenosine cobaltamine, and 10 mg of tramalin dissolved in 10 mL of saline, in the facet joints of the cervical spine to improve elbow pain. During injection therapy, the patient experienced chest tightness and difficulty breathing. The treating physician immediately stopped the injection and gave oxygen inhalation, mouth-to-mouth artificial respiration, injection of respiratory stimulants, and continuous-breathing mask pressure to maintain respiration. After extracorporeal cardiac compressions, an injection of 1 mg norepinephrine, continuous cardiac compression, balloon breathing, and other treatments, rescue continued for about 40 minutes. At last, the patient's breathing and heartbeat showed no signs of recovery, and the rescue was declared invalid. The disputes over the cause of death requested ropivacaine testing of the cerebrospinal fluid. The concentrations of ropivacaine and 3-hydroxy ropivacaine in the cerebrospinal fluid were 22.8 be toxic , whereasIn this study, a sensitive, rapid, and selective UPLC-MS/MS method was established for the determination of ropivacaine and 3-hydroxy ropivacaine in cerebrospinal fluid with a linear range of 0.2–2000 ng/mL. UPLC-MS/MS has a faster analysis time and higher sensitivity than traditional HPLC. It only takes 4 min to complete the analysis of one cerebrospinal fluid sample, which could save a significant amount of time and resources. It was also successfully applied to a ropivacaine poisoning case.


Material and Method. A retrospective, nonrandomized study was conducted in patients with macular edema (ME) due to retinal vein occlusion (RVO) who undertook intravitreal Ozurdex® as first-line treatment. We performed a complete ocular exam including macular OCT.  The purpose of the study was to assess the efficacy of intravitreal dexamethasone implant (IDI: Ozurdex®) injection in eyes with macular edema due to retinal vein occlusion. p < 0.0001 at each timepoint). Reinjection for recurrent macular edema after 18 weeks was indicated in five eyes (41.67%) in the BRVO group and in six eyes (25%) in the CRVO group. Cataract developed in two eyes (16.67%) in the BRVO group and in one eye (4.17%) in the CRVO group. The IOP was higher than 25 mmHg in two cases in the BRVO group (16.66%) and in three cases (8.33%) in the CRVO group.  The mean BCVA (logMar) improved from 0.420.42 ± 0.23 logMar at baseline to 0.21 ± 0.23 logMar at 48 weeks in the BRVO group and from 0.72 ± 0.16 logMar at baseline to 0.31 ± 0.23 logMar at 48 weeks in the CRVO group. In both groups, CFT values decreased significantly compared to baseline ( Ozurdex® injected intravitreally significantly improved the mean CFT and BCVA in eyes with macular edema due to retinal vein occlusion. It was approved for use in the treatment of RVO in the United States of America (USA), Europe, and Switzerland. Several studies showed that intravitreal steroid injections have anti-inflammatory, antiangiogenic, and antivascular permeability characteristics and are effective for treating RVO-related ME [Central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO) are between the most significant causes of decreased visual acuity due to the existence of macular edema (ME), whether the fovea is perfused or not . ME is tlated ME , 2, 6–8.Therefore, the purpose of the study was to evaluate the efficacy of intravitreal dexamethasone implant (IDI: Ozurdex®) injection in eyes with macular edema due to retinal vein occlusion.A retrospective, nonrandomized study was performed, based on the medical records of patients who had macular edema (ME) consequently to retinal vein occlusion (RVO) and had been treated as first-line treatment with Ozurdex intravitreal injection between September 2015 and December 2017 in Oculens Clinic, Cluj-Napoca, Romania. The study began after obtaining approval from the Clinical Ethics Committee.μ and visual acuity of +0.3 logarithm of the minimum angle of resolution (logMar) or worse were included. The exclusion criteria were coexisting retinal disease , or media opacities (cataract) that could decrease visual acuity (VA), and pregnancy. Patients who had previously received treatment for ME , with a history of ocular surgery (except cataract) and trauma, were excluded. All patients underwent standardized examination including measurement of best-corrected visual acuity (BCVA) using a projection chart at 5 m, slit-lamp biomicroscopy, fundus examination using a postdilation +90 diopter lens and a three mirror contact lens, measurement of intraocular pressure (IOP) via applanation tonometry, and color fundus photography. Fluorescein angiography (FA)  and optical coherence tomography (OCT) imaging  of the macula were performed prior to treatment initiation. At each visit, the aforementioned examinations were performed, with exception of FA. Macular optical coherence tomography (OCT) was used to measure central foveae thickness (CFT), which was defined as mean thickness of the neurosensory retina in central 1 mm diameter region, and was computed via OCT mapping software provided with the device. Fluorescein angiography was performed in order to establish capillary dropout zones at the fovea and peripheral retina, and for leakage, as causes of ME.Newly diagnosed naïve RVO patients who had macular edema  under 3 months at first presentation with a baseline central foveae thickness (CFT) of >300Written informed consent for treatment was obtained from all patients, and the study complied with the principles of the Declaration of Helsinki (1964) and its amendments .All injections were performed under sterile conditions in the operating room, after application of topical anesthesia  and of 10% povidone-iodine solution ; scrub was used on the lids and lashes, and 5% povidone-iodine was administered in the conjunctival sac. Intravitreal Ozurdex® 0.7 mg  was injected through the pars plana into the vitreous, at 3.5 mm posterior to the limbus with a customized, single-use 22-gauge applicator. After the injection, each patient was prescribed steroids and antibiotics five times a day for one week. Patients were instructed to return to the hospital if they experienced decreased vision, eye pain, or any new symptoms.All the patients were followed up for 48 weeks. During the study period, the patients were monitored for adverse effects . In the first year, the patients were examined the day after injection and 4 weeks, 8 weeks, 12 weeks, 24 weeks, and 48 weeks after injection.Panretinal or sectorial photocoagulation was applied to the patients who showed any kind of neovascularization during the follow-up. Panretinal photocoagulation was applied to the CRVO patients who showed neovascularization of the iris or on optic disc. Sectorial laser photocoagulation was applied to the BRVO patients who showed any kind of neovascularization, and the treatment area covered the entire ischemic area that was detected via FA.Data collected from patients' records included age, gender, type of RVO, ischemic status, types of RVO, associated risk factors, complications after injection, BCVA , intraocular pressure, and CFT. Visual acuity and CFT were measured at all timepoints .p value <0.05 was considered statistically significant.Visual acuity and the CFT values between baseline and the other timepoints were assessed with repeated measurement tests. Categorical variables were compared using the chi-square test. A p=0.33). The follow-up period was 48 weeks. Demographic characteristics of the patients are shown in Thirty-six eyes of 36 patients were included. The average age of the patients was 59.33 ± 15.74 years (range 22–89). Twenty-four of the patients (66.67%) had nonischemic CRVO, while 12 (33.33%) had nonischemic BRVO and received IDI injection as the first-line treatment for ME. No significant difference was seen between the two groups with respect to age and gender , ischemic heart disease (9%), and hypertension (19%). Primary open angle glaucoma was present in two cases of BRVO (16.66%) and in four cases of CRVO (16.66%). All these patients followed a topical treatment with fixed combination (timolol 0.5% and dorzolamide) or prostaglandin analogue, with well-controlled IOP. Small hyperopia was present in 16 cases (44.44%).p=0.0012 after 4 weeks;p < 0.0001 at 8 weeks, 12 weeks, 24 weeks, and 48 weeks).In the CRVO group, the mean BCVA (logMar) value was 0.72 ± 0.16 logMar at baseline and improved to 0.45 ± 0.19 logMar after 4 weeks, 0.36 ± 0.18 logMar after 8 weeks, 0.35 ± 0.24 logMar after 12 weeks, 0.33 ± 0.24 logMar after 24 weeks, and 0.31 ± 0.23 logMar at 48 weeks see . In the p=0.0017 after 4 weeks; <0.0001 at 8 weeks, 12 weeks, 24 weeks, and 48 weeks).In the BRVO group, the mean BCVA (logMar) value was 0.42 ± 0.23 logMar at baseline and improved to 0.26 ± 0.26 logMar after 4 weeks, 0.24 ± 0.23 logMar after 8 weeks, 0.22 ± 0.21 logMar after 12 weeks, 0.22 ± 0.22 logMar after 24 weeks, and 0.21 ± 0.23 logMar at 48 weeks see . In the p=0.0057), at 4 weeks (p=0.038), at 8 weeks (p=0.0248), at 12 weeks (p=0.0336), and at 24 weeks (p=0.0448), but there was no statistically significant difference at 48 weeks (p=0.1152)  see .μm at baseline and decreased to 366.58 ± 109.58 μm after 4 weeks, 322.13 ± 76.80 μm after 8 weeks, 288.25 ± 96.89 μm after 12 weeks, 277.92 ± 96.27 μm after 24 weeks, and 255.50 ± 67.86 at 48 weeks (see p < 0.0001 at each timepoint).In the CRVO group, the mean CFT value was 504.38 ± 112.91 eeks see . CFT valμm at baseline and decreased to 301.25 ± 66.30 μm after 4 weeks, 314.08 ± 102.30 μm after 8 weeks, 271.33 ± 59.78 μm after 12 weeks, 251.08 ± 64.85 μm after 24 weeks, and 250.80 ± 84.65 after 48 weeks .In the BRVO group, the mean CFT value was 430.25 ± 100.5 eeks see . CFT valp=0.0629 at baseline visit, p=0.0671 at 4 weeks, p=0.7929 at 8 weeks, p=0.5844 at 12 weeks, p=0.5519 at 24 weeks, and p=0.9393 at 48 weeks)  see .Reinjection for recurrence of CFT elevation demonstrated by the macular OCT at 18 weeks was indicated in 6 cases (25%) in the CRVO group and in 5 cases in the BRVO group (41.67%). These cases presented for a check-up at 18 weeks (even it was not the check-up timepoint) because they observed a significantly visual acuity decrease. These cases were treated using a second injection of anti-VEGF such as bevacizumab (Avastin). The switch had a good rationale due to the different mode of action of these agents (Ozurdex versus Becacizumab) and also because of financial reasons. In Romania, the intravitreal injection with Ozurdex is not covered by the National Health Care System.p=0.332 in the CRVO group and p=0.673 in the BRVO group). One patient required surgical treatment such as trabeculectomy.Intraocular pressure was measured in both groups in the first week after the injection. In the CRVO group and the BRVO group, the mean IOP value in the first week was 19.08 ± 2.95 mmHg and 18.75 ± 2.90 mmHg, respectively. The IOP was higher than 25 mmHg in three cases (8.33%) in the CRVO group and in two cases (16.66%) in the BRVO group two months after the intravitreal injection. IOP higher than 10 mmHg was present in 2 eyes (4.8%) in the CRVO group and in one eye (1.2%) in the BRVO group. Topical antiglaucomatous drugs were required in all these cases. Topical timolol 0.5% combined with dorzolamide in fixed combination was administered twice per day. Moreover, no statistical significant difference was shown between IOP values in the third and fourth month and baseline values (Cataract developed in one eye (4.17%) in the CRVO group and in two eyes (16.67%) in the BRVO group and required phacoemulsification with intraocular artificial lens implantation. Conjunctival hemorrhages occurred in five patients (13.8%). None of the patients developed endophthalmitis, vitreous hemorrhage, or retinal detachment.μg) dexamethasone in the Novadur solid polymer drug delivery system . It is a potent corticosteroid, which suppresses inflammation by inhibiting multiple inflammatory cytokines resulting in decreased edema, fibrin deposition, capillary leakage, and migration of inflammatory cells [Ozurdex®  is an intravitreal implant containing 0.7 mg . The results were similar to those of previous studies. In the GENEVA study, the improvement in BCVA by 15 letters or more was 29% at 60 days and 22% at 180 days [p=<0.001) after the second injection of Ozurdex intravitreal implant. Mayer et al. [Functional results from our study showed a statistical significant improvement in BCVA in both groups at each timepoint comparing with baseline  and was comparable with other studies. In the GENEVA study, it was shown a mean reduction of 119 μm at 180 days following treatment [μm. Shahina et al. [μm. Simsek et al. [p < 0.001) of CFT 4 months after intravitreal dexamethasone injection. Moreover, they observed the recurrence of CFT elevation in 65.3% of patients in the BRVO group and in 68.1% in the CRVO group 4 months after the second injection of intravitreal dexamethasone implant. In the present study, the recurrence after the first injection was present in 41.67% patients with BRVO and in 25% patients with CRVO. Bezatis et al. [p < 0.001) compared with the baseline at each follow-up visit.Mean reduction in central macular thickness in our study was significant in both groups at each time point  in the CRVO group and in five eyes 41.67%) in the BRVO group at 4.5 months from baseline. Published reports in which reinjections have been made after shorter intervals on an “as needed” basis are now available , 19. In 7% in theIn this study, cataract that decreased VA appeared in one eye 4.17%) in the CRVO group patients and in two eyes 16.67%) in the BRVO group during the follow-up. The results are similar with others studies % in the . Ozkaya .67% in thttp://ghrnet.org/index.php/IJOR/article/view/2513/2894 [In our study, intraocular pressure increased in three eyes 8.33%) in the CRVO group and in two eyes (16.66%) in the BRVO group. In 2 eyes in the CRVO group, the IOP increased more than 10 mmHg. These results were lower in comparison to those revealed by Schmitz et al. . In thei% in the 513/2894 . Françoi513/2894  and Arma513/2894  suggeste513/2894 –41.In our study, we did not have any endophthalmitis after Ozurdex intravitreal implantation. The results are similar with previous studies , 43.In our study, one eye (8.33%) with BRVO received a sectorial photocoagulation after 12 weeks and three eyes (12.5%) with CRVO received a panretinal laser photocoagulation 12 weeks after injection. In these cases, 12 weeks after the intravitreal treatment, the patients developed new vessels on the optic disc as a sign of ischemic form of retinal vein obstruction, even if they had a nonischemic form at the beginning of the study. The goal of the treatment was to decrease neovascular changes and prevent the development of neovascular glaucoma. There are studies that revealed that 30% of eyes with nonischemic CRVO at first may convert to ischemic type –47. TromThe present study has some limitation regarding the small sample size, the short period of follow-up and the absence of a control group. To our knowledge, this is the first Romanian study regarding the efficacy of intravitreal Ozurdex injection for ME after retinal vein occlusion. Nonetheless, further studies with active controls are needed to completely understand the efficacy and safety of intravitreal dexamethasone implant injection.Intravitreal Ozurdex® injection significantly improved mean BCVA and reduced CFT in eyes with macular edema due to retinal vein occlusion. The treatment is safe and effective. Cataract formation and increasing IOP demands regular visits in patients treated with intravitreal Ozurdex.