Listeria monocytogenes (Lm) pose a public health risk, recent studies suggest that Lm in its VBNC state remains virulent. VBNC bacteria cannot be enumerated by traditional plating methods, so the results from routine Lm testing may not demonstrate a sample’s true hazard to public health. We suggest that supplementing routine Lm testing methods with methods designed to enumerate VBNC cells may more accurately represent the true level of risk. This review summarizes five methods for enumerating VNBC Lm: Live/Dead BacLightTM staining, ethidium monoazide and propidium monoazide-stained real-time polymerase chain reaction (EMA- and PMA-PCR), direct viable count (DVC), 5-cyano-2,3-ditolyl tetrazolium chloride-4′,6-diamidino-2-phenylindole (CTC-DAPI) double staining, and carboxy-fluorescein diacetate (CDFA) staining. Of these five supplementary methods, the Live/Dead BacLightTM staining and CFDA-DVC staining currently appear to be the most accurate for VBNC Lm enumeration. In addition, the impact of the VBNC state on the virulence of Lm is reviewed. Widespread use of these supplemental methods would provide supporting data to identify the conditions under which Lm can revert from its VBNC state into an actively multiplying state and help identify the environmental triggers that can cause Lm to become virulent. Highlights: Rationale for testing for all viable Listeria (Lm) is presented. Routine environmental sampling and plating methods may miss viable Lm cells. An overview and comparison of available VBNC testing methods is given. There is a need for resuscitation techniques to recover Lm from VBNC. A review of testing results for post VBNC virulence is comparedThe detection, enumeration, and virulence potential of viable but non-culturable (VBNC) pathogens continues to be a topic of discussion. While there is a lack of definitive evidence that VBNC  Listeria monocytogenes (Lm) is a foodborne pathogen found in a variety of foods; outbreaks of listeriosis have been linked to the consumption of contaminated raw milk, ready-to-eat deli meats, cantaloupes, hot dogs, smoked fish, mushrooms, eggs, soft cheeses, frozen vegetables, packaged salads, ice cream, caramel apples, and bean sprouts [Listeria positive food contact surface results, the food product lots are put on hold. A representative sample of 25 g of the food product lots are taken daily and any product lots on hold are not released until three consecutive days of negative results are obtained. The European Union also regulates Lm, including the requirement that Lm counts must be absent in 25 g samples of food intended for infants and foods for special medical purposes. All other foods must contain less than 100 CFU/g in RTE foods unable to support the growth of Lm [ sprouts . The Foo sprouts . Using F sprouts . Adjusti sprouts . Due to  sprouts . In the th of Lm .Listeria control alternatives to comply with USDA-FSIS guidelines. These alternatives include using a post-lethality treatment, antimicrobial agents or processes, or strict sanitation standards. With each of these alternatives, regular food product and environmental samples are taken to validate that the current Lm controls continue to be effective. Depending on the Lm control option chosen, testing may be required yearly, quarterly, monthly, every two weeks, or weekly. If a sample is found to be positive for Lm, intensified sampling is conducted. Increased sanitization efforts are recommended with increased sampling to verify that the source of Lm contamination has been eliminated. If issues with Lm control continue to persist, the food products may be put on hold and the establishments may be found in non-compliance [In 2014, the USDA-FSIS published an updated guideline for the control of Lm in poultry and RTE products [Viable bacterial cells in a food processing plant are routinely exposed to stressful environments such as cleaning and sanitation operations, the depletion of available nutrients, or extended periods of desiccation. The loss of nutrients has been shown to be a trigger for viable bacterial cells such as Lm and other food pathogens to enter a viable but not culturable (VBNC) state ,8. In thVibrio using a rat ileal loop model. Thus, there may be a risk of Lm in the processing plant being undetected by traditional enrichment and plating techniques, being transferred to a RTE food and growing to a level that has a high probability of infecting a person when the food is consumed. Since regulators and the food industry are trying to minimize the risk of foodborne listeriosis, a new research focus is called for to understand the actual risks associated with Listeria’s VBNC state.The inability of VBNC Lm cells to grow on traditional plating media can lead to false negatives during routine testing of products or food contact surfaces. It is also possible that given the right conditions the VBNC bacteria can resuscitate in vivo and regain virulence, thereby leading to infections, as demonstrated by Baffone et al.  while exTo create a nutrient-deprived environment in the laboratory, that induces the VBNC state in Lm, microcosm water consisting of filter-sterilized water, sterile deionized water, and water containing various levels of minerals are usually used ,15. UsuaTM staining, (2) ethidium monoazide- and propidium monoazide-stained real-time polymerase chain reaction (EMA- and PMA-PCR), also known as viability PCR (v-PCR), (3) Direct Viable Count (DVC), (4) 5-cyano-2,3-ditolyl tetrazolium chloride—4′,6-diamidino-2-phenylindole (CTC-DAPI) double staining, and (5) carboxy-fluorescein diacetate (CDFA) staining although other more novel methods have been used to estimate the viability of Lm [To determine if Lm cells are completely non-viable or in a VBNC state, several laboratory analyses for viable cell detection have been developed. Each method attempts to assess some unique aspect of cell’s viability or metabolism. The five most widely used methods used are (1) Live/Dead BacLightty of Lm ,21. In tTM is a differential staining method used to detect viable, non-viable and total bacteria cells present in a sample. The kit uses two nucleic acid-binding stains, SYTO 9 and propidium iodide (PI), contained in a dimethylsulfoxide solution (DSMO) [BacLightn (DSMO) . SYTO 9 n (DSMO) ,23,24,25TM staining was run in conjunction with flow cytometry, high correlations of live and dead Lm cell counts were found (r2 = 0.97 and r2 = 0.99 respectively) between ratios of 10% to 100% (living to dead cells) when compared to known control populations of live and dead cells [TM stained Lm cells under microscopy also gave a more accurate estimation of viable cell counts when compared to EMA/real-time PCR [TM staining in conjunction with flow cytometry and comparing it with direct viable counts, the membrane integrity of VBNC Lm populations can be demonstrated, even as plate-count cultivability was reduced to nearly 0% [When BacLightad cells . BacLightime PCR . By usinearly 0% .TM kit, a statistically significant correlation, r2 ≥ 0.98, was found for A. hydrophila, B. subtilis, E. coli, P. aeruginosa, and S. epidermidis [TM produced equal to or better accuracy in detecting viable and total counts of E. coli in several tests [TM is seen in a wide range of bacteria, and it is also effective in the analysis of Gram-positive pathogens such as Lm. BacLightTM is commonly used to analyze Lm in biofilms [When the correlation between the percentage of metabolically active bacterial cells added to a sample was compared to the percentage of active cells measured by the BacLightdermidis . When coal tests . The accbiofilms ,29 and tHowever, clear bimodal (green and red) staining is not always possible. Frequently, a gradient from dual staining is observed, resulting in some difficulty interpreting the results ,31. At tQuantitative PCR (qPCR) targets the unique DNA sequences of foodborne pathogens such as Lm and amplifies these sequences to detectable levels. However, qPCR also has the potential to amplify intact DNA from dead Lm cells , and thuEthidium monoazide (EMA) and propidium monoazide (PMA) are both DNA-binding agents used in combination with Real-Time Polymerase Chain Reaction (RT-PCR) to prevent the DNA of dead bacterial cells from being amplified. EMA penetrates damaged membranes of dead cells  and, wheEscherichia coli O157:H7 [However, it is possible that EMA can penetrate viable cell membranes of certain bacterial species, including  O157:H7  and Lm [ O157:H7 ,41, whic O157:H7 . In conj O157:H7 .Concerns have been recently raised with the use of PMA-based RT-PCR in determining viable cell counts. When analyzing heat-treated Lm, PMA-based RT-PCR discrimination between viable and dead cells depended on the extent of the heat treatment applied. Specifically, in the study by Lovdal et al. , Lm cell6 bacteria ml−1), with DVC analyzing the cell elongation of Lm and CTC-DAPI measuring cell respiration of Lm cells through CTC fluorescence [6 bacteria ml−1 when exposed to 50, 80, and 100 ppm chlorine, while traditional culturable plate counts were below the limit of detection [The direct viable count (DVC) method was originally developed to enumerate bacteria in samples of seawater . Its userescence . Similarrescence . Highmorrescence  used a Detection . Care muCTC is a tetrazolium salt that is reduced to CTC-formazan by the electron transport system in actively respiring bacterial cells . The CTCStaining used in conjunction with DVC is a common method for evaluating metabolic activity in VBNC bacterial cells and works well to detect VBNC Lm with reasonable accuracy ,36. CTC-TM RedoxSensorTM Green may also be applied and analyzed using flow cytometry to confirm reductase activity of actively respiring cells [TM RedoxSensorTM Green penetrates the cell membrane, and the reagent is reduced by enzymes in the electron transport chain of actively respiring cells, producing a green-fluorescent signal. Like the CTC-formazan, the BacLightTM RedoxSensorTM Green does not fluoresce in dead cells with intact membranes due to the lack of enzymatic reduction of the dye within the dead cells. This signal can then be analyzed, and actively respiring cells subsequently counted using a flow cytometer [A similar stain, BacLightng cells . BacLighytometer .p ≤ 0.003) higher when compared to plate counts when analyzing VBNC contamination of cheese [p = 0.025) viable cell counts compared to control fluorescent labeling methods [L. innocua [L. innocua [CFDA is a colorless fluorogenic ester that enters bacterial cells through diffusion. CFDA is then enzymatically cleaved by bacterial cell esterase enzymes to produce a fluorescent product, carboxyfluorescein (cF). The fluorescence is then analyzed by epifluorescence microscopy or through a flow cytometer; the intensity of the signal determines the viable cell count of the sample ,50,51. Df cheese . When te methods . When us methods . Also, d methods . CFDA an innocua . The res innocua .p ≤ 0.003) difference in viable Lm counts when compared to direct plate counts over a period of 90 days regardless of storage conditions, suggesting that the Lm may have entered a VBNC state [Aeromonas hydrophila, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus epidermidis were compared, no significant differences (p > 0.05) of metabolically active bacteria were obtained by both the BacLightTM staining and CFDA staining in four of seven water samples when the differentially stained cells from both methods were compared using a flow cytometer [CFDA results can also be compared to plating on non-selective media to determine VBNC counts of Lm when evaluating food storage conditions. When evaluating the formation of VBNC Lm on packaged hard cheeses, CFDA-based fluorescent microscopy showed a significant (NC state . When meytometer .hly (which encodes for virulence protein Listeriolysin O) is an important virulence factor in Lm [hly and inlA expression continued after 27 days in VBNC Lm cultures starved in microcosm water [hly gene was demonstrated in VBNC Lm after starvation in microcosm water [There are currently several methods for the accurate and precise detection and enumeration of metabolically active Lm. However, in addition to determining that these metabolically active Lm cells are VBNC cells waiting for the appropriate environmental conditions that will allow them to begin replication, we must also understand the expression of virulence in VBNC Lm. The pathogenic gene or in Lm . Its expsm water . Continusm water .RAG1)-deficient mice were injected with 107 VBNC cells, sacrificed two weeks after injection, and their spleens harvested and plated on Brain Heart Infusion (BHI) plates. RAG1-deficient mice were chosen because RAG1 is required for somatic recombination of T-cell receptor (TCR) and Immunoglobulin (Ig) genes. Therefore, the absence of this protein resulted in immunodeficient mice [However, the expression of Lm virulence genes has yet to be unequivocally demonstrated to translate into Lm VBNC pathogenicity. Study results have been mixed in virulence models. VBNC Lm cells were tested in mouse models and were shown to be unable to colonize mouse spleens ,48. The ent mice . DespiteIn addition, VBNC Lm cells were not able to adhere to a HT-29 cell culture line. HT-29 was used as a human colon cancer cell line that can express characteristics of mature intestinal cells, giving an indication of microbial adhesion in the human gut ,48. CappIn contrast to these negative findings of VBNC virulence for Lm, we will now discuss two studies with results that suggest VBNC Lm cells’ avirulence may be either transitory, or in the case of the second study, simply require the right environment to become virulent. The first study  replicatCaenorhabditis elegans, a nematode model [C. elegans. Not only did C. elegans ingest the VBNC Lm, but Lm was also found outside of the intestinal lumen. Furthermore, the exposure to VBNC Lm produced the reduced nematodes’ lifespan by a similar length as consuming viable, culturable Lm did. The authors suggest that this reduction in lifespan may be due to continued expression of Lm virulence genes during the VBNC state.A second study used de model . VBNC LmTo determine how much of a risk VBNC Lm truly is, the likelihood of VBNC Lm resuscitation needs to be better understood. Therefore, the next research step is to look for effective ways of resuscitating the VBNC Lm into a culturable state. This is important for two reasons: (1) the ability to consistently resuscitate VBNC Lm would allow for its detection during routine in-plant environmental sampling and (2) research on resuscitation will hopefully shed light on under what conditions Lm VBNC are an increase public health food safety risk.The study using the embryonic egg model to resuscitate VBNC Lm raises questions of a public health risk regarding the VBNC state of Lm . Namely,Once we can answer these questions, we can better determine the health risks associated with the possibility of Lm resuscitating and subsequently expressing virulence in the environment or producing a food borne infection when consumed. We may also be able to develop standardized techniques for resuscitating VBNC Lm for food production plant and laboratory testing. We would adjust our testing methods so that a very small amount of Lm contamination could be detected, to meet the zero-tolerance requirements of the FDA. If the testing methods or the resuscitation requires many cells, then a potential infectious amount may be missed. Once we determine a proper testing method for VBNC Lm in the poultry processing plant, we may be able to adjust standard sanitization procedures to prevent the induction of Lm into VBNC state in the first place. If in fact the VBNC Lm cells can resuscitate and regain virulence in the environment or when consumed, this knowledge will better assist us in minimizing the risk of Lm associated foodborne illness.TM staining, and CFDA staining may allow for better detection/enumeration of total bacterial load and VBNC bacteria when compared to plate counts. Resuscitation using an embryonic egg model and the renewed expression of virulence in Lm has been proven possible [At this early stage, we are not able to confidently estimate the public health risks associated with the VBNC state in Lm, but if these cells are not able to revert to an infectious state unless under preferred environmental and nutrient conditions, then this public health risk would be minimal. However, one cannot assume based on current testing that in vivo conditions would prevent VBNC Lm from causing foodborne illness, even if little evidence supports its ability to resuscitate after entering VBNC state. Therefore, until it is proven that VBNC Lm cannot cause illness in vivo, Lm in the VBNC should probably be included in the current USDA zero tolerance regulations. Current plate count detection focuses on live Lm in food and environmental samples, with negative results giving confirmation that the sanitizing/cleaning process is working correctly. The use of viability RT-PCR, DVC, CTC-DAPI double staining, BacLightpossible , however


Anti-corrosion coating technology was developed in an effort to protect sewer pipes from degradation. This study evaluates a new class of relatively low-cost magnesium hydroxide-based coatings, regarding their ability to adhere efficiently onto the concrete surface, and offer efficient corrosion protection. Six magnesium hydroxide-based coatings were prepared with the addition of two different types of cellulose, used as adhesion additives, and these were applied on concrete specimens. Pull-off measurements showed that the addition of higher amounts of cellulose could improve the coating adhesion onto the concrete surface. An accelerated sulfuric acid spraying test was used to evaluate the consumption time of the applied coatings and their efficiency in maintaining over time slightly alkaline pH values (above 8) on the coated/protected surfaces. At the end of spraying test, a mineralogical analysis of surface samples was performed, indicating that the formation of corrosion by-products (mainly gypsum) was increased when the added amount of cellulose was lower. Hardness and roughness measurements were also conducted on the concrete surfaces, revealing that the coatings helped the concrete surface to preserve its initial surface properties, in comparison to the uncoated specimens. A SEM/microstructure analysis showed that aggregates were formed (possibly consisting of Mg(OH) Application of magnesium hydroxide-based coatings for the mitigation of corrosion in concrete sewer pipes.The addition of 1% cellulose (wt. of solids) in the applied coating showed great adhesion ability, and water and acid resistance properties.2S). This dangerous gas originates in the submerged portion of sanitary pipe sewers, where anaerobic conditions generally prevail, due to the presence of sulphur-reducing bacteria (SRB) located in the slime layer, i.e., the layer consisting of bacteria and inert solids, commonly existing in the flooded part of sewer pipes. The H2S (gas) subsequently can be diffused towards the upper  part of the sewer pipe  above the flowing wastewater surface, lowering the surface pH of exposed concrete walls/surfaces and favoring the development of sulfur-oxidizing bacteria (SOB). As a result, biogenic H2SO4 is ultimately produced, corroding the inner surface of concrete sewer pipes by reacting with the hydrated cement paste, and eventually producing (unwanted) gypsum, leading to the collapse of pipes [2S gas , high relative humidity, and rather high atmospheric oxygen content, MIC can take place. These conditions are commonly existent in the majority of wastewater transferring systems, at least several times yearly, depending also on seasonal parameters  [Microbiologically induced corrosion (MIC) or bio-corrosion in concrete sewerage systems, especially considering those with larger diameters, is a known problem , involviof pipes . In the ow rate) . It is aow rate) , or undeow rate) , using tow rate) , or by dow rate) . In the The economic losses due to MIC are considered to be very high worldwide . It is e2 has been previously studied as a neutralizing agent for acidic wastes [2-based coatings and by using common adhesion additives (celluloses). The spraying of the concrete pipes with an appropriate Mg(OH)2-based slurry could be a sustainable and economic solution for their protection from corrosion and it is considered among the most cost-effective strategies to mitigate MIC problems, especially in Greece [The various MIC mitigation strategies commonly applied, can be categorized into three main groups, i.e., (i) corrosion resistant concrete materials ,14, (ii)c wastes , as wellc wastes . The pren Greece .2-based consumption of coatings due to the reaction with sulfuric acid through accelerated tests on concrete specimens [2 coatings onto the concrete surfaces. However, the adhesion of the respective coatings onto the concrete surface is a crucial property that also needs to be examined carefully. There are many factors that can be studied, in order to evaluate the performance of coatings and the protection of the concrete’s surface against corrosion/degradation problems, such as the adhesion ability [2-based coatings, by using methyl-cellulose and/or carboxymethyl-cellulose as common adhesion additives. At the same time, this research evaluates the corrosion protection that these coatings can offer to the concrete surface, elucidating the respective coating–protection mechanisms. The amount of adhesive additives, influencing the ability for corrosion protection of the coating, was studied by monitoring the changes in the existing mineralogical phases. These changes refer mainly to the consumption of brucite (Mg(OH)2) and the formation of gypsum (CaSO4). The major characteristics and the different physicochemical properties of the six different coatings presented, selected among several others as bio-corrosion inhibitors, were evaluated by using different material characterization techniques. The ultimate aim of this paper was to determine the optimum cellulose combination and cellulose amount to be added in the Mg(OH)2-based coatings, and to examine the various important application properties, such as adhesion, water and acid resistance, and corrosion protection ability.Limited research has been performed so far regarding the evaluation of Mg(OH)pecimens ,21. The  ability , the thi ability , the sur ability , the con ability , the mas ability , etc. InThe specific concrete type used in the experiments was selected in order to appropriately simulate the respective concrete that is commonly used for the construction of sewerage pipes. According to the EN 1766:2000 Standard  and the 2 powder (MH), supplied by Grecian Magnesite S.A. . As adhesion promoters onto the concrete surface, methyl-cellulose (MC)   and carboxymethyl-cellulose (CMC)   were used. Deionized water was used for all coating preparations, resulting in slurries with 57.5% wt. content of solids. Dispersant polymer for mineral slurries (Acumer 9300), supplied by Dow  was added as a stabilizer for the slurries, and as viscosity reducer at 0.6% wt. of solids. The examined coatings were prepared by using a Mg(OH)Based upon preliminary experiments, two series of magnesium hydroxide slurries were synthesized by using methyl-cellulose as an adhesive in three different concentrations, i.e., 1%, 0.4%, and 0.1% wt. content of solids . The second series was prepared by using the combination of methyl-cellulose and carboxymethyl-cellulose at 1%, 0.4%, and 0.1% wt. content of solids of a mixture of both celluloses . The celluloses used in the second series of slurries were mixed in the ratio MC/CMC = 4/1.Deionized water was heated at 90 °C and then Acumer 9300 and celluloses were added into the hot water, under stirring until the solution reached room temperature. Magnesium hydroxide powder was added subsequently under mechanical stirring for 1 h, until the slurry become homogenous.2 and 0.0018–0.002 g/mm2 for the thin and the thick layer, respectively, according to our previous relevant experience and the respective preliminary experiments. The thickness of protective coatings may vary, depending on the various kinds of materials that can be used for this purpose. The organic coatings, usually applied for the protection of concrete surfaces, can be commonly applied with thicknesses between 0.1–1 mm; whereas the cementitious coatings and linings used on concrete surfaces may be applied at higher thickness, i.e., up to few a mm [The fast drying of applied coatings may lead to crack formation. In order to avoid this, the concrete surface was moistened with deionized water before the coating application. A spatula was used for the application of the coatings on the concrete specimens, in a way that a uniform thickness of the coating could be achieved. The coatings were applied on concrete blocks, in two different amounts (thickness). The amount of the applied coating was selected to be 0.0008–0.001 g/mmfew a mm . The thifew a mm , while afew a mm . The amoThe adhesion of the coatings onto the concrete substrate was studied by using the pull-off bond testing method. The test procedure was based on the respective standards, EN 1542:1999  and EN 1Coated cylindrical concrete specimens (with diameter 30 mm and height 60 mm) were immersed in a custom-made device (140 cm × 30 cm × 20 cm) in order to examine the adhesion properties of the examined coatings under the influence of different environments. The acrylic glass device was divided into 3 rows, allowing for the placement of 15 samples per row . After the liquid phase (water or sulfuric acid) passes through the first row, an opening for the flow enables it to pass through the second row and then to the third row, following an S-shaped route from the entry point to the final outlet and then, back to the pump. The coated specimens were immersed in water, as well as in 0.2 M sulfuric acid solution. The experimental device allowed the circulation of the liquid phase by the use of a peristaltic pump, applying an adjustable flow rate, corresponding to horizontal velocity equal to 0.05 m/s .In a combined sewage collection system of concrete sewer pipes, surface (mainly rain) runoff and sewage are simultaneously collected. The upper part of the concrete sewer pipes, which will be covered with the Mg-based coating, may be in direct contact with the water, e.g., in the case of sudden storm flushing, while during the dry periods wastewater flow is reduced, eliminating the contact of water with the applied coating. That is the reason why the authors have examined the water resistance properties of the coatings. Moreover, although several components can be transferred with the sewage along the sewer pipes, the authors focused their attention on the influence of sulfuric acid, since the biogenic-produced sulfuric acid is considered as the main corrosive agent inside the sewer pipe .1 = 14 m/s) were applied on the specimen surfaces for the duration of 20 min. The plastic tube (60 mm diameter) had two protrusions near the end, allowing the placement of one specimen at a time. However, for comparison reasons it should be noted that the maximum velocity of transferred wastewater in cement pipelines does not usually exceed 3 m/s [In addition to pull-off tests, supplementary tests were also carried out under high horizontal-velocity of the examined solution in order to examine the performance of coatings under rather extreme conditions of water flow (in order to simulate the sewerage network performance during a heavy storm incident in a combined sewer system). The concrete specimens were placed into a custom-made laboratory device  and highed 3 m/s . This exThis experiment was an accelerated procedure in order to evaluate the coatings’ consumption and protection ability, considering the real conditions existing in sewer pipes, which over time permit the application of much larger contact/reaction times.2SO4 solution (4 M) was used and sprayed with an appropriate handheld spraying device on the coated concrete specimens. The coatings’ mass that theoretically can react with the sulfuric acid was calculated, according to the respective magnesium and calcium content, when the later were expressed as oxides. The theoretically expected time of the coatings’ total consumption  was selected to be 6 days, according to previous experience, and based on preliminary relevant experiments and stoichiometric titrations. The total mass of the required acid solution for daily spraying was accordingly calculated, in order to achieve the daily consumption of a specifically pre-determined amount of coating, based upon the theoretical amount  sulfuric acid spraying tests. The coatings were applied on one side (50 mm × 50 mm) of the concrete blocks . Each daThe surface pH of coatings was measured daily with a flat surface pH electrode  . All measured pH values of the samples were recorded before spraying as the starting values. The specific spots to be measured were wetted with 1 mL of deionized water prior to the measurement. For comparison reasons, uncoated concrete specimens were also sprayed with sulfuric acid and measured. A climatic test chamber was used for the storage of samples throughout the duration of acid spraying experiments, simulating appropriately the conditions within the sewer pipes at the temperature of 20 ± 2 °C and 99% relative humidity.The spraying testing procedure was terminated when the respective coating was consumed, or when the extended formation of gypsum (a commonly formed by-product) was optically observed. The total consumption/reaction of coatings with the added sulfuric acid solution could not be achieved, due to the gypsum formation, which was initiated even when a small area of the concrete surface would be exposed to sulfuric acid . Gypsum is the product of the reaction between sulfuric acid and concrete constituents, and indicates that the applied coating cannot longer sufficiently protect the concrete surface.The degradation of concrete affects the concrete’s fundamental properties. The respective hardness and roughness measurements of the concrete surface can indicate the degree of protection that the applied coatings may provide to the concrete. The measurements of surface roughness and hardness were both conducted on uncoated specimens, as well as on sprayed-coated specimens. The surface roughness was measured with an electronic portable roughness gauge  , while the hardness measurements were carried out by a Shore A, hardness tester (Sauter) . Roughness was expressed in Ra (μm), with a cut-off length of 0.25 mm. The hardness tester was adapted on a test stand with a glass base plate, in order to conduct the respective measurements. The coated samples were measured after the acid spraying, as well as after the removal of remaining coatings from the concrete surface by using a spatula.The structural phases  of the samples were detected by the X-ray Diffraction analysis (XRD), using a PW 1840 Phillips diffractometer with CuKa radiation, step size of 0.02°, and step time of 0.4 s, operating at 30 kV and 10 mA. The obtained diagrams were quantified by following the Rietveld methodology, and by using the FullProf Suite Software on the basis of the respective PDF cards numbers #74-2220 (brucite) and #70-0982 (gypsum). The samples were scratched from the surface of the specimens and dried at 60 °C for 24 h. Afterwards the samples were ground and measured.Dried slurries mounted on aluminum stubs with sticky double-side carbon tape. No special treatment was applied to these samples  and no specific coating needed. Micrographs were obtained by using a Carl Zeiss EVO 50 VP scanning electron microscope . The examinations were performed at 5 kV accelerating voltage, under variable pressure mode, suitable for non-conductive specimens, and at a pressure of 30 Pa. A variable pressure secondary electron (VPSE) detector was used.The important property of adhesion, regarding the applied coatings on the concrete substrate, was evaluated in this study by using the pull-off technique. Magnesium hydroxide lacks adhesion ability onto a concrete surface, and celluloses were added in order to promote the adhesion. For each examined coating case, two different thicknesses were examined (thin or thick) in order to study the influence of the layer’s thickness on the property of adhesion. According to the aforementioned standards ,31 the lThe results of tensile bond strength between the coatings and the concrete surface are presented in The series of coatings with the methyl-cellulose additive presented a generally higher tensile bond strength than those with the mixture of both celluloses (methyl-cellulose and carboxy-methyl-cellulose) for the corresponding amounts. M1m showed better adhesion ability than M1mc, although both were synthesized with the total amount of 1% wt. celluloses. Therefore, it can be concluded that the adhesion ability of the examined coatings was reduced with the addition of  carboxy-methyl-cellulose adhesive.Additionally, it can be observed that the bond strength increased when the amount of cellulose was also increased, notifying that the M1m coating (1% wt. of methyl-cellulose) showed the best adhesion ability onto the concrete substrate, both in thin and thick layers. However, the bond strength values of the M2m and M1mc coatings (0.3 MPa), both for thin and thick layers, were also considered as satisfactory for the desired application on the concrete’s surface. The celluloses can enhance the adhesion of magnesium hydroxide coatings onto concrete; the respective adhesion mechanism is still under investigation, but it is considered possible that hydrogen bonds may be formed, between the hydroxyl groups of the celluloses and the surface of concrete.After the period of 1 month, during which the specimens were subjected to the influence of low water flow circulation (0.05 m/s), it was observed that the coatings showing the best adhesion performance were the M1mc and M1m; the M2m sample was next, presenting a slightly lower water resistance. Specifically, the M1m showed better water resistance, when compared to M1mc . The res2SO4 0.2 M), even under the low circulated experimental conditions, it was noticed that almost all coatings pulled-off almost immediately , except for the case of coatings M1m and M1mc, which pulled-off after a few minutes. According to the literature, different test methodologies have been used in order to study the degradation of concrete specimens through acid immersion. The standard methods, and the relevant studies, suggest the replacement of the acid solution, after each inspection period, or when the pH value exceeds a pre-selected value [However, when all samples were immersed in the acidic solution  enhanced the adhesion of magnesium hydroxide coatings on the concrete substrate, both in the cases of coatings with methyl-cellulose addition, and in the coatings with the combination of methyl-cellulose and carboxy-methyl-cellulose. The fact that the increased amount of additives can increase the adhesion properties of coatings onto the concrete surface is in agreement with the pull-off tests.After immersion for 20 min under the influence of higher water circulation velocities, the sample M1m showed still near-perfect coating adhesion performance (only some black spots can be observed), while in the case of M1mc much larger parts of the coating were removed . The larIn sewer pipes, the reduction of pH values on the surface of concrete can create the ideal conditions for acidophilic microorganisms to develop, eventually leading to the degradation of the concrete substrate. The produced biogenic sulfuric acid reacts with common calcium compounds of the cement paste, resulting in the formation of gypsum. The coatings suggested in this study can offer corrosion protection in two ways, i.e., (a) by retaining the alkaline surface pH values , and (b) by reacting with  the biogenic sulfuric acid. The ability of coatings to maintain higher  pH values, and resistance to pH (acidic) lowering, was evaluated by using an accelerated method of spraying an appropriate sulfuric acid solution on the surface of the coated concrete specimens. As a result, the sprayed sulfuric acid can react  with the  coating, before reacting with the concrete surface and provide protection, whereas the coating is consumed.2-based coatings lie in the neutralization reaction with sulfuric acid. The fundamental reaction between the magnesium hydroxide and the sulfuric acid is given below:The anti-corrosive properties of the examined Mg(OH)Based on this reaction, 1 mol of magnesium hydroxide reacts with 1 mol of sulfuric acid, producing 1 mol of magnesium sulfate and 2 moles of water. As a result, the concrete surface is protected, because the sulfuric acid cannot reach its surface, as it reacts with the coating. It is important to note that the celluloses were added as adhesion additives and not as reactant agents. However, the effect of cellulose addition amount on the anti-corrosive properties of magnesium hydroxide was studied by using three different cellulose amounts  and two different cellulose combinations  coatings with methyl-cellulose, and (b) coatings with methyl-cellulose and carboxy-methyl-cellulose).The coated and uncoated concrete specimens were sprayed with a concentrated sulfuric acid solution (4 M), as described in For the thick coatings, the two specimens containing the highest amount of methyl-cellulose  showed lower consumption rates , while regarding the coatings containing the mixture of both celluloses, the M2mc coating exhibited the slowest consumption rate . In both series of thick coatings, those with the smallest cellulose amount  exhibited the shortest consumption time . Regarding the examined cellulose amounts in the prepared coatings, the coatings containing 1% and 0.4% wt. of celluloses were found to last longer than the coatings containing 0.1% wt. of cellulose. However, in order to achieve a more concrete conclusion considering the relation between the consumption time of the coatings and the amount of added cellulose, more experiments with different cellulose amounts should be performed. In contrast, the thin layers of coatings did not present any difference between the coating samples. These results indicate that the coating thickness affects the diffusion of acid through the coating (reaching the concrete surface), and consequently the coatings’ consumption/neutralization rate/time.2/100 mL H2O). The acid is added at the rate of one drop (0.5 mL) per minute.The fundamental surface properties of hardness and roughness were presented as relative values, in respect to the initial ones of concrete specimens, i.e., those measured before acid spraying. Studies have shown that concrete’s hardness and roughness can change after it is subjected to sulfate attack ,36. In tThe uncoated concrete specimens exhibited about 19% reduction from the initial hardness value of the concrete surface, while the coated specimens showed a substantially lower reduction of hardness. This indicates that the coatings seemed to protect sufficiently the concrete surface and its original properties. The hardness of the concrete surface was generally reduced, due to the formation of gypsum onto the surface, which is produced when the sulfuric acid reacts with the cement paste, neutralizing the alkaline components. In the case of the coated concrete specimens, the coating can react with the sulfuric acid ; therefore, it can preserve (protect) the concrete’s initial surface hardness.When the cement paste reacts with sulfuric acid and gypsum is formed, the inert aggregates content is left exposed on the surface, or they can even remove from the concrete substrate when they become fully exposed. Some researchers showed that chemical attack can lead to an increase of concrete surface roughness . The resThe coatings (and the formed by-products) were scratched/removed from the concrete specimens after they were sprayed with 4 M sulfuric acid solution, using the daily stoichiometry 17% of the respective reaction, according to the procedure described in 4) formation for all the examined coatings, are presented in The series of coatings containing both celluloses (mixture) presented more gypsum formation, than the coatings containing only methyl-cellulose, when using similar coating amounts. Apparently, the addition of carboxymethyl-cellulose enhanced the formation of gypsum. For example, despite the same consumption time, the M1mc coating exhibited a lower brucite amount formation and a higher gypsum amount than the M1m coating. This result indicates that the optically observed coating  which was collected for the M1mc coating, provoked the increased gypsum formation, with respect to the brucite amount. A possible explanation is that the inner morphology of the M1mc coating could permit the diffusion of sulfuric acid through the coating mass; hence, reaching the concrete surface and leading to the formation of gypsum on the coating–concrete interface. Therefore, and despite the fact that both coatings presented the same consumption time, they exhibited different corrosion protection abilities.The M3mc coating presented the greatest decrease in the brucite phase among all the examined coatings, whereas the M3m exhibited the greatest gypsum formation of the coatings containing methyl-cellulose. These results showed that the amount of added cellulose also affects the consumption of coatings, as well as the formation of corrosion (by-)products . AccordiThe micrographs of four selected dried coatings are presented in 2 aggregates, which may slow down its rapid reaction with sulfuric acid. The formation of large aggregates  may lead to a smaller exposed surface of magnesium hydroxide that is available to react, than when smaller aggregates are formed  [The observed different surface characteristics may affect the reaction between the coatings and the sulfuric acid. The addition of celluloses seemed to lead to the creation of Mg(OH)nd M3mc) . This faHowever, the aggregates of the M1mc coating presented a different morphology than the M1m coating. Probably, this morphology affected the reaction mechanism of the M1mc coating, leading to increased gypsum formation, according to the aforementioned XRD quantitative results. As explained in 2-based coatings were examined, regarding their application onto the surface of concrete sewer pipes, acting as potential bio-corrosion inhibitors. Their adhesion ability was found to depend on the amount of cellulose addition. The magnesium hydroxide-based coatings seemed to protect the concrete surface longer than the respective theoretically calculated consumption time, considering the neutralization reactions with sulfuric acid, and seemed to maintain the surface pH towards alkaline values; therefore, inhibiting the action of acid-producing bacteria.In this research, six different Mg(OH)Among the various coatings, containing methyl-cellulose, that with 1% wt. content of celluloses showed better adhesion ability, water resistance, and acid attack resistance properties. According to the quantitative evaluation of the XRD results, the coatings with 1% and 0.4% wt. of methyl-cellulose  exhibited the lowest formation of gypsum, after spraying with sulfuric acid between all examined coatings, indicating better corrosion protection. Furthermore, the corrosion protection ability of the studied coatings was supported by the respective hardness and roughness results of concrete’s surface, in comparison with the uncoated (but sprayed) concrete specimens.SEM micrographs showed the different surface roughness among the examined coatings. In addition, the coatings with the higher cellulose content exhibited the formation of aggregates that may affect the reaction  between magnesium hydroxide and sulfuric acid. These aggregates may block the (otherwise) intense reaction activity, and could lead to the decrease of the coating’s relevant consumption rate .Regarding future research studies, the authors will examine the effectiveness of the most promising applied protective coatings, but used to protect a regular size concrete pipe under realistic sewer network conditions. Moreover, raw materials, also based upon magnesium hydroxide but with different physical properties, will be studied, in order to evaluate the effect of these properties on the protection ability for concrete corrosion.


Immunoglobulin G4-related disease (IgG4-RD) is a multi-organ disorder predominantly occurring in middle-aged to elderly male patients characterized by multi-organ fibrosis, specific pathological findings of storiform fibrosis with IgG4-positive plasma cell infiltration, and elevated serum IgG4 level. We herein report a rare presentation of IgG4-RD forming an isolated mass in the middle mediastinum mimicking a mediastinal tumor and discuss the clinical significance of mediastinal IgG4-RD.An 82-year-old male patient without any symptom was referred due to left middle mediastinal mass (3.8 × 2.4 cm). Because of suspected lymphoma, Castleman’s disease, and lymphangitis due to tuberculosis, we performed a thoracoscopic resection for diagnosis and treatment. The mass was yellowish white with well-encapsulated, and storiform fibrosis with plasma cell infiltration, and obliterative phlebitis were observed microscopically. Additional immunohistochemical stain revealed IgG4-RD. Other radiological findings and serological results did not show evidence of other organs being affected from IgG4-RD nor autoimmune diseases. He is now followed at outpatient clinic without additional treatment for over a year, and an enhanced computed tomography does not show any recurrence.It was a rare presentation of IgG4-RD forming isolated middle mediastinal mass, which suggests that we might suspect IgG4-RD for undetermined mediastinal mass in case of middle to elderly male patient. Immunoglobulin G4-related disease (IgG4-RD) was firstly reported as a sclerosing pancreatitis, which was characterized by a storiform fibrosis with IgG4-positive plasma cell infiltration and elevation of serum Immunoglobulin G4 . Till daAn 82-year-old male patient without any symptoms was referred to our institute due to an abnormal shadow detected by chest X-ray in his annual health examination. An enhanced computed tomography showed a homogenous round-shaped mass 3.8 × 2.4 cm in diameter located in left middle mediastinum Fig. a–c. Howe4 (14–34 × 104); eosinophil count, 172 (21–252/µL); myeloperoxidase-anti-neutrophil cytoplasmic antibody (MPO-ANCA), < 0.5 (< 3.5 IU/mL); anti-Scl-70 antibody, < 1.0 (< 10 U/ mL); anti-Ro/SSA antibody, < 1.0 (< 10 U/mL); and anti-La/SSB antibody, < 1.0 (< 10 U/mL), which did not meet the exclusion criteria. Therefore, the diagnosis was probable IgG4-RD. He was discharged at 6 postoperative days uneventfully. He is now followed at outpatient clinic without additional treatment for over a year, and an enhanced computed tomography does not show any recurrence or any other malignancy. The survey for possible another systemic disease using F-18–2-fluoro-2-deoxy-D-glucose positron emission tomography computed tomography postoperatively did not detect other affected organs from IgG4-RD nor autoimmune diseases  met the criteria of a “probable” case of IgG4-RD . FurtherIgG4-RD is a multi-organ affected disorder predominantly in middle to elderly male patient characterized by multi-organ fibrosis, specific pathological findings of storiform fibrosis with IgG4-positive plasma cell infiltration and elevation of serum IgG4 , 2. AmonAs for the etiology of IgG4-RD, a recent investigation has revealed that an imbalance of immune regulatory system, which activates Th2-dominant regulatory T cell, was associated with specific fibrotic change in affected organs . AlthougConsidering the treatment for IgG4-RD, immunosuppressive agents, such as glucocorticoids, are quite effective in sclerosing pancreatitis and another IgG4-RD , 2. In pWe experienced a rare presentation of IgG4-RD with mass formation in the middle mediastinum, requiring correct pathological findings and several serological examinations to distinguish from lymphoproliferation diseases and autoimmune disorders. More numbers of underdiagnosed IgG4-RD in mediastinum might be seen in clinical practice, and those case studies and longer follow-up could clarify the nature of the disease, treatment, and prognosis in future.


In this research, the influence of reinforcement bars on concrete pore structure and compressive strength was experimentally investigated. Concrete samples with two mixture ratios and nine reinforcement ratios were provided. Tests were conducted on concrete pore structure and compressive strength at three ages . It was found that reinforcement bars changed the concrete pore structure. In terms of size, the pore structure of concrete increased with the increase of reinforcement ratio. At the same age, concrete compressive strength in reinforced concrete specimens saw a gradual reduction when reinforcement ratio increased. A formula was proposed to calculate the compressive strength of concrete in reinforced specimens according to the strength of unreinforced concrete. Concrete is a cement-based material, whose strength is affected by pores and microcracks, especially under the action of thermal loads ,2,3,4,5.In engineering practices, concrete is often combined with reinforcement bars to form a reinforced concrete structure. The existing studies showed that there are interfacial transition zones (ITZ) in reinforced concrete structures between reinforcement bar and concrete. The porosity of concrete in ITZ area is higher than that in non ITZ area , therebyBased on the previous findings about the connection between concrete strength and porosity and changes in concrete pore structure by restraints, it could be reasonably inferred that the pore structure of reinforced concrete would be greater than that of unreinforced concrete in the shrinkage process of concrete because of the restraint effect of reinforcement, which caused the strength of reinforced concrete to be lower than that of unreinforced concrete. However, this reference remains to be confirmed by research.In the early phase of construction, it is necessary to carry loads by scaffolding because of low concrete strength. According to Chinese standards , the remThis research experimentally investigated the influence of reinforcement bars on concrete pore structure and compressive strength. Concrete samples with two mixture ratios and nine reinforcement ratios (0–6.56%) were given. At different ages , the mercury intrusion method was adopted to test the pore structure of concrete samples with two mixture ratios and a reinforcement ratio of 0%, 1.14%, 3.24%, and 6.56%. Moreover, the compressive strength of all types of samples was also tested. All specimens were well vibrated before testing. A comparison was made between concrete pore structure and compressive strength with different reinforcement ratios to study the changes in concrete pore structure and compressive strength after reinforcement.3, a fineness modulus of 3.0, and a particle size distribution of 0.2–4 mm. Crushed limestone was used as coarse aggregate with a specific gravity of 2.8 g/cm3 and a particle size range of 5–20 mm. The superplasticizer used is polycarboxylate superplasticizer with a water-reducing rate of 30–35%. The information of reinforcement bars used in this study is shown in Two different concrete mixing ratios were applied in this study . Cement Produced by a Teflon mold, specimens whose dimension is 200 mm × 200 mm × 500 mm were adopted in the present study  were used in the present investigation . ReinforAfter 3 d, 7 d, and 28 d, three cylindrical samples whose diameter is 100 mm and height is 200 mm (height/diameter = 2) were drilled from each type of specimen to measure the concrete compressive strength (according to the JGJ/T384 standard ) Figure. The tesConcrete pore structure with a reinforcement ratio of 0%, 1.14%, 3.24%, and 6.56%  was tested. The remaining specimens after drilling were used as the samples for concrete porosity tests. The concrete samples were first crushed and then filtered by a sieve to collect concrete particles with a size of 2.5–5 mm. Acetone was applied to prevent concrete hydration, and a vacuum dryer was used to dry the samples. At last, concrete pore structure was tested by an automatic mercury porosimeter that has an aperture measurement range of 0.003–1000 μm, low-pressure analysis pressure range of 0–345 kPa, high-pressure analysis pressure range of 101.325–414000 kPa, and a resolution ratio of 69 Pa.It was also observed that the placement of reinforcement bars led to a significant change in concrete pore structure whose size saw a great increase when reinforcement ratio increased. For example, the critical diameter of C60-R8 concrete with a reinforcement ratio of 6.56% was 44.30 nm at 28 d, which is higher than those of C60-R5 (ρ = 3.24%), C60-R1 (ρ = 1.14%), and C60-R0 (ρ = 0%) specimens . Similar trends were also observed in C30 concrete specimens, other ages, and pore structure parameters.ρS is the parameter of concrete pore structure corresponding to the reinforcement ratio ρ; S0 represents the parameter of concrete pore structure corresponding to the reinforcement ratio of 0%; n is the increase in the parameters of pore structure for each unit of increase in reinforcement ratio.n , a decline appeared in concrete pore structure because of filling by hydrated products and capillary stress, as shown in From Equation (2), it can be seen that restraint stress saw an increase when reinforcement ratio increased, thereby reducing concrete pore structure because of capillary stress. Therefore, the experimental results indicated that the pore structure of concrete increased when reinforcement ratio increased.However, compressive strength gradually decreased when reinforcement ratio increase in specimens, which are the same in strength but different in reinforcement ratio. For specimens with the same strength grade, the highest compressive strength was always obtained in specimens whose reinforcement ratio is 0%. For example, the compressive strength of C30-R8 (ρ = 6.56%) at 28 d was 24.6 MPa, which is lower than that of C30-R7 (ρ = 5.16%), C30-R6 (ρ = 3.95%), C30-R5 (ρ = 3.24%), C30-R4 (ρ = 2.61%), C30-R3 (ρ = 2.05%), C30-R2 (ρ = 1.56%), C30-R1 (ρ = 1.14%), and C30-R0 (ρ = 0%) .The relationship between concrete compressive strength and pore structure (mainly porosity) could be described ideally in a linear, exponential, or polynomial form ,11,12. IThis study showed that reinforcement bars would reduce the decline of concrete pore structure caused by capillary stress, thereby causing reinforced specimens to be inferior to unreinforced ones in compressive strength. Concrete compressive strength gradually decreased when reinforcement ratio increased. In engineering practices, the risk of accidents may be increased if the removal of scaffolding is determined by the compressive strength of unreinforced concrete.cρf and fc0 are the compressive strength of concrete with a reinforcement ratio of ρ and 0% respectively; b refers to a calculation parameter and is equal to 5.1 in this study.It is difficult to directly test the pore structure parameters of concrete at the construction site. Therefore, the connection between the reinforcement ratio and compressive strength of concrete is established in Equation (3), which can be used to evaluate the strength of reinforced concrete according to the known strength of unreinforced concrete.(1)The size of concrete pore structure gradually decreased during cement hydration because of the filling of hydrated products and capillary stress. The proportion of pores whose diameter ranges from 0 to 50 nm increased, whereas that of pores whose diameter is above 50 nm declined noticeably.(2)The size of concrete pore structure gradually increased when reinforcement ratio increased. A linear relationship was found between pore structure parameters  and reinforcement ratio.(3)At the same age, the compressive strength of reinforced concrete specimens was inferior to that of unreinforced ones and gradually declined with the increasing reinforcement ratio.(4)In engineering practices, the risk of accidents may be increased if the removal of scaffolding is determined by the compressive strength of unreinforced concrete. A formula was proposed in the present study to forecast the strength of reinforced concrete using unreinforced concrete strength and reinforcement ratio. The predicted results well agreed with the measured ones.The present study experimentally investigated the effects of reinforcement bars on concrete pore structure and compressive strength that were tested at three ages in the case of various reinforcement ratios. The main observations are presented below:


Limosilactibacillus reuteri LRE02-DSM 23878 and Lacticaseibacillus rhamnosus LR04-DSM 16605) on the prevention of AAD in an outpatient pediatric setting. Questionnaires were delivered to pediatricians by each patient/parent during the visits after antibiotics and probiotics treatment to monitor physiological parameters. The primary outcome of both groups (probiotics and no probiotics treated) was the evaluation of the prevalence of AAD between the two groups. Evaluation of stool consistency using the Bristol Stool Scale (BSS) score was performed, as well as the evaluation of AAD duration, frequencies of daily evacuation, and the beginning of diarrhea and weight loss during AAD in both groups and related to antibiotic categories. Results indicated that probiotics, at the recommended dosage of 1.2 × 109 CFU (Colony Forming Unit) per day for 30 days, are associated with lower rates of AAD and a decreased number of days with diarrhea, independent of the type of antibiotic used. Moreover, the use of probiotics resulted in a normal stool consistency in a shorter time period, as evaluated by the BSS.Probiotics are living microorganisms, which, upon oral ingestion, may prevent antibiotic-associated diarrhea (AAD) through the normalization of an unbalanced gastrointestinal flora. The objective of this study was to evaluate the benefits of a probiotic combination ( Antibiotic-associated diarrhea (AAD) is defined by the presence of diarrhea with three or more soft/liquid stool evacuations within 24 h or an acute and evident change of fecal pattern during antibiotic treatment or also a few weeks later, without other demonstrable causes .Clostridium difficile [Different mechanisms may determine AAD: a direct toxic effect of antibiotics on the intestine, an altered digestive function secondary to reduced concentrations of gut bacteria, and the over-growth of pathogenic microorganisms . Mostly,ifficile .AAD occurs in about 5–30% of patients either early during antibiotic therapy or up to two months after the end of the treatment ,5,6.The frequency of antibiotic-associated diarrhea depends on the antibiotic type , dosage, number of antibiotic prescriptions as well as by host factors  .In the pediatric population, AAD represents a healthy concern being that the burden and costs are also not well documented by national surveillance studies . The AADRecent trials and metanalysis suggest that specific probiotics are useful in AAD prevention in children ,11. Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus) or Saccharomyces boulardii at 5–40 billion colony forming units (CFUs)/day appeared to improve NNT [Goldenberg et al. (2015) performed a systematic literature review, which reported moderate evidence for a protective effect of probiotics in preventing AAD, with an NNT (number needed to treat) of 10. Among the various probiotics evaluated, rove NNT . In a recent review, Guo et al. (2019) added valuable information on AAD prevention by probiotics. In particular, dosage appeared to account for the substantial reduction in the NTT. The authors also reported the subgroup effect based on high dose probiotics (≥5 billion CFUs per day), which revealed an NNT of six . OverallLimosilactobacillus reuteri LRE02–Lacticaseibacillus rhamnosus LR04   was chosen respecting the new rules recently established by a new European Society of Gastroenterology, Hepatology, and Pediatric Nutrition (ESPGHAN) position paper [The present work aims to evaluate, in a primary care setting, the effect of a combination of two microencapsulated probiotics on AAD in a vast cohort of children who underwent antibiotic therapy. The probiotic mixture  of on paper , i.e., dA survey methodology was conducted for collecting data reported by all the Surveyflor Group, which includes 68 general pediatricians located at the national territory . The study had been previously approved by the Institutional Review Board, in which many pediatricians in the national territory were included. The survey consisted of a specific questionnaire administered to each patient/parent and delivered to the medical doctor (MD) who collected the data in an electronic online repository. Based on the anamnestic, clinical status, and the outcome of the survey described below, MDs recruited and collected information from the parents and during the visits of each patient enrolled. Inclusion criteria are the following: age 3 years or less, antibiotic therapy less than 24 h ago, written informed consent. Exclusion criteria: acute and/or chronic diarrhea before enrollment, diagnosis of chronic gastrointestinal diseases or diagnosis of other chronic diseases, immunodeficiencies, use of probiotics in the previous two weeks before enrollment, administration of antibiotics 4 weeks before enrollment, prematurity.Limosilactobacillus reuteri LRE02  and Lacticaseibacillus rhamnosus LR04  (Antibiotic–Probiotic group: A), or only antibiotics (Antibiotic group: B). The probiotic combination manufacturer recommended a dosage of five drops/day  for 30 days. The AAD episodes in group A and group B were recorded for one month. The questionnaires were delivered by each patient/parent during the visits after 5 (Time 0: T0) and 30 days (Time 1: T1) from the end of antibiotic administration. The primary outcome was the evaluation of the prevalence of AAD between the two groups. Secondary outcomes were: (1) evaluation of stool consistency using the Bristol Stool Scale (BSS) score [Type 1: Separate hard lumps, like nuts (difficult to pass and can be black);Type 2: Sausage-shaped, but lumpy;Type 3: Like a sausage but with cracks on its surface (can be black);Type 4: Like a sausage or snake, smooth and soft (average stool);Type 5: Soft blobs with clear cut edges;Type 6: Fluffy pieces with ragged edges, a mushy stool (diarrhea);Type 7: Watery, no solid pieces, entirely liquid (diarrhea).Longitudinal study design, administered online, included patients who underwent antibiotic therapies for different infections see . All patS) score , in bothTypes 1 and 2 indicate constipation, with 3 and 4 being the ideal stools as they are easy to defecate while not containing excess liquid, 5 indicating lack of dietary fiber, and 6 and 7 indicating diarrhea.®, San Diego, CA, USA). The following tests were performed to check the normality  of the distributions: D’Agostino and Pearson omnibus normality test, Shapiro–Wilk normality test, and Kolmogorov–Smirnov normality test. Following these tests, one-way ANOVA and multiple comparisons (Tukey’s multiple comparisons test) were done to compare groups. For non-parametric distributions, the Mann–Whitney test was used. Otherwise, the standard Student’s t-test was performed. The Chi-squared test was used to compare the prevalence between the groups. A p-value less than 0.05 was considered statistically significant.The statistical analyses were performed with GraphPad Prism version 8.01 for Windows  of 28 ± 21 months. Group A (antibiotics plus probiotics) included 5048 patients , while group B (antibiotics only) included 4912 patients (49.3%). Infections requiring antibiotic prescription are reported in p value: 0.0357), otitis (Ot) , and gastroenteritis (Ge) .p value < 0.0001). Thirty days after (T1), the mean value in group A was 3.7 ± 1.2 , and in group B was 4.2 ± 1.4  (p < 0.0001).Stool consistency results are reported in p < 0.0001, Mann–Whitney test). Besides, the number of days with diarrhea in group A (2.8 ± 1.3 days) was significantly lower than those reported in group B (3.2 ± 1.4 days) (p < 0.001)  A. The nu< 0.001) B. The di< 0.001) C. Group Among all the three physiological parameters reported in In AAD patients (N = 4897), stool consistency was monitored all throughout the antibiotic administration and also at T0 and 30 days after (T1). The results, summarized in The prevalence of prescribed antibiotics among group A and group B is summarized in p < 0.001)  . For morRegarding stool frequency, the BSS showed at T0 and T1, for all the three main antibiotic categories, a higher score in group B than in group A  except fp < 0.001 for penicillins and cephalosporins, p = 0.017 for macrolides); macrolides showed the maximum average of duration  compared to penicillins (3.1 ± 1.4) and cephalosporins (3.2 ± 1.4). The number of stools/day (p = 0.035). Diarrhea in group A started earlier compared to group B regarding penicillins (p = 0.01) and cephalosporins (p = 0.013), while no differences were found for the macrolides (na = not applicable) (The duration (in days) of diarrhea A was higools/day B was simlicable) C.t-test.The stool consistency in AAD-positive patients (N = 4897) at T0 and T1 shows statistical differences between the two groups with respect to the three antibiotic categories . CompariThe World Health Organization defines probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” . Dr. Metchnikoff opened the “era” of probiotics because he was the first to propose that ingesting certain bacteria could help replace harmful microbes in the body .9 live cells per day [9 live cells of at least one of the strains, including Saccharomyces cerevisiae, or lactic acid fermenters, such as Streptococcus thermophilus or Lactobacillus bulgaricus [As reported in the literature, the minimum quantity sufficient to obtain temporary colonization of intestines by a microbial strain is 10 per day . Therefolgaricus ,19.Lactobacilli, recently reclassified in 23 different genera  and bifiThe use of probiotics is now widespread both for intestinal and extraintestinal disorders, especially in pediatrics ,22,23. AChildren are estimated to use three times more antibiotics than adults  and AAD It is also noteworthy that dysbiosis related to antibiotic use in the first years of life is a risk factor for obesity , functioLacticaseibacillus rhamnosus is a probiotic strain, which when compared to other probiotics, is one of the most appropriate for preventing AAD due to its modest NNT [Limosilactobacillus reuteri has even been extensively studied in several intestinal conditions, and its therapeutic and preventive effects have been documented  [A Probiotics’ mechanism of action is exerted through the modulation of the content of gut microbiota, maintenance of the integrity of the gut barrier, prevention of bacterial translocation and the modulation of the local immune response by the gut-associated immune system. In particular, dest NNT . Limosilressors) .Limosilactobacillus reuteri (formerly Lactobacillus reuteri LRE02) 2 × 108 CFU and Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus LR04) 1 × 109 CFU on the prevalence of AAD. These probiotic strains are free of plasmids that carry antibiotic resistance, are microencapsulated, and have a shelf life  that is guaranteed for 24 months; moreover, in vitro studies have demonstrated a synergistic ability of these two strains when simultaneously cultivated. The product is “allergen-free” [In our randomized intervention study, we evaluated the efficacy of a new double-strain probiotic formulation which included en-free”  and withen-free” ,34. At ten-free” . One of en-free” . As staten-free” ,34.In our study, the prevalence of AAD was about 50%, regardless of the antibiotic used. This high prevalence, however, previously reported , could bThe most prescribed antibiotics, to treat a range of infections reported in Our results showed a significant reduction in AAD prevalence in group A (probiotic plus antibiotics) (38.5%), compared to the control group B  (59.9%).The type of antibiotics and the reason for their administration do not appear to affect the outcome of the probiotic on AAD, as previously reported for one of two probiotics recommended by ESPGHAN for AAD prevention ,38.L. reuteri and different Lactobacillus species were shown to moderate jejunal motility within minutes [L. rhamnosus GG and found a significant shortening of smooth muscle cells that had an impact on motility [Stool consistency, evaluated with BSS score, was significantly better in group A (mean BSS score 4.5 ± 1.5) than in group B (mean BSS score 4.9 ± 1.6), for all antibiotic categories, both at the end of antibiotic therapy (T0) and after 30 days (T1). In AAD patients, the mean BSS score at T0 was not different between the two groups, regardless of the antibiotic categories used; however, at T1, the BSS score was significantly better in group A than in group B. Surprisingly, in AAD patients, diarrhea started earlier in group A than in group B, regardless of the type of antibiotic used. This effect could be related to the influence of gastrointestinal commensals on motility as previously observed in vivo and ex vivo in mice and rat models, in which the administration of  minutes ,40,41. Amotility . Evidencmotility .However, the number of days with diarrhea was significantly lower in group A than in group B (2.8 ± 1.3 days vs. 3.2 ± 1.4 days), depending on the antibiotic used, as macrolides showed the worst duration. We did not find any difference in the number of stools/day and weight loss in AAD patients in either of the two groups. This is in line with the other studies that have shown that probiotics are associated with a reduction in the mean duration of AAD of 18 h, without having an effect on the number of stools per day ,23.Our study is not a placebo-controlled study, and a limitation could be the self-reported symptoms made by parents. However, the large number of patients enrolled, and the utilization of the BSS certainly helps to reduce possible biases.This study has demonstrated that using our probiotic combination from the beginning of antibiotic therapy is also helpful in reducing the mean duration of diarrhea  as well as in obtaining a normal stool consistency in a shorter time.Limosilactobacillus reuteri LRE02–Lacticaseibacillus rhamnosus LR04, at the recommended dosage of 1.2 × 109 CFU of both microorganisms daily for 30 days, is associated with lower rates of AAD in children (aged one month to 18 years). A reduction in AAD prevalence, the reduced number of days with diarrhea and a better stool consistency were demonstrated regardless of the type of antibiotic used or the reason for their prescription. However, a double-blinded clinical trial is required to further confirm these results.The probiotic mixture of


BDNF gene that affects neuroplasticity and has been associated with cognition, brain structure and function. The aim of this study was to clarify the relationship between BDNF Val66Met polymorphism and neuronal oscillatory activity, using the electroencephalogram (EEG), in a normative cohort. Neurotypical (N = 92) young adults were genotyped for the BDNF Val66Met polymorphism and had eyes open resting-state EEG recorded for four minutes. Focal increases in right fronto-parietal delta, and decreases in alpha-1 and right hemispheric alpha-2 amplitudes were observed for the Met/Met genotype group compared to Val/Val and Val/Met groups. Stronger frontal topographies were demonstrated for beta-1 and beta-2 in the Val/Met group versus the Val/Val group. Findings highlight BDNF Val66Met genotypic differences in EEG spectral amplitudes, with increased cortical excitability implications for Met allele carriers.The brain-derived neurotrophic factor (BDNF) protein is essential for neuronal development. Val66Met (rs6265) is a functional polymorphism at codon 66 of the  The BDNF Val66Met (rs6265) polymorphism is a functional polymorphism in the BDNF gene where a valine  to methionine (Met) amino acid substitution at codon 663 occurs. The BDNF Val66Met polymorphism causes impaired activity-dependent secretion of BDNF in individuals carrying one or more copies of the Met allele relative to non-Met carriers 4. The Met allele has been associated with cognitive impairment, particularly memory5, reduced hippocampal volume6, increased risk of depression8, anxiety-related behaviors9, neurodegenerative disorders10, particularly Alzheimer’s disease (AD), and psychiatric disorders such as schizophrenia11. Due to its neuroprotective effects, BDNF has also been implicated as a potential therapeutic agent for AD12.Brain-derived neurotrophic factor (BDNF) is an essential protein widely expressed in the brainBDNF Val66Met polymorphism has been linked to memory function in healthy adults, with young adult Met carriers  exhibiting poorer episodic memory performance  than their Val homozygous peers12. Young adults (aged 24.0 ± 0.8 years) heterozygous for the Val/Met genotype also showed lower working memory ability (as measured by a 2-back task) when compared to the Val/Val homozygotes4.The BDNF Val66Met polymorphism, white and grey matter structural alterations, and functional hemodynamic brain changes in healthy individuals13. Structural magnetic resonance imaging (MRI) studies have reported a reduction in the volume of temporal and occipital lobe grey matter14, hippocampus15, amygdala16, and prefrontal cortex15 in healthy Met carriers  compared to Val/Val homozygotes of the BDNF Val66Met polymorphism. Healthy middle-aged adults (46.4 ± 6.8 years) carrying one or more copies of the Met allele showed lower hemodynamic responses (blood oxygen level dependent [BOLD]) in the right superior frontal gyrus and the middle occipital gyrus than the non-Met carriers  during n-back working memory tasks, as measured by functional MRI (fMRI)17. Alterations in hippocampal and lateral prefrontal activation in Met/Met homozygotes, and a localised reduction in hippocampal grey matter volume compared to Val carriers , as measured by fMRI and structural MRI (respectively) have also been demonstrated18.There is also an established relationship between the BDNF Val66Met polymorphism on waking resting state EEG activity in adults across several conditions. In healthy adults (aged 24.0 ± 0.8 years), EEG alpha power (9.75–11.75 Hz) in prolonged (40 h) wakefulness was roughly doubled in Val/Val homozygotes compared to the heterozygous counterpart 4. In another study of participants assessed for trait depression, homozygous Met allele carriers (aged 36.9 ± 12.6 years) exhibited higher delta (1.5–3.5 Hz) and theta (4.0–7.5 Hz) power, and lower alpha (8–13 Hz) and beta (14.5–30 Hz) power in both eyes-open (EO) and eyes-closed (EC) resting-state EEG, relative to participants homozygous for the Val allele7; this Met/Met-related modulation of neuronal activity was associated with trait depression. Similarly, another study found a negative correlation between parietal-occipital alpha power (8–13 Hz) and depression severity, which was maximal in the Met/Met genotype group8. To date, all prior studies investigating the effects of the BDNF Val66Met polymorphism on EEG frequency spectra have done so in a clinical context , with no studies characterising BDNF Val66Met genotypic differences in EEG spectra in a neurotypical context. It should be noted that a range of other studies have investigated BDNF Val66Met polymorphism-related differences in EEG-derived event-related potentials (ERPs)20, EEG recorded during sleep4, or under general anesthetic21, magnetoencephalography (MEG)22 and non-invasive brain stimulation such as transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS)25; these are beyond the scope of the current study and will not be reviewed further.Resting state electroencephalographic (EEG) spectral activity provides a measurement of the intrinsic activity of neuronal populations and large-scale neuronal networks, and is linked to a range of factors such as cognitive function, task performance, clinical phenotypes, and arousal states. Several studies have explored the effects of the BDNF Val66Met polymorphism and EEG brain activity in neurotypical young adults. It was hypothesised that compared to the Val/Val genotype group, individuals carrying one or more copies of the Met allele  would have greater delta and theta, and lower alpha activity7, and that the Val/Met genotype group would have greater beta activity than the Met/Met genotype group7. To improve comparability with previous EEG studies and test key hypotheses19, the three genotype groups were compared, rather than testing Met allele carriers versus non-Met carriers.The aim of this study was to further understand the relationship between variants of the Individuals enrolled in the School of Psychology at the University of Wollongong were recruited to participate in the study in return for course credit. Written informed consent was obtained, followed by completion of a demographic and screening questionnaire. Saliva samples were collected using the OrageneDNA collection kit , and EEG measures were recorded. Participants were instructed to refrain from consuming psychoactive substances for at least 12 h prior to EEG testing, and from consuming tea, coffee, alcohol, and cigarettes for at least 2 h prior to saliva collection. Eligibility criteria were self-reported, and included no prior use of psychotropic/central nervous system medication, no neurological or psychiatric illnesses, normal hearing, and normal or corrected-to-normal vision. Ethical approval for this study was obtained from the University of Wollongong and Illawarra and Shoalhaven Local Health District Health and Medical Human Research Ethics Committee. This research was conducted in accordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects prepared by the Council for International Organisations of Medical Sciences (CIOMS) in collaboration with the World Health Organisation (WHO), and the Australian National Statement on Ethical Conduct in Research Involving Humans. Written informed consent was obtained from all study participants, and participants were free to withdraw at any time without penalty.20, following sample collection, saliva was stored at room temperature until DNA was extracted as per the manufacturer’s recommendations . After extraction, DNA samples were quantified using a NanoDrop ND-1000, and stored at − 20 °C. High-throughput SNP genotyping was performed using the MassARRAY genotyping assay , with the analysis performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Extension primer design, and selection were performed using MassARRAY Designer Software . Control samples were run in parallel with the tested samples (on each plate), allowing for accurate evaluation and quality control checks of each of the steps involved . Each participant was genotyped as homozygous Val/Val (GG), heterozygous Val/Met (GA), or homozygous Met/Met (AA)20.As per Steiner et al.21. Participants were then instructed to fixate on a 10 × 10 mm gray cross centered on a black background, while EO resting EEG activity was recorded for 4 min.As per Steiner et al., 2018, participants were seated in an air-conditioned room 600–800 mm in front of a 19″ Dell LCD monitor, and completed an electrooculogram (EOG)/EEG calibration taskN = 33) sampled at 2000 Hz. In both setups, EOG was recorded using tin cup electrodes placed 2 cm above and below the left eye for vertical movements, and on the outer canthus of each eye for horizontal movements. Impedance was < 10 kΩ for all electrodes.Continuous EEG data were recorded DC–30 Hz with a Neuroscan Synamps 2 digital signal-processing system and Neuroscan 4.3.1 Acquire software  from 30 scalp sites , grounded by an electrode located midway between Fp1, Fp2, and Fz. Two recording setups were combined to obtain data: one used electrode caps with tin electrodes referenced to A1 and included A2 as a separate active channel for offline re-referencing (N = 59), sampled at 1000 Hz; the second setup was recorded using sintered Ag/AgCl electrodes referenced to the nose and recording M1 and M2 as active channels . Data were re-referenced to digitally linked ears (N = 59) or mastoids (N = 33) and extracted offline using Neuroscan Edit software ; the dataset recorded at 2000 Hz was resampled to 1000 Hz. The 4-min of data were segmented into 2000 ms epochs, each DC-corrected by baselining across the entire epoch, and any epochs containing artifact exceeding ± 75 μV were rejected. A visual inspection was conducted to further remove artifact-contaminated trials, and interpolate channels containing excessive artifact (> 70% epochs retained) with unaffected data from immediately surrounding sites. Data were quantified in MATLAB and EEGLAB (Version 14.0.0b). For each subject and epoch, spectral amplitudes were computed using Discrete Fourier Transforms with a 10% Hanning window; correction for the window was subsequently applied. Frequency data from 0.0 to 29.5 Hz were extracted with a resolution of 0.5 Hz before averaging across epochs. The spectral band amplitudes were calculated by summing the activity across the frequency bins for delta: 0.5–3.5 Hz; theta: 4.0–7.5 Hz; alpha-1: 8.0–10.5 Hz; alpha-2: 11.0–13.5 Hz; beta-1: 14.0–20.5 Hz; and beta-2: 21.0–29.0 Hz. This resulted in one datapoint for each subject, electrode, and band.EEG data were corrected for all eye-movement types using the Revised Aligned-Artifact Average (RAAA) EOG Correction Programversus Met/Met, Val/Val versus Val/Met, and Val/Met versus Met/Met. All F-tests reported were one-tailed  and had  degrees of freedom unless otherwise specified; α was set at 0.05 for all tests. It should also be noted that as this paper details results for multiple dependent measures , the frequency of Type I errors increases. However, this increase in frequency of Type I errors cannot be controlled by adjusting α-levels, because the probability of Type I error remains the same26. Mixed-model 3 × 3 × 3 MANOVAs with the within-subjects factors of sagittal  and coronal topographies  and between-subjects factor of genotype  were conducted for each EEG band using IBM SPSS Statistics, v22. To facilitate topographic analyses of EEG bands, data were pooled across the 30 electrodes to form 9 regions: frontal-left , frontal-midline , frontal-right ; central-left , central-midline , central-right ; posterior-left , posterior-midline , posterior-right . These regions are represented diagrammatically in Fig. 28 was used to examine the sources of each EEG frequency band. Source localisation was conducted on the grand mean EEG band amplitudes with the aim of complementing the statistical analyses of their scalp topography and assist with the neurobiological interpretation of the results.eLORETA  in collaboration with the World Health Organisation (WHO), and the Australian National Statement on Ethical Conduct in Research Involving Humans.All participants were provided with a copy of the participant information sheet and consent form, and written informed consent was obtained from all participants prior to ocmmencing data collection.All participants consented to the publication of their data in the deidentified aggregate form in which it is presented.Mage = 21.4, SD = 5.0 years, 67% female, majority Caucasian ethnicity). Table BDNF Val66Met genotype groups . Data were normally distributed based on the skewness and kurtosis of histogram plots. Genotype breakdowns across the sample were as follows: Val/Val n = 61 (66.3%); Val/Met n = 25 (27.2%); Met/Met n = 6 (6.5%). The allele frequency for G  was 74.6%, and A (encodes Met) was 25.4%. This did not differ from the Hardy–Weinberg equilibrium .The sample included 92 undergraduate students (r(180) = 0.80, p < 0.001); thus data were pooled and not treated separately for any further analyses. EEG spectra for each of the three BDNF Val66Met genotype groups at Fz, Cz, and Pz are shown in Fig. EEG spectral data for the two lab setups across the 30 scalp sites and six EEG bands were highly similar , larger on the right than the left , and in the midline compared with the hemispheres . Frontal amplitudes were greatest in the midline , and fronto-posterior amplitudes were greatest on the right . As shown in Fig. F = 7.101, p = 0.009, ηp2 = 0.07; Val/Met < Met/Met × L < R: F = 8.732, p = 0.004, ηp2 = 0.09), as was the right fronto-posterior enhancement . To improve readability, statistical effects are omitted from the remaining text; please refer to Table Delta amplitudes were greater frontally than parietally (F > P: p < 0.001) as well as midline enhancements (p < 0.001); these effects interacted, with frontal amplitudes greatest in the midline (p < 0.001). Theta was greater on the right fronto-posteriorly (p = 0.006). There were no main effects or interactions involving BDNF Val66Met genotype.Theta amplitudes showed frontal (p < 0.001), in the right hemisphere (p = 0.029) and in the midline (p < 0.001), with the midline enhancement largest frontally (p < 0.001). Alpha-1 was smaller centrally on the right (p = 0.001). This right-central reduction in alpha-1 was greater for Met/Met than for both Val/Val and Val/Met genotype groups .Alpha-1 amplitudes were largest in the posterior region (p < 0.001) that was greatest on the right (p = 0.009). There was a midline enhancement (p = 0.005) that was largest frontally (p < 0.001) and centrally (p < 0.001); central alpha-2 was larger in the left than right hemisphere (p = 0.026). The Met/Met group showed a reduction in this left hemispheric enhancement observed for both Val/Val and Val/Met genotype groups .Alpha-2 showed a parietal topography (p < 0.001), and fronto-parietally compared to centrally (p = 0.019); this was greatest in the midline (p = 0.044). Frontally, the interaction was largest in the midline compared to the right and left hemispheres (p < 0.001). The Val/Met genotype group showed greater frontal beta-1 activity compared to the Val/Val genotype group (p = 0.008).For Beta-1, amplitudes were maximal in the parietal region (p = 0.001) and fronto-parietally (p < 0.001), with the frontal enhancement being greatest in the midline (p = 0.022). As with beta-1, beta-2 amplitudes were larger frontally in the Val/Met genotype group compared to the Val/Val genotype group (p = 0.038).Beta-2 was enhanced frontally  have been linked to schizotypal personality disorder and schizophrenia29. Further work is required to ascertain whether the left hemispheric delta reduction observed here in healthy young BDNF Met homozygotes is indeed linked to such left anterior cingulate abnormalities, and whether this confers increased psychosis risk11. Of note, the presence of the Met allele variant has also been shown to have functional consequences in individuals with chronic aphasia; it may play a role in speech and language, which is largely laterialised. In a sample of individuals with chronic aphasia as a result of left hemisphere stroke, a naming task revealed a greater number of activated voxels in the right hemisphere of Val/Val homozygotes than Met/Met individuals30. Future studies may seek to characterise whether healthy Met allele carriers demonstrate lateralised differences in speech and language processing mechanisms.Right hemispheric and fronto-parieto-right delta amplitudes were greater for the Met/Met group compared to both homozygous Val/Val and heterozygous Val/Met genotypes. These genotype-related topographic differences seem to be driven by a right-parieto enhancement and a left hemispheric reduction for the Met/Met genotype group see Fig. . This is7. One previous study reported elevated theta power in the Met/Met genotype group as opposed to Val/Val homozygotes7, while another showed that theta is reduced in Met allele carriers during REM sleep and resting state EC conditions4. However, these prior studies were in the context of depression and sleep deprivation and elicited theta amplitudes during sleep states and a memory task, where theta has been shown to be maximal31. This suggests that a resting-state, eyes-open paradigm in healthy individuals may not be optimal to observe theta wave amplitude differences associated with BDNF rs6265.Theta amplitudes showed the typical midline frontal distribution, and this was reflected in the eLORETA source plots with diffuse sources in the cingulate gyrus and medial frontal gyrus. Counter to our hypothesis, there were no effects of genotype on theta amplitudes7. eLORETA analyses matched the dominant alpha-1/2 midline parietal distribution, with maximal sources in the cuneus, the paracentral lobule, and the postcentral gyrus. Alpha amplitudes largely originate in default mode network (DMN) functional hubs within the parietal and occipital lobes32 and have been linked with inhibition of active occipital cortical processing. The lower alpha-1 and alpha-2 amplitudes observed here in the Met/Met genotype group may point to dysregulation of occipital network processing, with less deactivation of active processing in DMN occipital hubs during an eyes open resting state33. Given that Met allele carriers of the BDNF Val66Met polymorphism have an increased risk of AD, it is important to note that reductions in alpha power (7.5–12.5 Hz) have also been linked to an earlier age of AD onset34, reflecting dysregulated cortical excitability most likely associated with excitotoxicity35. Future work is necessary to understand if reduced alpha power associated with the BDNF Val66Met polymorphism is also an early risk factor for cognitive impairment.Consistent with our hypothesis, the Met/Met genotype group exhibited reduced centro-parietal alpha-1 and left hemispheric alpha-2 amplitudes relative to both the Val/Val and Val/Met genotype groups. These results align with prior research which demonstrated that the Met/Met genotype group had reduced alpha power (8–13 Hz) compared to the Val/Val genotype group for temporal and parieto-occipital regionsn = 13 per group), however our investigation did not replicate these findings7. This may suggest an additive effect of Met allele frequency on BDNF activity and beta, although this is speculative and requires replication in a study with balanced genotype groups. Beta activity reflects a state of cortical activation via excitatory pyramidal cells mediated by inhibitory interneurons gated by γ-aminobutyric acid type A (GABAA) receptors that act as ‘pacemakers’ to maintain beta and gamma rhythms38, suggesting that Met allele carriers may have a disequilibrium of excitatory/inhibitory homeostatic inputs in these frontal neural networks39. eLORETA analysis demonstrated maximal beta-1 sources in the superior and middle gyrus, and maximal beta-2 sources in the superior, middle, and inferior gyrus, suggesting that these may be key areas of interest for future studies investigating altered GABAA receptor expression/activity in Met allele carriers, particularly in light of BDNF being shown to reduce GABA-ergic function via downregulation of GABAA receptor expression40. This relationship may be further mediated through impairment of synaptic transmission and plasticity in BDNF Met/Met genotypes, as prior research has demonstrated a decrease in GABAA receptor-mediated synaptic transmission in the pyramidal neurons of BDNF Met/Met mice41. Future work is required to understand if these findings are specific to particular brain regions and if there are functional effects of this altered receptor status. Greater beta amplitudes observed in the Met/Met genotype group may have functional implications in low-arousal states, and serve as a means of compensation for impaired task performance in a state of low arousal42. Although our study was conducted in a resting state, this finding could highlight a functional consequence of altered beta-1 and beta-2 activity in the Met-carrying groups.Our study was the first to observe greater frontal beta-1 and beta-2 amplitudes for the Val/Met genotype group than for the Val/Val genotype group. Prior research has revealed enhanced beta power in the Met/Met genotype group compared to the Val/Met genotype group (N = 92), and no differences in age, sex, or data recording experimental set-ups. Given that our sample consisted of healthy neurotypical adults, rather than individuals that are part of a clinical group, normative data for resting-state EO EEG is presented by our study. Additionally, we conducted eLORETA source localisation for each EEG band, which adds depth and breadth to our EEG spectral findings. Nevertheless, our study had several limitations. We chose to sum across narrow frequency bands to form each of the “classic” EEG bands, with the additional subdivisions of alpha-1/2 and beta-1/2. Although these band limits aligned with previous work, subdivision selection nonetheless remained arbitrary. Future work should seek to use a more data-driven approach by utilising principal components analysis (PCA) to decompose the EEG frequency spectra43. Further, data-driven approaches such as cluster-based permutation can be applied to identify significant clusters across both the spatial and frequency domains44. We did not measure cognition in this cohort, so findings can only be interpreted in the context of intrinsic brain function. Although our study did not utilise a clinical sample, it does provide useful insights that may pinpoint early differences in brain function at a single timepoint amongst (currently) neurotypical individuals, that could help predict future changes in brain function and disease risk; this should be researched in future longitudinal studies. Finally, our study chose to isolate the homozygous Met/Met group from the heterozygous Val/Met group, rather than pooling the two together as ‘Met allele carriers’. Although this reduced study power due to the low sample size of the Met/Met genotype group (n = 6), it increased our study’s comparability to existing literature that chose to isolate Met/Met separately and had similar genotype group sizes to ours19. Obtaining a larger sample of participants who are homozygous for the Met allele in an effort to balance the sample size of each genotype group may further strengthen the power and significance of future studies. Such research would require a minimum genotype group size of 23 to detect a moderate to large effect size similar to prior studies19. From an epidemiological perspective, the population prevalence of Met/Met genotypes is relatively low: ~ 4.5% in prior studies, ~ 6.5% here, which would translate to a sample size of > 500.Factors that point to increased generalisability of our study include a robust sample size (BDNF Val66Met polymorphism and neuronal oscillatory activity. Findings demonstrate enhanced delta, reduced alpha-1 and alpha-2 in Met/Met homozygotes relative to Val allele carriers, enhanced beta-1 and beta-2 amplitudes in the Val/Met genotype group compared to the Val/Val genotype group, and an absence of any genotypic association with theta. Our findings confirm that varied BDNF genotypic presentation is important in understanding resting-state EO oscillatory EEG activity in healthy, neurotypical adults. Findings suggested dysregulated cortical excitability/inhibition in Met allele carriers; future work is required to further understand these physiological mechanisms and establish an association with cognition. This study highlights the importance of genetics as a factor that influences intrinsic brain activity, and confirms the utility of investigating genetics and neurophysiology in a neurotypical sample in future studies.This study examined the relationship between the