The alarming number of recently reported human illnesses with bacterial infections resistant to multiple antibacterial agents has become a serious concern in recent years. This phenomenon is a core challenge for both the medical and animal health communities, since the use of antibiotics has formed the cornerstone of modern medicine for treating bacterial infections. The empirical benefits of using antibiotics to address animal health issues in animal agriculture (using therapeutic doses) and increasing the overall productivity of animals (using sub-therapeutic doses) are well established. The use of antibiotics to enhance profitability margins in the animal production industry is still practiced worldwide. Although many technical and economic reasons gave rise to these practices, the continued emergence of antimicrobial resistant bacteria is furthering the need to reduce the use of medically important antibiotics. This will require improving on-farm management and biosecurity practices, and the development of effective antibiotic alternatives that will reduce the dependence on antibiotics within the animal industry in the foreseeable future. A number of approaches are being closely scrutinized and optimized to achieve this goal, including the development of promising antibiotic alternatives to control bacterial virulence through quorum-sensing disruption, the use of synthetic polymers and nanoparticles, the exploitation of recombinant enzymes/proteins , and the use of phytochemicals. This review explores the most recent approaches within this context and provides a summary of practical mitigation strategies for the extensive use of antibiotics within the animal production chain in addition to several future challenges that need to be addressed. The rising number of recently reported human illnesses with bacterial infections resistant to multiple antibacterial agents has become a fundamental concern in recent years , 2. ThisCampylobacter spp. found within the beef food system in Malaysia were resistant to tetracycline (76.9%) and ampicillin (69.2%), respectively  and one synthetic [(5Z)-4-bromo-5-(bromomethylene)-2(5H)] furanones [Artemia from the V. harveyi strain BB120 and V. campbellii strain LMG21363 in a concentration-dependent manner.More recently, an autoinducer 2 (AI-2) that is produced in several pathogenic uranones . The incV. harveyi, the pathogen responsible for vibriosis outbreaks in Macrobrachium rosenbergii (giant freshwater prawn), was targeted recently with three quorum-sensing disruptors including cinnamaldehyde, (Z-)-4-bromo-5-(bromomethylene)-2(5H)-furanone and (Z)-4-((5-(bromomethylene)-2-oxo-2,5-dihydrothiophen-3-yl)metoxy)-4-oxobutanoic acid [M. rosenbergii larvae growth yet they were all able to positively increase the larvae survival rates in response to V. harveyi challenge when added to the culture water .Moreover, oic acid . None ofStaphylococcus aureus were suggested as good targets for possible virulence control [hla gene encoding α-hemolysin as well as the regulatory RNAIII of the agr quorum-sensing system in S. aureus 8325-4 and USA300 strains. Upon close inspection, norlichexanthone was found to reduce S. aureus toxicity towards human neutrophils; to interfere with AgrA, an agr two component response regulator, binding to its DNA targets; and to reduce staphylococcal biofilm formation [Other cellular targets such as the SaeRS two-component systems in  control , 21, as ormation . A furthormation .While the scientific community has developed a good grasp of the mechanisms involved in bacterial quorum-sensing and revealed many groups and classes of either naturally-available or synthetic compounds that can be utilized in pioneering this approach of controlling bacterial pathogenesis, multiple challenges still exist for its wide-spread implementation –25. BeyoFurthermore, the initial promise of disrupting mechanisms involved in bacterial quorums is based on the assumption that such anti-virulence drugs will minimize any resistance-development issues within the targeted bacterial species; such claims were considered unfounded most recently through the emergence of reports showing that resistance towards anti-virulence compounds does exist . DelineaFor quite some time, organic acids have been known for their ability to prevent food spoilage and extend the shelf-life of many perishable commodities . This caEscherichia coli F4 (K88)+. The retrieved results suggested that dietary organic acids can restore the growth and immunity parameters of E. coli-challenged broilers to the original levels noticed in non-challenged birds [A recent study inspected the effects of three commercial formic and propionic acid preparations on the growth performance, cecal microbiology, and immunity of broiler chickens challenged with ed birds .E. coli and Salmonella while the used dietary antibiotic, enramycin in this case, was efficient in decreasing Salmonella counts only. Differences, however, were noticed among individual organic acid mixtures that were tested for either their influence on birds’ performance or intestinal bacterial counts [Another experiment was conducted using Ross 308 broiler chicks to study the effect of using two commercial mixtures of organic acids as a substitute for a commercially available antibiotic growth promoter. Birds that were fed the organic acids-supplemented diets had gains of 3–16% in comparison to the negative control group (which had no organic acids or antibiotic supplements) with improved feed conversion ratios . Furtherl counts .Salmonella Enteritidis to invade avian intestinal epithelial cells was investigated at an earlier time [Salmonella Enteritidis with various concentrations of propionate or butyrate resulted in decreased invasions compared to the control group. Later, butyrate was found to specifically down-regulate Salmonella pathogenicity island 1 (SPI1) genes expression including hilD and invF [How short-chain fatty acids might influence the ability of ier time . The preand invF .Salmonella enterica infections in a very cost-effective way. Alternatively, the addition of acids to the feed or drinking-water could be another effective approach to control these infections [Based on the research outlined above, it was suggested that diets that stimulate organic acid production (short and medium-chain fatty acids) within the caecum can possibly help control fections .Salmonella Enteritidis involved feeding them with short-chain fatty acids encapsulated in mineral carriers, resulting in a slow release during the transport of these carriers throughout the intestinal tract. This treatment was able to significantly decrease the colonization and the invasion capacity of this pathogen [Recent animal trials conducted on young chickens challenged with pathogen .Lactobacillus species [The protection of the organic acids through the use of lipid matrixes, polymers, and/or enteric coating was shown to prevent their dissolution or disintegration within the gastric environment and to maximize their efficacy and bio-potency. An experiment conducted to investigate the effects of protected essential oils and organic acids in poultry (as antibiotics replacement) produced encouraging outcomes showing that the protected essential oil and organic acid mixtures could indeed serve as efficient growth-promoters and antibiotic alternatives . Surpris species . This sulongissimus muscle area. Interestingly, these acids were associated with positive changes in the color and firmness of the resulting meat. Finally, the protected organic acid blends caused a reduction in the total fecal E. coli counts while increasing Lactobacillus counts in a fashion that was dependent on the dosage of the organic acid blend. The 0.2% level of protected organic acids showed more positive effects on the above production parameters and was found to be superior to the lower levels of inclusion (0.1%) [Blends of protected organic acids were used in a 2-week trial to evaluate their effects on growth performance, nutrients digestibility, intestinal microbiota, and fecal gas emission. The dietary inclusion of protected organic acid blends (at 0.1–0.2% levels) linearly improved average daily gains and nutrients digestibility while decreasing fecal ammonia and acetic acid emissions. Furthermore, the supplementation with protected organic acids affected meat quality leading to a significant increase in the n (0.1%) .One emerging concern related to the use of organic acids as antibiotic alternatives is their ability to enhance the survivability of acid-sensitive pathogens exposed to low pH by the induction of an acid tolerance response , which mSalmonella Typhimurium [Salmonella growth/expansion was only restored after implementing a treatment with tributyrin, an ester composed of butyric acid and glycerol. The above study suggests that increasing intestinal short- and medium-chain fatty acid levels (e.g. butyrate) might indirectly limit pathogen expansion by limiting epithelial oxygenation and the aerobic growth of pathogens.The possibility of using short chain fatty acids as alternative growth promoters is indirectly supported by the results of numerous research studies aimed at understanding the course of antibiotic treatments in animals. For example, the effect of streptomycin on intestinal microbial communities was recently tracked . The reshimurium . The resBased on the above hypothesis, a dietary intervention was optimized and recently investigated to inspect the effect of administrating protected calcium butyrate on nutrient digestibility and growth parameters in broiler chickens . The incBacillus populations, decreases in E. coli counts, and higher Lactobacillus counts within the ileum and caecum of treated animals. Similar outcomes with regard to the intestinal microbial communities’ composition and the influence of medium-chain fatty acids  on Salmonella Typhimurium and coliforms were reported by Messens et al. [A similar study was conducted to investigate the effects of benzoic acid on the intestinal development and growth parameters of farm animals. A basal diet was supplemented with either 2000 or 5000 mg/kg of benzoic acid . Improves et al. . At the s et al. .− CD8− γδ T cells within the jejunum epithelium and confirmed that the majority of the intra-epithelial lymphocytes were expressing the surface marker CD3. Hence, the study concluded that short-chain organic acids were indeed exerting a beneficial effect on the local immunity of animals by merely evoking mechanisms known to be involved in defeating infections [Salmonella Enteritidis in chickens. Low concentrations of these acids inhibited bacterial growth and invasion in in vitro models, with caproic acid showing the highest potency. The conducted mechanistic studies indicated that these acids suppressed the expression of hilA, a key regulator related to the invasive capacity of Salmonella. The addition of caproic acid to chicken starter feed in a subsequent animal trial (in the amount of 3 g/kg) indicated a substantial decrease in Salmonella Enteritidis colonization levels inside the ceca and other internal organs. This result provided direct evidence of the potential use of such acids in reducing Salmonella colonization and the number of contaminated eggs entering the food-supply chain [The impact of medium-chain fatty acids and short-chain organic acids on animal gut morphology and its immune system was specifically established through a study that investigated the effect of using 0.41% fumaric and 0.32% lactic acids (short-chain organic acids) alone or in combination with 0.15% caprylic and capric acids (medium-chain fatty acids). Tissue samples were used to investigate the potential impact of these additives on villus length and crypt depth of the jejunum and to quantify intra-epithelial lymphocytes. While this study did not report any significant changes in the morphometric data, it noticed that the short-chain organic acids significantly increased the quantity of CD2fections . The capSalmonella Typhimurium field-strains were recently determined [fimA and hilA (hence strain invasivity) were significantly lowered in the presence of these acids, especially in the case of caproic or caprylic acids . Furthermore, serving the animals feed supplemented with coated butyric acid decreased the levels of fecal shedding and intestinal colonization of Salmonella Typhimurium. Surprisingly, the uncoated fatty acids did not influence the noted fecal shedding or intestinal colonization [The minimal inhibitory concentrations (MIC) of formic, acetic, propionic, butyric, caproic and caprylic acids against 54 porcine termined . These vtermined . The expnization .Salmonella growth. The above benefits were confirmed in piglets recently by adding up to 15% medium-chain triglycerides to feed mixtures without detecting any negative impact on feed intake or any sensory/organoleptic issues associated with the finished feed. These diets resulted in lower mortality rates and better development of newborn animals, particularly underweight ones [Besides the ability of medium-chain triglycerides and fatty acids to provide instant energy and physiological benefits, they were also found to affect and stabilize the composition of intestinal microbiota and inhibit coliforms and ght ones .Poor development and growth, diseases, morbidity and mortality of animals are often caused by poor gut health associated with a leaky gut, intestinal atrophy, infections and inflammation, particularly when the animals are young . In respEnterobacteriaceae demonstrating the ability of zeolites to decrease poultry pathogen loads without disturbing the natural beneficial gut microbiota of the animals [Zeolites, porous-, and cation-rich alumino-silicates were suggested as poultry feed additives with multiple productivity benefits. Most recently, diets of laying hens were supplemented with zeolites at 1, 2, or 4% (w/w) for 23 weeks. Cloacal swabs followed by next-generation sequencing of 16S rRNA genes reflected a significant reduction in  animals . Similar animals . Such a Salmonella Typhimurium through administering 1% or 2% (w/w) biotite and bentonite mixtures in feed [Salmonella Typhimurium infection and the numbers of viable bacterial cells retrieved from feces/tissues samples were significantly decreased within the treatment groups, with the 2% (w/w) supplementation level being the most promising one.Biotite and bentonite, two phyllosilicate minerals with industrial applications, were reported with several beneficial activities as antimicrobial agents. The bacterial clearance capabilities of a mixture of biotite and bentonite were investigated on experimental infection models of  in feed . The outThe effectiveness of replacing antibiotics (serving as growth promoters) with tribasic copper chloride and copper sulphate pentahydrate in duck diets was recently investigated . After aEnterobacteriaceae and the Escherichia groups while Bifidobacterium, Enterococcus, Streptococcus, Weissella spp. and Leuconostoc spp. did not reflect any influence due to high-dietary zinc concentrations throughout the 5-week trial period.Among the many studied metal antimicrobials , only zinc oxide (ZnO) has been successfully used in animal feed at a large-scale to boost growth. The use of ZnO in these applications with much higher doses  surpasses its role as a micronutrient for optimal animal growth. The used pharmacological doses of ZnO (within the 2000–3000 mg/kg range) have proven to be effective in reducing the symptoms of post-weaning diarrhea in pigs , 53. TheZinc oxide is currently used extensively in Europe as an alternative for colistin in weaned piglets and not just as a feed ingredient to improve feed conversion, as previously mentioned. In fact, new formulations of zinc oxide nanoparticles are being investigated as substitutes for the commonly used colistin sulfate. Most recently, a study indicated that the supplementation of 1200 mg/kg nano-ZnOs (approximately 30 nm in diameter) significantly increased the final body weight and average daily gain of treated animals in comparison to the colistin sulfate group (20 mg/kg). This treatment also improved the intestinal morphology, significantly decreased total aerobic bacterial populations in mesenteric lymph nodes, and significantly decreased plasma and tissue zinc concentrations (liver and tibia) .S. aureus (MRSA) associated with farmed animals including pigs and poultry [Despite the promising outcomes of using high concentrations of mineral within the animal production chain, some major criticisms directed towards the use of such metals exist. The fact that many of the used heavy metals tend to accumulate in the soil resulting in serious environmental and health consequences is at the top of this list , 57. Fur poultry . Other a poultry  and muta poultry . The emePhytochemicals are natural compounds/secondary-metabolites derived from botanicals and medicinal plants and generally used in complementary or alternative medicine to improve human health and/or treat human diseases. Certain phytogenic compounds (and their blends) have been reported to have promising anti-microbial properties  because E. faecalis and E. coli growth [Escherichia and Enterococcus genera and improved the total counts of Lactobacillus within the jejunal digesta of weaning animals [E. coli showed a substantial decrease in numbers when animals were fed a diet supplemented with 100 or 150 mg/kg essential oils containing 14.5% thymol and 3.5% cinnamaldehyde, respectively, while the proportion of Lactobacillus species was increased [Lactobacillus and E. coli species in broilers after 41 days of feeding [Recently published in vitro studies have shown that thymol, carvacrol, cinnamaldehyde (or combinations) have the ability to inhibit i growth . Further animals . In anotncreased . Similar feeding .Aeromonas hydrophila and 63 μg/mL against Streptococcus iniae. The utilized preparations showed a high toxicological effect on tilapia (Oreochromis niloticus) fingerlings while adult tilapia could tolerate the aforementioned toxic effect [Allicin is another potent phytochemical with potential applications within the animal production field. Its possible use in aquaculture against fish pathogens was recently investigated. A 10% (v/v) solution of allicin in dimethyl sulfoxide showed MICs of 125 μg/mL against c effect . FurtherThe effect of dietary allicin on the health and growth performance of weanling piglets was explored by comparing a control group that was fed a diet supplemented with antibiotics with multiple treatment groups that were fed diets supplemented with allicin  at concentrations of 0.10, 0.15, 0.20 and 0.25 g/kg, respectively. The results showed that the average daily weight gain increased as the level of dietary allicin increased while the incidents of diarrhoea in treatment piglets decreased. Interestingly, an improvement in the local farm environment was also reported as the feces produced by the animals receiving the allicin treatment was less attractive to flies .E. coli F4 (K88)+. Allicin protection was not due to its antibacterial activity, since enterotoxigenic Escherichia coli growth was unaffected by the presence of allicin [Mechanistically speaking, allicin was reported to protect intestinal cells (such as IPEC-1) from membrane damage and from the increased membrane permeability associated with infections caused by enterotoxigenic  allicin .Allium sativum) and allicin both showed significant in vitro and in vivo antibacterial activities against isolates of multiple drug-resistant Shigella dysenteriae 1, S. flexneri Y, S. sonnei and enterotoxigenic E. coli. Allicin aided in fully curing the infected rabbits within 3 days eliminating the challenging bacteria, judged by rectal swabs within the 2nd day of treatment. Moreover, allicin did not show any adverse effects on standard blood parameters/animal performance when used at the reported therapeutic doses [The efficacy of garlic and allicin in treating experimental shigellosis in rabbits has also been tested. Aqueous extracts of garlic  on animal productivity especially in broiler chickens , a few ohemicals .The variation of animal response toward dietary phytochemicals might be explained by the dependence of outcomes on many factors including treatment duration, the administered dose, and how these phytochemicals affect nutrient digestibility and/or feed intake. For example, feeding animals a diet supplemented with a 100 mg/kg phytochemical blend  increased their daily weight gain significantly in comparison to the un-supplemented basal diet . A similSimilar to the duration factor above, another study showed the influence of the administered dose on the varying responses of animals. Feeding a diet supplemented with a 2000–3000 mg/kg phytochemical blend (containing 60 mg/kg carvacrol and 55 mg/kg thymol) for 3 consecutive weeks increased feed intake and the reported average daily gain of weaning pigs in comparison to animals that were fed with un-supplemented basal diet . HoweverIn addition to their antimicrobial capabilities, some phytochemicals exert anti-oxidative and anti-inflammation functionalities . A commeThe future development and use of integrated genomic, meta-genomic, transcriptomic and proteomic approaches will help researchers to better understand the modes of action of many phytogenic compounds and possibly lead to the development of feasible additives/treatments that can be used as alternatives to antibiotics in animal feeds.In our battle against antibiotic resistance, polymer and material scientists have an important role to play. These scientists are contributing to the mitigation of antibiotic resistance through the use of major strategies that include: (a) developing antibiotic alternatives using antimicrobial metal-nanoparticles and antimicrobial-polymers which bacteria are less likely to develop resistance to and (b) fabricating new nano-carriers to boost the therapeutic efficacy of existing antimicrobial molecules with an increased selectivity to inhibit bacterial cells rather than host eukaryotic cells.Scientists have made rigorous attempts to increase zinc bioavailability as well as its influence on growth performance and immune response by using lower doses/concentrations of zinc in feeds and more recently by utilizing ZnO nanoparticles (NPs) –87. WhilCationic antimicrobials have been circulating within general-use in clinical and domestic settings since 1955. The most well-known cationic antimicrobials are quaternary ammonium compounds (QACs). These cationic surfactants contain one quaternary-nitrogen associated with at least one major hydrophobic substituent. The mode of action of QAC against bacterial cells is hypothesized to involve a general perturbation of the lipid bilayer membranes . Following a tight ionic association between the positively charged quaternary-nitrogen in QAC and the negatively charged phosphate groups in membrane phospholipids, the hydrophobic tail of QAC intertwines into the hydrophobic membrane core to decrease membrane fluidity and disturb its osmo-regulatory functions. Since the above mechanism involves QACs, penetration into the core of bacterial membranes to impact their structures, QACs can practically be pumped out by bacterial ATP-dependent efflux pumps (as a defence counter response) and be less efficient in killing bacteria in some cases.The most recently developed and reported cationic polymers can impact the fluidity of bacterial membranes  by interacting specifically with the phospholipid bilayer without being susceptible to the action of efflux pumps-mediated resistance mechanisms. Poly(hexamethylene biguanide) (PHMB) is a good example of this as it has been used extensively for skin disinfection, wound-infection control, and swimming pool disinfection for several decades, and so far no bacterial resistance to PHMB has been ever reported . To becoS. aureus (ATCC 29213) and an extended-spectrum beta lactamase-producing Escherichia coli (ATCC 35218) were unchanged indicating a lack of induction of any antibiotic resistance genes. In contrast, the MIC values of classical antibiotics  were increased by 8- to 64-folds after the 15 successive subcultures were conducted reflecting the emergence of resistant mechanisms due to the bacterial passaging/adaption.Xue et al.  tested tS. aureus over red blood cells.In order to have value as therapeutic agents, in addition to the absence of resistance induction, cationic polymers must selectively target pathogens rather than cells belonging to the host. The selectivity of an antimicrobial agent is generally defined as the ratio of its hemolytic concentration (the concentration that kills 50% of red blood cells) to its MIC against a chosen pathogen. Lienkamp et al.  used a mBased on the results of the above numerous studies and in view of their low potential for inducing bacterial resistance, antimicrobial polymers hold the promise as key alternatives to antibiotics within veterinary medicine/animal production applications in the coming decades.P. aeruginosa [Nano-carriers can be used to not only boost the therapeutic efficacy of existing antimicrobials  but alsoruginosa . Howeverruginosa .E. coli K12, demonstrating the versatility of this strategy.In a MRSA and fibroblast co-culturing assay, 2 mg/mL of encapsulated and antibody-conjugated C17 nanoparticles wiped out all MRSA cells in 2 h while preserving 50% of total fibroblast cells in comparison to >98% reduction of both fibroblast and MRSA cells in the case of free C17 at an equivalent concentration. In the above example, a nanoparticle assembly exhibiting selective antibacterial activity towards a specific pathogenic bacterium has been successfully developed starting from a non-selective and potentially non-resistance-inducing biocide, C17. The authors suggested that the binding of C17-SLN-Ab to MRSA cell-surface is most likely facilitating its endocytosis hence the later release of C17 inside MRSA cells, leading to a subsequent effective and selective inactivation of MRSA. Importantly, by switching the conjugated antibody, they were able to turn the same nano-assembly to be more specific towards another target bacterium, The above concept can be extended to other potent antimicrobial agents, against which bacterial resistance is unlikely to develop. Because the antibody itself does not impact the viability of the bound bacterium and the encapsulated biocide kills the bacteria in a non-specific way; it is intrinsically less likely to induce bacterial resistance or select any resistant mutants. Overall, this study opens a new door of killing bacterial pathogens while potentially bypassing bacterial resistance mechanisms by using the most recent advances in nanotechnology.In essence, understanding the mechanism(s) of bacterial growth-inhibition of many novel synthetic polymers and nanoparticles should provide a safe, effective, and inexpensive way to control the prevalence of antibiotic resistance in the animal production chain.The increasing acceptance of using recombinant proteins/enzymes to enhance animal productivity  has led 2O2) in the surrounding environment by a glucose oxidase-mediated conversion of glucose  was shown to inhibit a number of pathogenic bacterial species and prevent biofilm formation of sa cells . The res2O2 generation system using a glucose oxidase and glucose to target Propionibacterium acnes. The results indicated the effectiveness of such treatment in reducing the observed inflammation over both short and long-term applications at 68 and 56% rates, respectively [Another study looked into developing a Hectively .The aforementioned examples show that a recombinant glucose oxidase can be utilized in the inhibition of bacterial biofilm(s) formation and any associated symptoms (such as inflammation), as well as demonstrating the potential of this treatment for generating topical ointments for animal treatment purposes. The ability to apply such intervention through oral routes needs to be further investigated and developed.The digestive-system microenvironment plays a pivotal role in sustaining animal health and productivity. Many factors including the consumed feed, gut microbiota, and the animal’s intestinal immune response interrelate to influence this localized environment. Among the recently highlighted risk factors that influence the homeostasis of such an environment are bacterial lipopolysaccharides (LPS) that have long been recognized as potent pro-inflammatory mediators/toxins that constitute a challenging-factor for growing animals , 98. AlkIn animals, the endogenous levels of IAP are reported to decrease at the weaning stage; hence pathogenic gram-negative bacteria (through an LPS-mediated mechanism) can up-regulate inflammatory responses leading to a symptomatic diarrhea . To addrIn a recent study the effect of an intestinal alkaline phosphatase (IAP) and sodium butyrate on LPS-induced intestinal inflammation was evaluated in pigs. The exogenous IAP was able to complement endogenous IAP levels and down-regulate LPS-induced inflammatory responses via the RelA/p65 (NF-κB) route demonstrating that such a treatment may indeed be beneficial in attenuating LPS-induced intestinal inflammation .Ctenopharyngodon idella) after challenging with A. hydrophila [Flavobacterium columnare in another study [Increasing evidence points to the use of exogenous protease(s) to reduce the protein content of diets and improve nutritional and health outcomes of farm animals. This practice is hypothesized to lower the indigestible part of feed, to limit subsequent hindguts fermentations and the potential growth of enteric pathogens, and to improve the gastrointestinal tract overall health . Furthermore, it is predicted that the use of proteases can indirectly reduce the usage of antibiotics by improving gut and animal health and reducing stress-related hormones. This idea is supported indirectly by a recent study that investigated the effects of dietary protein levels on disease-resistance and immune function of grass-carp  activities, plasma total proteins, intestinal villus heights, intestinal morphology and the expression levels of specific genes. Significant increases in growth performance have been observed which were attributed to better intestinal development, enhanced protein digestibility, and improved nutrient transport efficiencies . The supThe influence of a commercial acid-stable protease mixture on the utilization of corn and soybean meal-based diets was evaluated most recently. The grouped animals had free access to a mash diet and the ileal content was collected for digestibility determination after 14 or 29 days of the protease-mixture administration. The results collectively showed that the apparent ileal digestibility of individual amino acids was increased during the supplementation course (29 days of treatment) while the apparent ileal digestibility of the indispensable amino acids, methionine and cysteine, and branched-chain amino acids was increased only by the end of the study period but not in an earlier phase. The presented data argues for a continuous supplementation regimen of proteases during the entire production phase to maximize the associated benefits and overall animal productivity .A further exploration of the effects associated with supplementing broilers diets with exogenous proteases to enhance nutrient digestibility in low-protein poultry by-product meal based diets was conducted. The continuous dietary supplementation of these poultry by-product meal diets with exogenous proteases for 35 days increased feed intake and body weight gain in treatment animals. The overall improvements in feed intake and body weight gain were associated with the provided levels of crude proteins during that period which also influenced nitrogen retention rates. The presented results suggested that the addition of enzymes not only improves production parameters, but crude protein levels can also be safely reduced while using poultry by-product meals supplemented with exogenous proteases for the same production outcomes .3 emissions were enhanced. Blood samples profiling indicated decreased creatinine levels without any changes in the lymphocytes or red/white blood cells counts. Fecal microbiota (particularly E. coli and Lactobacillus) were also similar between all groups. From the aforementioned studies [The use of protease supplementation for enhancing the nutritional and production outcomes of a diet reduced in crude protein as well as digestible amino acids and energy was recently reported. The suggested protease supplementation (at 125 g/ton) showed the potential to maintain the live weight and feed conversion rate of pigs up to the level of a matching regular diet . Further studies , 109, thThe use of non-starch polysaccharide (NSP) hydrolytic enzymes in boosting antibiotic treatments within the digestive systems of mono-gastric animals that cannot ferment and utilize such polysaccharides is another promising approach. This technique is similar, in principle, to the use of proteases. In essence, diets with high concentrations of rye and barley have high viscosity when passing the alimentary canal due to their high content of non-starch polysaccharides (NSP)/fibers . The eleAn earlier study established the effects of an exogenous supplementation of non-starch polysaccharides hydrolytic enzymes on the activities of endogenous pancreatic and small intestinal digestive enzymes in animals fed with high percentages of barley. Such supplementation (at the 0.15% w/w level) improved the growth performance of the studied animals and had no effect on the digestive-enzyme activities within the pancreas but at the same time the treatment affected the duodenal contents where the proteolytic enzymes, trypsin, amylase, and lipase activities decreased by 57.56, 76.08, 69.03 and 40.22%, respectively .In the past decade, mannan-oligosaccharides have been reported as alternatives to antibiotics with benefits that include an improvement in the animal’s performance, an enhancement in the feed conversion efficiency, and a boost of the gastrointestinal health. Despite these benefits, the molecular mechanisms behind such functionalities are not yet clear.To elucidate the effects of these additives on the intestinal gene expression and characterize the involved biological pathways, a recent study was conducted in which young broilers were fed a diet supplemented with 2.2 g/kg of mannan-oligosaccharides for 3 consecutive weeks followed by jejunal gene-expression profiling . The obtC. jejuni and C. coli [Salmonella Enteritidis infections [Another study reported the beneficial effect of mannan-oligosaccharides on inhibiting the adherence of bacterial cells including  C. coli . Other pfections .E. coli and S. enterica [Among the good (and cheap) sources of mannan-oligosaccharides is dried brewer’s yeast with contents close to 5.2%. The dried brewer’s yeast was assessed most recently as an alternative to the antimicrobial carbadox for young pigs. Agglutination tests confirmed the ability of the yeast product to sequester/bind several serovars of enterica . Pigs thenterica .The results of these studies collectively suggest that mannan-oligosaccharides could be used as a feed supplement for livestock to control enteric infections, to improve performance, and to minimize the use of antibiotics as growth-promoters throughout different cellular mechanisms.The development of novel agents for pathogenicity control, especially anti-virulence ones, requires an in-depth understanding of the unique roles that such virulence factors have in disease/infection progression. Without such knowledge, the possibility of off-targets cannot be ruled out. The lengthy process of conceptualizing a detailed mechanistic understanding of virulence factors and the research resources needed for such conceptualization are sometimes considered to be major limiting factors.2O2 content, accumulation, or diffusibility within the matrix will retrospectively affect the treatment/usage outcomes.Another challenge related to the use of recombinant enzymes (e.g. glucose oxidases) is the fact that such enzymes exhibit considerable variations in their antibacterial activity/efficiency based on the surrounding environment. Any factor that influences the overall HP. aeruginosa isolates significantly correlated with H2O2 content and more importantly, with the glucose oxidases levels. Higher glucose oxidases levels led to better outcomes. In response to this finding, the authors proposed the breeding of novel honeybee lines that express higher levels of glucose oxidases in order to increase the antibacterial efficacy of the final products [In a recent study, the antibacterial activity of honey samples (a rich source of glucose oxidases) against products .While the use of proteases is surely a promising way of inhibiting bacterial biofilm(s) formation and accelerating infection clearance, the use of proteases is by itself a controversial topic. Some recent reports indicated unwanted roles of bacterial proteases in extending bacterial infections within chronic wounds environments . The eleThe use of polymers and surface-coating technologies for quorum-sensing manipulation is considered to be very potent technology but attention should be paid to the conjugation of the involved compounds in a way that retains their ability to influence bacterial hosts/colonies . InvestiWhile the rapid evolution and spread of antibiotic resistance mechanisms among animal pathogens is outpacing the discovery of new antibiotics, there is an increasing number of published studies clearly showing that alternatives do exist. The optimization of many of the approaches outlined in this review coupled with a deep understanding of their underlying molecular mechanisms will eventually lead to strong and empirical strategies for mitigating antibiotics usage in the field of animal production and husbandry.In the past 2 decades, many promising approaches have been reported for addressing bacterial resistance and antibiotics over-usage in the fields of animal farming and livestock production Table . While t


In the current era of expedited orthodontics, among many clinicians, tertiary care hospitals and patients, surgery first orthognathic approach (SFOA) has gained popularity. The advantages of SFOA (face first approach) are the reduced overall treatment duration and the early improvement in facial esthetics. In SFOA, the absence of a presurgical phase allows surgery to be performed first, followed by comprehensive orthodontic treatment to achieve the desired occlusion. The basic concepts of surgery early, surgery last, SFOA and Sendai SFOA technique along with its variations are reviewed in the present article. The recent advancement in SFOA in the context of preoperative preparation, surgical procedures and post-surgical orthodontics with pertinent literature survey are also discussed. Conventional orthognathic surgery (COS) requires certain duration of presurgical orthodontics to alleviate the dental crowding, level the curve of Spee, decompensate the dental inclinations, remove any occlusal interferences and coordinate the upper and lower arches.,In addition to prolonged treatment duration, other disadvantages of presurgical orthodontics include gingival recession, gingival hyperplasia, dental caries, root resorption, deterioration in occlusal function, masticatory and speech discomfort and subsequent psychological problems due to delay in resolution of patients’ chief complaint.The alternatives to COS include surgery early, surgery last and surgery first orthognathic approach (SFOA). Hernandez-Alfaro and Guijarro-MartınezIn SFOA, there is no presurgical phase; surgery is performed first followed by comprehensive orthodontic treatment to achieve the desired occlusion . This ap11 in their systematic review, have reported a mean total treatment duration of 14.2 months with a range of 10.2-19.4 months for SFOA. With regard to complications, SFOA and COS are both comparable.Recently among many clinicians, tertiary care hospitals and patients, SFOA has gained popularity due to no presurgical phase and reduced overall treatment duration. Peiro-Guijarro et al,16 were among the first to actually carry out SFOA using miniplates for post-surgical orthodontic treatment. The increased range of orthodontic tooth movement in all dimensions is helpful in even correction of relapse that may have occurred post-surgically. Justus et al11 However, it should not be considered for patients with cleft related deformities, patients with high probability of development of CR-CO discrepancy and unilateral or bilateral cross-bite or scissor bite post-surgery.The SFOA is indicated in highly motivated patients with minimal arch length discrepancy, mild to moderate transverse, vertical and sagittal discrepancies, with normal incisor inclinations and minimal dental compensations, to avoid interferences during the surgical correction.,SFOA requires efficient treatment planning, skilled model surgery and meticulous post-surgical orthodontics.a. Preoperative:» Diagnosis: The appropriate treatment goals for an individual are determined using the dental casts, radiographs and photographs as diagnostic aids. » Bonding and stabilization wire: The Sendai SFOA recommends bonding 0.022-in brackets one week prior to the surgery. The 0.018 x 0.025-in stainless steel wires are bent passively and are inserted followed by soldering of surgical hooks, to facilitate intermaxillary fixation during the surgery.» Model surgery: The traditional facebow records are obtained and models are simulated according to the set treatment goals as determined from the prediction tracings. The surgical splint is then fabricated to maintain the interim transit malocclusion (ITM) post-surgically. Sendai SFOA does not recommend achieving three point occlusal contact during mandibular surgery as this may result in posterior lengthening of the ramus, which has a high relapse tendency.» Surgical splint: The surgical splint may be placed in the mandibular arch especially in cases of maxillary surgery. It consists of four ball hooks and a lingual arch.b. Surgical:19 This design prevents the condylar dislocation due to a buccal step adjacent to mandibular second molar area, hence minimizing the relapse tendency. The titanium miniplates are placed at appropriate locations to facilitate the dental movements. Sendai SFOA recommended the modified bilateral sagittal split osteotomy combined with a T-shaped miniplate fixation for mandibular surgery.c. Post-surgical orthodontics:Removable Gelb-type splint is maintained for about four to six weeks after the surgery. Various dental movements in sagittal, vertical and transverse planes are achieved using SAS after the removal of splint.Over the period of years, various clinicians have modified the original SFOA, according to their clinical expertise, skills and convenience:» Diagnosis: Various technologies such as CBCT, intraoral scans and combining these to form a 3D virtual model are being utilized to facilitate the diagnostic procedure. Swennen et al22 have recommended the usage of 3D models to simulate the jaw and future dental movements. These may also determine various interferences that may occur during the surgical procedure. » Computer-aided surgical simulation: Ima et al,» Splint fabrication: The splints may be fabricated directly on the models» Orthodontics preparation: As opposed to the original protocol with no presurgical orthodontics, a minimal duration of presurgical orthodontic treatment may be indicated to avoid interferences during the surgical procedure.Yu et al,The original Sendai SFOA has recommended leaving stainless steel wires in place post-surgically, while the stability of determined positions of the jaws have been achieved.» Post-surgical splint: While some advocate the use of the splint only during surgery, others have advocated its use anywhere between one to four weeks after surgery. Sugawara et al27 while others suggested a delay of two-three weeks.16,18 Kim et al» Post-surgical orthodontics: The post orthodontic treatment may be initiated immediately post-surgery as proposed by Leelasinjaroen et al,The occlusion cannot be used as a guide during the surgical procedure in SFOA. The following should be considered during the treatment planning phase to maximize the stability of the corrected jaw position:» The model surgery should result in an ITM comprising of two occlusal stops in the posterior and one in the anterior region.» The surgical movement of the jaws should be greater as compared to the conventional orthognathic surgery, to allow for decompensation of teeth post-surgery.» The molar relationship may be used as a guide for ITM.29 suggested that extraction should be done if the angulation of the upper incisor to occlusal plane is less than 53 degrees. Moreover, distalization or angulating the maxillary segment during the surgical procedure may also be used to improve the teeth inclinations.» Extractions may be indicated for correction of crowding, inclinations and improvement of facial profile. Sharma et al» The transverse discrepancy can be resolved either during surgery or post-surgery with archwires and elastics.» In Class II division 2 cases, a short term period of minimal orthodontics to upright the incisors or to overcorrect the jaw deformity to Class III relations is indicated to provide sufficient overjet for surgical correction.» In Class III cases with moderate to severe crowding and retroclined incisors, the jaw deformity should be overcorrected to a Class II jaw relationship. ,» In subjects with hypodivergent skeletal pattern, the deep bite can be corrected during surgery by bringing the anterior teeth into edge to edge bite with no contact between the posterior teeth. The posterior teeth are then extruded postsurgically to correct the bite.» In subjects with hyperdivergent skeletal pattern, the anterior open bite is corrected by clockwise rotation of maxilla and anticlockwise rotation of mandible to counter postsurgical relapse.34 have reported increased counterclockwise rotation while Kim et al17 found clockwise rotation of mandible in SFOA group as compared to COS group. For sagittal plane, Kim et alBaek et al,,,38 Oh et alThe introduction of virtual treatment simulation and planning softwares utilizing 3D imaging techniques and virtual models have greatly improved the orthodontic diagnosis and predictability of the expected outcome.SFOA is an efficient and time saving technique, but it is limited to patients with minimal arch length discrepancy, normal incisor inclination and mild-moderate sagittal, vertical and transverse discrepancies. Hence, the patient selection is critical. In addition, passive wire bending is cumbersome and time consuming. The occlusion cannot be used as a guide and the entire occlusal stability is dependent upon the surgical splint. These drawbacks may be easily overcome with proper case selection, vigilant treatment planning and effective communication between the orthodontist and maxillofacial surgeon.


Commensal gut microbiota exerts multifarious effects on intestinal and extra-intestinal immune homeostasis. A disruption in the microbial composition of the gut has been associated with many neurological disorders with inflammatory components. Here we review known associations between gut microbiota and neurological disorders. Further we highlight the emerging role of diet and microbiota interrelationship in regulating neuroinflammation. Inflammation in the nervous system, termed neuroinflammation, is now recognized as a cardinal feature of many neurological disorders. In central nervous system (CNS) autoimmune diseases such as multiple sclerosis, there is a conspicuous infiltration of immune cells from the periphery into the CNS leading to demyelination and neuronal damage . HoweverOver the years, our understanding of the initiation and regulation of neuroinflammatory processes has been revised. In particular, bidirectional communication between the immune system and the nervous system has been given greater emphasis. Evidence has been mounting in recent years supporting the substantial contribution of several environmental factors including diet and the gut microbiota besides genetic factors in neuroinflammatory diseases. Once thought to be important primarily for conferring nutritional benefits, the gut microbiota has now been implicated in various physiological and pathophysiological responses through direct and indirect interactions with the host . Recent MS and NMO are the chronic inflammatory demyelinating disorders of the central nervous system that involve the brain, optic nerve and spinal cord with distinct immunological and pathological features. Compelling evidence for the role of the gut microbiota in MS has largely emerged from animal models. Early observations in a spontaneous experimental autoimmune encephalomyelitis (EAE) animal model—a mouse model for MS—suggested a microbial role in initiation of neuroinflammatory disease processes. Goverman et al. showed that transgenic mice harboring high frequencies of myelin basic protein (MBP)-specific T cells in their immune repertoire showed higher spontaneous EAE incidence in conventional dirty facilities compared to clean housing conditions . EmpiricIn 2011, using a transgenic mouse model for relapsing-remitting MS (RRMS) under germ free (GF) conditions i.e., mice devoid of microorganisms, a clear role for the gut microbiota in triggering CNS inflammation was established. The relapsing remitting (RR)-MS model—a T cell receptor transgenic mouse strain that is prone to develop spontaneous EAE under specific pathogen free (SPF) housing conditions, remained largely protected from EAE development under GF conditions . SimilarClostridia and Bacteroidetes species  spectrum disorders stemmed from observations that antibodies against intestinal antigens have been found in NMO patients . One stufringens . More dipatients .H1 and TH17 cells to the CNS. Clinching the effect of microbial dysbiosis in this process was that GF mice receiving microbiota from filamentous middle cerebral artery occlusion (fMCAO) operated mice showed larger infarcts post middle cerebral artery occlusion (MCAO) induction in addition to higher levels of pro-inflammatory cytokines IFNγ and IL-17. Moreover, fecal microbial transplantation restoring a healthy microbiota was neuroprotective , the second most common age-associated neurodegenerative disorder, has pathological hallmarks of chronic neuroinflammation with features of activated glial cells and increased in pro-inflammatory cytokines. The gut microbiota has also been implicated in PD. It is interesting to note that PD is accompanied in many instances by GI symptoms. A study by Devos et al. on PD patients showed pro-inflammatory cytokine signature in the colon corresponding to disease duration. The mRNA levels of TNFα, IL-1β, IFNγ, and IL-6 were increased in colon biopsies from PD patients as compared to healthy controls . Anothercontrols . Similarcontrols –32. Withcontrols .Firmicutes and Actinobacteria at the phylum level with increase in abundance of Bacteroides in AD patients as opposed to healthy controls. A deeper analysis in AD participants showed reduced abundance at the family level for Ruminococcaceae, Turicibacteraceae, Peptostreptococcaceae, Clostridiaceae, Bifidobacteriaceae, and Mogibacteriaceae, and increased abundance of Gemellaceae, Bacteroidaceae, Rikenellaceae and the genus Bilophila in the phylum Proteobacteria. Correspondingly, correlations between relative abundance of aforementioned bacteria and CSF biomarkers like Aβ42/Aβ40 and p-Tau showed consistent trends, with genera identified as more abundant in AD being associated with greater AD pathology, even in non-demented participants (Increasing evidence suggests that Alzheimer's disease (AD) pathogenesis involves strong interactions with innate immunological mechanisms in the brain. In an AD disease mouse model, antibiotic treatment was reported to perturb microbial diversity, alter circulating cytokine and chemokine levels and curtail amyloid plaque deposition . One stuicipants .Bacteroides fragilis was shown to improve gut barrier integrity, restore specific microbiota changes, modulate serum metabolome profile and ameliorate anxiety-like, communicative and sensorimotor behaviors. Interestingly, this improvement was not dependent on Polysaccharide A (previously identified as responsible for immunomodulatory effects mediated by B. fragilis). Moreover, no evident colonization by the B. fragilis was observed in the GI tract, nor was there change in systemic immunity. Besides, the same behavioral improvements were possible by treatment with Bacteroides thetaiotaomicron too but not with Enterococcus feacalis  effects on neurological disorders. Research in this direction has expanded in recent times producing a multitude of results. The role of diet in neurological disorders has been reviewed elsewhere , 42. HerMicroglia and astrocytes are the key local resident cells which critically contributes to the inflammatory processes in the CNS. Indeed, it has been shown that the microbiota influences microglial maturation and function . GF miceThe gut microbiota and microbial metabolites have also been shown to affect astrocyte activity through aryl hydrocarbon receptors (Ahr) , 46. DieH17 lymphocytes. IL-17 mediated suppression of endothelial NO via Rho kinase (ROCK)-dependent inhibitory phosphorylation of endothelial NO synthase (eNOS) leads to a cerebral hypoperfusion and alteration in the endothelial regulation of cerebral microcirculation  promoted EAE (H17 cells through the NFAT5/SGK1 dependent cascade (Lactobacillus murinus in the gut and tryptophan metabolites indole-3-lactic acid (ILA) and indole-3-acetic acid (IAA). Mono-association of GF mice with L. murinus increased fecal ILA and IAA as well as ameliorated EAE even under high salt diet conditions , a product of choline metabolism, has been recently correlated with increased levels of pro-inflammatory monocytes in the blood and bone marrow of stroke patients . AnotherAs reviewed, gut microbiota contributes to the inflammatory processes in neurological disorders. Associating specific microbial communities in patients with neurological disorders has been made easy with improvement in sequencing tools. But the functional relevance of these associations and the mechanisms through which they cause or ameliorate neurological disorders are unexplored. Diet being the primary factor regulating microbial community structure and diversity, further research is needed to find specific dietary factors that shape the commensals and the resulting bioactive metabolites. This will help us in designing new therapeutic interventions through dietary modifications which serve several advantages like being cost effective, simpler to implement and can be personalized therapeutics.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The efficacy, safety, and clinical importance of extended-duration thromboprophylaxis (EDT) for prevention of venous thromboembolism (VTE) in medical patients remain unclear. We compared the efficacy and safety of EDT in patients hospitalized for medical illness.Electronic databases of PubMed/MEDLINE, EMBASE, Cochrane Central, and ClinicalTrials.gov were searched from inception to March 21, 2019. We included randomized clinical trials (RCTs) reporting use of EDT for prevention of VTE. We performed trial sequential and cumulative meta-analyses to evaluate EDT effects on the primary efficacy endpoint of symptomatic VTE or VTE-related death, International Society on Thrombosis and Haemostasis (ISTH) major or fatal bleeding, and all-cause mortality. The pooled number needed to treat (NNT) to prevent one symptomatic or fatal VTE event and the number needed to harm (NNH) to cause one major or fatal bleeding event were calculated.p = 0.002). EDT increased risk of ISTH major or fatal bleeding  in both meta-analyses and trial sequential analyses. Pooled NNT to prevent one symptomatic VTE or VTE-related death was 250 (95% CI: 167–500), whereas NNH to cause one major or fatal bleeding event was 333 . Limitations of the study include variation in enrollment criteria, individual therapies, duration of EDT, and VTE detection protocols across included trials.Across 5 RCTs with 40,247 patients , the duration of EDT ranged from 24–47 days. EDT reduced symptomatic VTE or VTE-related death compared with standard of care  prophylaxis among hospitalized patients with an acute medical illness until discharge. However, the risk of VTE persists and is cumulative in the postdischarge phase over the subsequent 4 to 6 weeks.Several randomized clinical trials have evaluated the therapeutic effects of extended-duration thromboprophylaxis (EDT) in attenuating the accumulated VTE risk. Although reduction in VTE was seen in these trials, none of them individually demonstrated superiority of EDT over standard of care.Our principal aim was to evaluate the aggregate efficacy of EDT on clinically relevant endpoints and to ascertain the robustness of efficacy signals balanced against the safety of the EDT strategy.We performed a systematic review, trial sequential analysis, and cumulative meta-analysis to identify all randomized clinical trials (RCTs) that assessed EDT in medically ill patients and evaluate the aggregate efficacy of EDT on clinically relevant endpoints. We assessed the robustness of efficacy signals balanced against the safety of the EDT strategy.We identified 5 RCTs that compared EDT with standard of care in medically ill patients requiring hospitalization, most commonly for heart failure.We observed that EDT reduced symptomatic VTE or VTE-related death compared with standard of care at the expense of an increased risk of major or fatal bleeding in both trial sequential and cumulative meta-analyses.A post-hospital discharge EDT strategy of anticoagulation for a 4–6 weeks period reduces symptomatic or fatal VTE events in patients hospitalized for acute medical illness at the expense of increased risk of major or fatal bleeding. Further investigations are required to define risks and benefits as well as cost-effectiveness within specific populations of medically ill patients. Current guidelines advocate for the use of venous thromboembolism (VTE) prophylaxis in hospitalized patients with an acute medical illness until the time of discharge . HoweverPrevious meta-analyses have shown that EDT is associated with a reduction in VTE risk, largely driven by a reduction in asymptomatic VTE events, a finding that is counterbalanced by an increased propensity for bleeding complications –8. PriorTrials that measure treatment effects can demonstrate exaggerated effect sizes early in the chain of evidence, a phenomenon referred to as the “proteus effect” ,11 of seBecause this was a systematic review and meta-analysis of published trial results, institutional review board and ethics committee approval was not required.Our review was registered with PROSPERO with the registration number CRD42018109151.This study was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines .We included RCTs enrolling adult patients (>18 years of age) hospitalized for acute medical illness and compared EDT with standard of care. We did not have any language exclusions. The search strategy is detailed in the supporting information .A systematic PubMed/MEDLINE, EMBASE, Cochrane Central, and ClinicalTrials.gov search was performed from inception through March 21, 2019 using prospectively established criteria  for RCTsMV and AQ independently extracted data, and any inconsistencies were resolved by a third investigator (NSB).Data items extracted from each study included study characteristics, risk of bias (RoB) items, demographic information, treatment details, follow-up, and outcomes of interest.RoB was assessed using the Cochrane RoB tool modified to capture the components of random sequence generation, allocation concealment, blinding of participants, blinding of outcome assessment, and analysis of incomplete outcome data.The principal efficacy endpoint examined in this analysis was symptomatic VTE or VTE-related death . We also2 statistic proposed by Higgins and Thompson; 95% confidence intervals (CIs) around I2 statistic were also estimated [p < 0.05 was considered statistically significant. We intended to assess small study treatment effects using funnel plot techniques, Egger’s regression test, and Duval and Tweedie trim and fill methods as appropriate, given the known limitations of these methods [Random effects modeling was used to estimate summary risk ratios (RRs) for all outcomes. Data were analyzed for heterogeneity using the Istimated . The Coc methods ,15. Cumu methods . Trial s methods . RoB for methods .Most meta-analyses lack sufficient statistical power to detect treatment effects even when they are large . When thWe calculated the pooled number needed to treat (NNT) to prevent one symptomatic or fatal VTE event and the number needed to harm (NNH) to cause one major or fatal bleeding event. To estimate pooled NNT and NNH, random effects meta-analyses of risk difference were performed, and the pooled estimates derived from these analyses were inverted . All anaThe 5 RCTs –5,9 in tp = 0.002) with moderate heterogeneity  (p < 0.001).Traditional and cumulative meta-analysis showed that EDT significantly reduced symptomatic VTE or VTE-related death alone across the 5 trials when compared with standard of care  . The hetd = 0.0) . When thSmall study treatment effects were not assessed because the number of included trials was inadequate to properly create a funnel plot or employ more advanced regression-based assessments.As new trial data became available, the cumulative meta-analysis showed increasing precision of treatment effect, with narrowing of 95% CI . The cump < 0.001) in both cumulative meta-analysis and trial sequential analysis  as compared with standard of care in both cumulative meta-analysis and trial sequential analysis. The heterogeneity estimates for all safety outcomes are presented in EDT also significantly increased risk of ISTH-criteria–based major or fatal bleeding , whereas pooled NNH to cause one major or fatal bleeding event was 333  . When trIn this study, we observed that EDT reduced the risk of symptomatic or fatal VTE when compared with standard-duration thromboprophylaxis  among medically ill patients after hospitalization. The observed benefits across trials were directionally consistent and of a similar magnitude and less likely to be due to type I or type II error because cumulative treatment effect from our analyses crossed both statistical and trial sequential boundaries of benefit. The benefits accrued with VTE prevention were, however, counterbalanced by an increased rate of major or fatal bleeding with EDT. The overall risks and benefits observed across the 5 trials were modest, as evidenced by high summary NNT and NNH.Patients discharged from hospital after medical illness face an ongoing risk for development of VTE for up to 6 weeks. These thrombotic events that develop within the postdischarge vulnerable period confer clinical morbidity, mortality, and excess healthcare expenditure because of the high rate of short-term readmission . Five clClinical interpretation of these aggregate findings must account for variation in the definitions of trial enrollment criteria, individual trial definitions of primary efficacy and safety endpoints, and EDT approaches in each trial using different agents with distinct pharmacological profiles and doses. The typical patient enrolled in these trials was relatively similar across trials—older, relatively immobile, with acute medical illness. However, some important differences deserve discussion. In 3 trials, certain risk-enrichment strategies were applied based on clinical parameters , age, anThe results of this meta-analysis provide a more comprehensive understanding of the use of EDT as a therapeutic strategy. For instance, the US Food and Drug Association recently approved the factor Xa inhibitor betrixaban based on exploratory analyses of aggregate data from the APEX trial despite it not having met its prespecified primary efficacy endpoint . The recNonetheless, our review is not without limitations. Amalgamation of data in the form of meta-analyses has well-recognized limitations . The incIn summary, our meta-analysis demonstrated that use of a post-hospital discharge EDT strategy for a 4-to-6-week period reduced symptomatic or fatal VTE events. These modest benefits were observed at the expense of increased risk of major or fatal bleeding events. Given the relatively infrequent occurrence of these events, we estimate that 250 patients would need to be treated with EDT to prevent one symptomatic or fatal VTE event, and 333 patients would need to be exposed to EDT to cause one major or fatal bleeding event. Further investigations are required to define risks and benefits within specific and discrete populations of medically ill patients, evaluate the cost-effectiveness of EDT, and develop pathways for targeted implementation of this postdischarge strategy in appropriately selected patients.S1 PRISMA checklist(DOCX)Click here for additional data file.S1 Search Strategy(DOCX)Click here for additional data file.S1 TableVTE, venous thromboembolism.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Sensorineural hearing loss is the most common form of hearing loss in humans, and results from either dysfunction in hair cells, the sensory receptors of sound, or the neurons that innervate hair cells. A specific type of sensorineural hearing loss, referred to as auditory synaptopathy, occurs when hair cells are able to detect sound but fail to transmit sound stimuli at the hair-cell synapse. Auditory synaptopathy can originate from genetic alterations that specifically disrupt hair-cell synapse function. Additionally, environmental factors such as noise exposure can leave hair cells intact but result in loss of hair-cell synapses, and represent an acquired form of auditory synaptopathy. The zebrafish model has emerged as a valuable system for studies of hair-cell function, and specifically hair-cell synaptopathy. In this review, we describe the experimental tools that have been developed to study hair-cell synapses in zebrafish. We discuss how zebrafish genetics has helped identify and define the roles of hair-cell synaptic proteins crucial for hearing in humans, and highlight how studies in zebrafish have contributed to our understanding of hair-cell synapse formation and function. In addition, we also discuss work that has used noise exposure or pharmacological mimic of noise-induced excitotoxicity in zebrafish to define cellular mechanisms underlying noise-induced hair-cell damage and synapse loss. Lastly, we highlight how future studies in zebrafish could enhance our understanding of the pathological processes underlying synapse loss in both genetic and acquired auditory synaptopathy. This knowledge is critical in order to develop therapies that protect or repair auditory synaptic contacts. Figures 1A,B). Hair cells transduce stimuli when apically localized mechanosensitive channels are activated, leading to graded depolarization of the hair-cell membrane . Hair-cells transmit stimuli at the hair-cell synapse. At the synapse, hair-cell depolarization opens voltage-gated calcium channels; calcium influx through these channels drives synaptic vesicle fusion and glutamate release onto innervating afferent nerves . If either the hair cells or downstream afferent nerves are damaged or dysfunctional, the pathological consequence is sensorineural hearing loss. Sensorineural hearing loss can be caused by genetic factors, infections, toxins, age and excessive noise, and is the most common form of hearing loss in humans (90% of cases) (Sensory hair cells in our inner ear must both reliably transduce and transmit auditory and vestibular stimuli (f cases) .Figure 1C). Ribbon synapses are required for transmitting stimuli in a fast and sustained manner needed for precise sensory encoding, and are structurally and functionally unique from classic neuronal synapses . Neuromast hair cells are particularly advantageous for hair-cell assessment because these cells are located superficially just beneath the fish skin, with their apical hair bundles protruding into the aqueous environment. This access makes it relatively straightforward to apply pharmacological agents, to stimulate the hair cells with fluid-flow, and assess hair-cell structure and function in vivo. Moreover, relative to mammals where hair cells mature over several weeks . For example, in zebrafish, the synaptic ribbon can be seen clearly in TEM as an electron-dense region that is adjacent to the postsynaptic density on the innervating afferent neuron . TEM is the most accurate way to determine the size of the synaptic ribbon. TEM can also be used to visualize the synaptic vesicles tethered to the synaptic ribbon and near the active zone . Currently TEM is the only method able to quantify the number and distribution of these synaptic vesicles populations. While these ultrastructural measurements are valuable, preparing, sectioning, imaging and analyzing TEM samples requires considerable time and effort. Moreover, in most cases, TEM is only able to capture a subset of synapses within each hair-cell organ.Similar to work in mammals, precise ultrastructural measurements can be obtained from zebrafish hair-cell synapses using TEM , synaptic ribbons , postsynaptic densities , afferent  and efferent  lateral-line neurons, and glutamate receptor subunits (Summary of Abs: Table 1). Immunolabels can be used in combination with transgenic lines that specifically mark cell types of interest such as hair cells, glia-like supporting cells, as well as the innervating neurons . Each afferent will respond to a single direction along the neuromast’s axis of sensitivity . Once this pairing is identified, many features relevant to hair-cell synapse function can be quantified using these recordings. For example, during fluid-jet stimulation, the timing and number of afferent spikes can be recorded. While spike number can provide important information on vesicle release and replenishment . Phase-locking assesses how consistently the afferent neuron spikes during a particular phase of the sine stimulus and reflects the fidelity of neurotransmission. Overall, afferent recordings have been able to resolve subtle yet important differences in zebrafish mutants that alter hair-cell synapse function . An important advantage of functional imaging over electrophysiogical recordings is the ability to resolve activity spatially among multiple cells and subcellularly within individual cells. In contrast, microphonics or extracellular afferent recordings are the readout of many hair cells within a neuromast, and whole-cell recordings examine activity from one hair cell at a time.Because the zebrafish model system is genetically tractable, work in the last decade has expanded toward using genetically encoded optical indicators to examine hair-cell function in zebrafish . These sFigure 1C). Initial work primarily used genetically encoded calcium indicators localized to the cytosol of lateral-line hair cells. During fluid-jet stimulation, it was shown that calcium signals could be reliably measured in the cytosol . To determine the location of synaptic ribbons and measure presynaptic calcium signals at sites of release, calcium indicator lines can be combined with an additional transgenic line that marks synaptic ribbons via a Ribeye-mCherry fusion protein . In response to fluid-jet stimulus, afferent calcium signals can be detected at sites adjacent to synaptic ribbons that are marked using a Ribeye-mCherry transgenic line  results in Sinoatrial Node Dysfunction and Deafness (SANDD), a disorder whereby affected individuals have abnormal heart rhythms and severe deafness  and subsequent loss of synaptic juxtaposition (at day 5) in zebrafish V1.3aca mutants is one example of how quickly phenotypic differences in synapse development and maintenance can be assayed in zebrafish. By taking advantage of these distinct developmental time points, this study also found that transiently (1 h.) treating the developing hair cells of 3 day-old zebrafish with dihydropyridine agonists and antagonists was able to rapidly decrease or increase presynaptic-ribbon size respectively , it possible that the release of trophic factors from the synapse is required for maintenance of hair-cell synaptic connections   is a progressive, high frequency non-syndromic hearing loss caused by a heterozygous mutation in the (VGLUT3) . Vglut3—(VGLUT3) . These ssduction . Additio neurons . Similarocytosis .vglut3 zebrafish mutants, afferent innervation appears relatively normal  localization at the base of hair cells , lysotracker brightly labeled rings around synaptic ribbons that likely correspond to ribbon-associated vesicles. The intensity of lysotracker labeling was dramatically reduced in rbc3α mutants, indicating reduced acidification of organelles and vesicles surrounding synaptic ribbons.This study found that zebrafish hair cells deficient in Rbc3α impacted Vacuolar-type Hir cells . V-ATPasrbc3α mutant synaptic vesicles contain less glutamate. Accordingly, afferent recordings revealed reduced glutamate-dependent evoked release from rbc3α mutant hair cells. In addition, rbc3α mutants showed loss of fidelity of phase-locked spiking at higher frequencies (20 vs. 60 Hz)  or the membrane (V0) subunits of the V-ATPase in wild-type and Rbc3α deficient hair cells revealed that Rbc3α is required to traffic or assemble the V1 subunit at the base of hair cells . Therefo. 60 Hz) . CurrentSynj2 mutants appears to be due to degeneration or loss of hair bundles, and sunken appearance of cell bodies, followed by hair-cell death. These observations support that Synaptojanin 2 plays an important role in hair-cell survival, but the process by which it contributes to cell survival is not understood.Proteins in the Synaptojanin family are lipid phosphatases that play an important role in endocytosis and vesicle recovery at synapses . AlthougSynj1 zebrafish mutants, similar to rbc3α zebrafish mutants, have moderate auditory and vestibular defects. These behavioral defects were accompanied by morphological disruptions in Synj1-defficient hair cells; specifically, basal membrane protrusions, or blebbing. These protrusions were dependent on functional Cav1.3 channels and were observed in 1/3 of synj1 mutant hair cells. In contrast to mice Synj2 mutants, other aspects of hair-cell morphology in zebrafish synj1 mutants appeared normal. Transmitted electron micrographs revealed fewer vesicles at synj1 ribbons, indicating vesicle recycling was impaired. Reduced vesicles and basal membrane protrusions in mutant synj1 hair cells led to deficits in synapse function; afferent recordings in synj1 mutants revealed a delay in afferent spike timing and impaired phase-locking in response to high-frequency stimuli  resulted in zebrafish with impaired acoustic startle response, vestibular abnormalities and defective optokinetic response (Pwi encodes tryptophan-rich basic (WRB) protein, a small transmembrane protein found in the endoplasmic reticulum that is a receptor for insertion of tail-anchored (TA) proteins. Disruption of TA-protein membrane insertion would likely result in disruption of TA-protein trafficking, and numerous hair-cell vesicular proteins are TA, including Otoferlin. A subsequent report further examined zebrafish pwi mutants, and verified that Wrb is necessary for normal Otoferlin protein levels in hair cells and hearing in zebrafish  in order to release neurotrophic factors including BDNF , Neurotrophin-3 (NT-3), Brain-derived neurotrophic factor (BDNF) may be important for the survival and recovery of afferent terminals and synapses . More sping BDNF . Future N-methyl-D-aspartate (NMDA) to mammalian inner ears or hair-cell explants has also been used to mimic glutamate excitotoxicity associated with noise exposure. In mammals, application of AMPA or KA results in overactivation of the iGluR receptors mediating neurotransmission on postsynaptic afferent terminals  was examined . This trA second study used similar methodology but applied iGluR agonists for longer durations . Here thCurrently there are no published zebrafish studies in adults or larvae using noise exposures to model acquired auditory synaptopathy in the ear. In the future, it will be useful to create protocols to modulate the intensity and duration of noise exposures in order to define the pathological changes in the zebrafish inner ear that are associated with moderate noise exposure. By modulating the intensity and duration of noise exposures in zebrafish, it will be possible to examine the dynamic progression of damage following moderate noise exposure, including loss of afferent fibers and synapses . In addiAfter establishing both moderate and intense noise exposure methods in zebrafish, it will be informative to apply the same tools and assays that have been used to understand the effects ototoxic drugs on hair-cell pathology. For example, the zebrafish lateral line has been used to screen for compounds that protect hair cells or promote hair-cell regeneration during and after ototoxic insult . Based oFigure 3), can be used to image hair cells in vivo. Using live imaging, it will be possible to examine the morphology of hair cells, ribbon synapses and afferent nerve terminals during noise exposure, as well as during repair and regeneration. Currently, it is not possible to visualize these changes in the hair-cell organs in living mammals. The ability to visualize morphological changes during and after insults in whole animals is an important advantage to using zebrafish for these studies. These live imaging approaches could reveal the specific pathological changes accompanying both moderate and intense noise exposures. While imaging morphological changes accompanying noise exposure or lateral-line over stimulation will be invaluable, it will also be interesting to explore the functional consequences to the synapse during the noxious insult, as well as during recovery. These studies could be accomplished using electrophysiology and imaging-based methods that have been established in the zebrafish lateral line .These studies will be particularly advantageous in larval zebrafish where transgenic lines (Finally, in addition to examining the morphological and functional consequences of noise exposure or lateral-line over stimulation in zebrafish, it will also be worthwhile to explore the downstream molecular mechanisms underlying the observed pathologies, as well as the recovery. In recent years, several models and methods have been developed to profile gene expression changes in specific cell types, including zebrafish hair cells . It willKK and LS wrote the review article and made the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mitochondrial genome-wide association studies identify mitochondrial single nucleotide polymorphisms (mtSNPs) that associate with disease or disease-related phenotypes. Most mitochondrial and nuclear genome-wide association studies adjust for genetic ancestry by including principal components derived from nuclear DNA, but not from mitochondrial DNA, as covariates in statistical regression analyses. Furthermore, there is no standard when controlling for genetic ancestry during mitochondrial and nuclear genetic interaction association scans, especially across ethnicities with substantial mitochondrial genetic heterogeneity. The purpose of this study is to (1) compare the degree of ethnic variation captured by principal components calculated from microarray-defined nuclear and mitochondrial DNA and (2) assess the utility of mitochondrial principal components for association studies. Analytic techniques used in this study include a principal component analysis for genetic ancestry, decision-tree classification for self-reported ethnicity, and linear regression for association tests. Data from the Health and Retirement Study, which includes self-reported White, Black, and Hispanic Americans, was used for all analyses. We report that (1) mitochondrial principal component analysis (PCA) captures ethnic variation to a similar or slightly greater degree than nuclear PCA in Blacks and Hispanics, (2) nuclear and mitochondrial DNA classify self-reported ethnicity to a high degree but with a similar level of error, and 3) mitochondrial principal components can be used as covariates to adjust for population stratification in association studies with complex traits, as demonstrated by our analysis of height—a phenotype with a high heritability. Overall, genetic association studies might reveal true and robust mtSNP associations when including mitochondrial principal components as regression covariates.  Mitochondrial genome-wide association studies (MiWAS) are used to identify mitochondrial single nucleotide polymorphisms (mtSNPs) that associate with disease or disease-related phenotypes. A human mitochondrion has several copies of a condensed circular genome that encodes 13 large proteins, 22 tRNAs, 2 rRNAs, and many peptides  ,5,6. GenSeveral mtSNPs have been associated with metabolic disease and neurodegenerative disease risk. For example, Kraja et al. reported that two mtSNPs significantly associated with metabolic outcomes in a MiWAS that included ~170,000 individuals from 45 cohorts . AdditioHowever, mitochondrial and nuclear genetic association studies are prone to confounding associations because of population substructures embedded within the studied samples if drawn from multi-ethnic or admixed groups . In ordeThe purpose of analyses presented here is three-fold: Use a large cohort with a high-level of admixture to (1) assess whether White, Black, and Hispanic individuals can be grouped into ancestral clusters using principal components derived from array-based mtSNPs, (2) compare nucDNA and mtDNA representation of ethnic variation, and (3) examine effects of principal components derived from mtPCA on height—a phenotype with a high heritability—in White, Black, and Hispanic Americans .Data from the Health and Retirement Study (HRS), an on-going nationally representative study of United States adults over 50 years-old, was used for all analyses. The goal of HRS is to track changes in aging-related outcomes over time. HRS has genotyped nearly 16,000 individuals using either the Illumina HumanOmni2.5-4v1 and HumanOmni2.5-8v1 arrays for samples collected in 2006, 2008, and 2010. Genotyping was performed by the NIH Center for Inherited Disease Research  with standard quality control procedures implemented by the University of Washington Genetic Coordinating Center ,22. The Principal component analysis was conducted separately using mtSNPs and nucSNPs in ethnic-stratified and ethnic-combined analysis . For nucSNPs , the PLINK 2.0 pca command was used to extract principal components. In this process, nucSNPs are used to calculate eigenvectors using a variance-standardized genetic relationship matrix between individuals, which is similar to that implemented in EIGENSTRAT software . No clumn = 4584) by using 20 nuclear and/or 20 mitochondrial principal components. The optimal model was selected using the largest accuracy value derived from the train function (rpart method and a tune length of 10) and subsequently predicted self-reported ethnicity on the remaining 70 percent of the data using the predict function. Plots were generated using the prp function of the rpart.plot R package. The caret R package, which contains a group of functions to create predictive models, was used to generate a cross-validated  decision-tree training algorithm on 30 percent of the data  and in a combined ethnicity model using the lm function in R. The dependent variable was height (in centimeters) and the predictors included a total of 20 principal components, biological sex, and centered age. The purpose of these analyses was to (1) understand whether reducing mitochondrial genetic variation with principal components could explain the variation in height within and across ethnic groups and (2) serve as proof-of-concept for using mtSNPs to characterize genetic ancestry in association studies. The race/ethnic makeup of the study sample is presented in Nuclear PCA revealed that the first three principal components captured the highest amount of variance and, when these three were plotted, reflected an expected pattern of ancestry. As shown in Mitochondrial PCA revealed an expected pattern of ancestry when plotting the first three principal components. Also shown in The analysis of intra-ethnic mtPCA revealed population substructures. As seen in The amount of variation captured by nucPCA and mtPCA was examined by self-reported ethnicity. While inter-ethnic nucPCA captured much more variance in fewer components than mtPCA, intra-ethnic mtPCA captured similar to slightly more variance compared to nucPCA . In partWe assessed how well nuclear and mitochondrial principal components classified broader ethnic sub-groups when defined by self-report. Using optimal decision tree algorithms derived from a 30% training sample and then separately implemented on the remaining 70% of the data, we found that nuclear and mitochondrial principal components comparably classified individuals into ethnic sub-groups to a high degree: at a 94.9 percent and 92.0 percent rate, respectively. Combining both nuclear and mitochondrial principal components increased statistical classification accuracy into self-reported ethnic subgroups to 96.8 percent. This analysis is notable because the nuclear and mitochondrial misclassification error suggests controlling for genetic ancestry within self-reported ethnic analyses or assigning individuals into genetically homogenous groups for analyses is necessary. Cutoffs and nodes are illustrated in In order to evaluate the utility of mitochondrial principal components as covariates to adjust for population stratification in association studies, we tested its association with height , which iOur analyses demonstrated the utility of mtPCA for mitochondrial and nuclear genetic association studies. First, we showed genetically admixed substructures from mtDNA in all ethnicities in HRS. Second, we illustrated that the amount of variance captured by mitochondrial principal components in Hispanics and Blacks is similar to slightly greater than that captured by nuclear principal components, whereas nuclear principal components captured substantially more variance in combined ethnic analysis and in Whites. Third, using mitochondrial and nuclear principal components to train a decision tree for self-reported ethnicity classification showed high statistical accuracy yet similar misclassification error between mitochondrial and nuclear analyses. This misclassification rate suggests that conducting MiWAS by ethnic-specific stratification without adjusting for genetic ancestry might not be a sufficient way to control for genetic admixture. Hence, we showed that factoring in principal components during stratified analysis can provide an analytic approach to further address the more complex admixture. Our analysis shows that mitochondrial principal components associated with a high heritability phenotype, height, when evaluated across ethnicities and intra-ethnically. One novel aspect of our analyses is that mtSNPS derived from an array capture within ethnic variation, which could be critical when designing analytic strategies to minimize confounding due to admixture. In the absence of nuclear DNA data during mitochondrial gene association studies , controlling for genetic ancestry using mitochondrial principal components could reduce type one error and provides a solution for analyses lacking nuclear DNA. As nationally representative cohorts continue to grow larger, it is likely that research groups will attempt to identify the effects of mtSNPs on a variety of phenotypes. Based on previous publications, groups might design their analytic strategies by assigning terms to mitochondrial haplogroups or single mtSNPs while controlling for genetic ancestry. The former is limited by reference group classification and the latter is limited by no standard method to control for genetic ancestry. Notably, Biffi et al. examined mtSNPs derived from commercially based arrays—similar to that used by HRS— and showed that mitochondrial haplogroup analysis was inferior to mtPCA for discovery of true associations and nucPCA had little effect on mitochondrial association testing . Since pFuture mitochondrial gene studies might reveal true and robust mtSNP associations by controlling for mitochondrial genetic principal components. Moreover, it is plausible that there is significant interaction between nuclear and mitochondrial SNPs, and that controlling for mitochondrial genetic ancestry to identify such nuclear and mitochondrial genetic associations might be an important consideration when defining the analytic strategy. However, subpopulation genetic architecture will vary from cohort-to-cohort due to SNP-based array and sample variation. Therefore, before conducting genetic association studies, comparing nuclear and mitochondrial genetic substructures by ethnicity could guide analytic plans.