Twelve evolutionarily unrelated oxidoreductases form enzyme complexes that catalyze the simultaneous coupling of exergonic and endergonic oxidation–reduction reactions to circumvent thermodynamic barriers and minimize free energy loss in a process known as flavin-based electron bifurcation. Common to these 12 bifurcating (Bf) enzymes are protein-bound flavin, the proposed site of bifurcation, and the electron carrier ferredoxin. Despite the documented role of Bf enzymes in balancing the redox state of intracellular electron carriers and in improving the efficiency of cellular metabolism, a comprehensive description of the diversity and evolutionary history of Bf enzymes is lacking. Here, we report the taxonomic distribution, functional diversity, and evolutionary history of Bf enzyme homologs in 4,588 archaeal, bacterial, and eukaryal genomes and 3,136 community metagenomes. Bf homologs were primarily detected in the genomes of anaerobes, including those of sulfate-reducers, acetogens, fermenters, and methanogens. Phylogenetic analyses of Bf enzyme catalytic subunits (oxidoreductases) suggest they were not a property of the Last Universal Common Ancestor of Archaea and Bacteria, which is consistent with the limited and unique taxonomic distributions of enzyme homologs among genomes. Further, phylogenetic analyses of oxidoreductase subunits reveal that non-Bf homologs predate Bf homologs. These observations indicate that multiple independent recruitments of flavoproteins to existing oxidoreductases enabled coupling of numerous new electron Bf reactions. Consistent with the role of these enzymes in the energy metabolism of anaerobes, homologs of Bf enzymes were enriched in metagenomes from subsurface environments relative to those from surface environments. Phylogenetic analyses of homologs from metagenomes reveal that the earliest evolving homologs of most Bf enzymes are from subsurface environments, including fluids from subsurface rock fractures and hydrothermal systems. Collectively, these data suggest strong selective pressures drove the emergence of Bf enzyme complexes via recruitment of flavoproteins that allowed for an increase in the efficiency of cellular metabolism and improvement in energy capture in anaerobes inhabiting a variety of subsurface anoxic habitats where the energy yield of oxidation-reduction reactions is generally low. Table 1) and each of these comprise multiple protein subunits that form a complex . A common theme among bifurcating (Bf) enzymes is the involvement of ferredoxin (Fd) as a substrate as well as coordination of at least one flavin, the proposed site of bifurcation (+) and exergonic reduction of NAD+ via oxidation of NADPH  and NADP(H) and is thus a key regulator modulating the favorability of catabolic and anabolic reactions (Flavin-based electron bifurcation (FBEB) involves the simultaneous reduction of two electron acceptors using a single electron donor in an enzyme complex, whereby a thermodynamically favorable exergonic reaction drives a thermodynamically unfavorable endergonic reaction . A totalurcation . Studiesof NADPH , revealsn by Fd+ . Thus, aeactions .+ and balancing the redox state of the pyridine nucleotide pool, FBEB has been suggested to improve the efficiency of cellular metabolism by allowing for more complete capture of energy released during substrate oxidation (+ or NAD+) for those compounds. For example, glucose fermentation in Clostridium pasteurianum generates 3.0 mol ATP per glucose when non-Bf butyryl-CoA dehydrogenase (Bcd) is involved whereas 3.3 mol ATP per glucose is generated when Bf Bcd is involved, an increase of ∼11% (-) are generated. All three Fd- are shuttled toward hydrogen (H2) generation, while all three NADH are used to produce butyryl-CoA when non-Bf Bcd is involved. However, when Bf Bcd is involved, one NADH is used to reduce Fd+ that eventually yields an additional H2, one NADH is used to reduce two acetyl-CoA to crotonyl-CoA, and the other NADH is used to further reduce crotonyl-CoA to butyryl-CoA, which generates additional ATP. The reader is referred to several recent reviews  available in the National Center for Biotechnology Information (NCBI) database as of March 2016 were compiled. This compiled database included complete genomes of Archaea (n = 230), Bacteria , and Eukarya (n = 15). In addition, the protein sequences encoded in each of the environmental metagenomes  in the Department of Energy’s Integrated Microbial Genomes and Microbiomes (DOE-IMG) (All complete genomes (DOE-IMG)  as of ApTable 1) was identified based on literature surveys and empirical bioinformatics analyses, as described below for each Bf system. Representative sequences of each subunit were used as bait sequences to extract all the homologs of each subunit for each enzyme complex from the complete genome database (described above) using the phmmer program via the HMMER (ver. 3) software package (Table 1). Importantly, since assembled genomes from natural environments are often incomplete , and since our criteria required the presence of genes encoding the minimum complement of proteins that constitute a given Bf enzyme complex to be present for it to be counted as a homolog, the distribution of Bf enzyme homologs reported here for metagenomic sequences is likely to be a conservative estimate of their actual abundance.The minimum number of subunits that comprise a Bf enzyme complex . A custom python script was used to assess the presence of companion subunits in each case, and the number of open reading frames that were surveyed for companion subunits are provided for each catalytic subunit in Table 1. If the genes encoding subunits necessary for bifurcation capacity were identified within these open reading frames, the enzyme homolog was considered as a putative Bf enzyme, whereas putative non-Bf enzyme homologs were identified by the absence of the specified subunits in the flanking gene regions. We also screened metagenomes for homologs of genes coding for putative Bf enzymes using the same approaches outlined briefly above and as described for each system below in more detail.For each Bf enzyme homolog, companion subunits for the catalytic subunit were identified in flanking gene regions based on the expected gene distributions from empirical analyses  and non-Bf [FeFe]-hydrogenases as monomeric or dimeric  cluster(s)  (hydABC (minimum among both trimeric and tetrameric forms) and these genes tend to be co-localized in genomes , we first screened our databases using HydA from Chlamydomonas reinhardtii (AAL23572) as a query since it does not encode N- and C-terminal FeS cluster binding motifs (Our previous bioinformatics work classified putative Bf [FeFe]-hydrogenases (Hyd) as multimeric ( dimeric . Putativgure 2A) . In addigure 2A) . Therefo genomes . Becauseg motifs . Extractg motifs  and wereg motifs .2 oxidation occurs), MvhG, and MvhD; both MvhG and MvhD harbor FeS cluster binding motifs  (Table 1), aligned and screened for distal and vicinal cysteine pairs that delineate [NiFe]-hydrogenase from paralogs such as MbxL, FuoD, and NuoD  harboring homologs of Bf enzymes . Mvh comg motifs . Mvh ass 1B, 2B) . Therefoand NuoD . MvhA haand NuoD . TherefoMethanothermobacter marburgensis where Mvh-Hdr was first described  that distinguishes it from its paralog FdhF2; FdhF2 is involved in another Bf complex that is described below  that include FdhA that contains a conserved [4Fe-4S] binding motif and HytA that is the catalytic site for H2 oxidation . HytA is homologous to HydA and contains key active site motifs that are largely the same as those in HydA, including the L1 (TSCCPxW), L2 (MPCxxKxxE), and L3 (ExMACxxGCxxGGGxP) motifs. Likewise, HytB and HytC are homologous to HydB and HydC, respectively. Homologs of HytA were aligned and were screened for the L1, L2, and L3 motifs, as defined above; those sequences that did not contain these motifs were discarded. HytE1, HytE2, and HytABCD are co-localized in the genome (Supplementary Table Table 1).The NADP(H)-dependent formate dehydrogenase (Hyt) complex, first identified in xidation . In addiClostridium kluyveri comprises two subunits: the small subunit (NfnS) that contains one FAD and one [2Fe-2S] cluster and the large subunit (NfnL) that contains one FAD and two [4Fe-4S] clusters   . Several 1E, 2E) . NfnI ha 1E, 2E) . NfnII r, 2E)    .There are several classes of electron transfer flavoproteins, including those that bifurcate and those that do not, as discussed previously . Non-Bf gure 1F) . Fix frolectrons . FixA, Flectrons . FixX coC. kluyveri .Butyryl-CoA dehydrogenase (Bcd) is typically involved in fatty-acid metabolism  and has Acetobacterium woodii . In addition to containing a flavin binding domain, CarD also contains two [4Fe-4S] cluster binding motifs .To our knowledge, caffeyl-CoA reductase (CarC) has not yet been reported to function on its own (see caveat below). Rather, characterized enzymes form a complex that consists of three subunits: CarC, EtfA (CarD), and EtfB (CarE). This complex was first identified in m woodii . All thrClostridium acidurici  . In addition to putatively binding FeS clusters, HylB also contains a flavin binding motif . HylA is homologous to HydA but lacks conservation in the three aforementioned active site motifs, L1, L2, and L3 (The NAD(H)-dependent formate dehydrogenase (Hyl) complex is tetrameric and was first identified in gure 1I) . The tet, and L3 . ExtractFigure 1J; A. woodii and Ldh and EtfAB contain flavin binding motifs  (Table 1).Lactate dehydrogenase (Ldh) is involved in the reduction of pyruvate to lactate . Ldh cangure 2J) . In addigure 2J) , which gMethanosarcina acetivorans (420H2 with the reduction of Fd+ and heterodisulfide from coenzyme M (CoM) and coenzyme B (CoB) (termed Hdr2) . Like HdrABC, the catalytic subunit of Hdr2A is not co-localized  cluster and a [2Fe-2S] cluster, respectively  (Escherichia coli which does not form a Bf complex (Methylene-tetrahydrofolate (Hrom NADH . It can rom NADH . Furtheroacetica . In addigure 2L) . Importa complex . Hence,  complex  were retA binary table was created to represent the presence or absence of specified Bf enzymes in each genome and metagenome. The binary table was used as input to generate an abundance plot and was also subjected to co-occurrence analysis using the co-occur package in R .All 16S ribosomal RNA (rRNA) genes from genomes that encoded at least one homolog of a Bf enzyme were compiled via BLASTn. The extracted sequences were subjected to multiple sequence alignment using the SILVA rRNA gene database  as an alTo further define the potential taxonomic origin of the 12 Bf enzyme complexes among extant organisms with available genome sequences, and to identify environment types that harbor organisms with the most deeply rooted homologs of each of the 12 Bf complexes, we also subjected our curated database of oxidoreductase catalytic subunit homologs  from complete genomes and metagenomes to phylogenetic analysis. We did not conduct phylogenetic analysis of the catalytic subunit of Hdr homologs  identified in metagenomes because of difficulty in identifying whether the subunits of Hdr2  were from the same genome and if they function alone or in association with Met, Mvh, or Fdh, as described above. Likewise, while phylogenetic analyses were conducted on HdrA, we did not assign homologs as belonging to Met, Fdh, Mvh, or Hdr2 complexes since it is not possible to link these functions based on genome context. Briefly, paralogs (described below) of each of the aforementioned catalytic subunits were identified and the curated putative Bf homologs were aligned using Clustal Omega . The aliParalogs of the catalytic subunits for each Bf enzyme complex have already been identified in previous studies for many of the enzymes and these served as outgroups in phylogenetic reconstructions. These include the eukaryotic Nar-like protein that lacks the conserved L1, L2, and L3 motifs present in HydA/HytA , non-Bf The numbers of Nfn and Met homologs in metagenomes were much larger  than those associated with other classes of Bf enzymes. Therefore, to reduce the computational time necessary to analyze the evolutionary history of homologs of these two enzymes, we first clustered them into homolog ‘bins’ using CD-HIT  at the 6To determine the extent to which individual subunits comprising each of the Bf complexes have co-evolved, we first aligned homologs of individual subunits using Clustal Omega . Pairwis4 and are presented as this transformed value. High E-values indicate that homologs exhibit a lower diversity and thus are more similar phylogenetically. Low E-values indicate that homologs are more diverse and are less similar phylogenetically. We then subjected homologs of the catalytic subunits of Bf complexes identified among the metagenome datasets to pairwise sequence comparisons against the dataset comprising the homologs identified in genome sequences using phmmer. This analysis was performed to assess the extent that homologs identified in the complete genome dataset adequately captured the natural diversity detected in metagenomes for each Bf enzyme. Homologs identified in metagenomes that exhibit a lower average or range of E-values than those identified in complete genomes are underrepresented in complete genome databases .Lastly, we determined the variance in amino acid identities of homologs of the catalytic subunits of Bf complexes identified among the complete genome datasets as a proxy for the functional diversity of those homologs. Pairwise sequence comparisons of the homologs of each Bf enzyme catalytic subunit identified in complete genomes were used to calculate pairwise E-values using phmmer. E-values identified for each pairwise comparisons were used as a proxy for amino acid identity differences. E-values were normalized by multiplying by -10n = 126 genomes), Crenarchaeota (n = 42 genomes), and the candidate division Korarchaeota (n = 1 genome). The majority (83%) of Bf enzyme homologs were identified in genomes from the bacterial phyla Firmicutes (n = 184 genomes), Proteobacteria (n = 147 genomes), Bacteroidetes (n = 44 genomes), Thermotogae (n = 25 genomes), and Spirochaetes (n = 22 genomes)  coded for at least one homolog of a Bf enzyme (Supplementary Table 2) into their energy metabolism based on information acquired from the DOE-IMG database and previous physiological characterizations. Of the 681 identified organisms whose genomes encoded at least one Bf enzyme homolog, 16% (n = 110 genomes) were aerobes, 9% (n = 59 genomes) were facultative anaerobes, and 74% (n = 503 genomes) were strict anaerobes. The prevalence of Bf enzyme homologs among obligate anaerobes is consistent with our hypothesis suggesting the importance of FBEB for organisms inhabiting energy limited environments.We also classified organisms whose genomes coded for Bf enzyme homologs as a function of their ability to integrate oxygen . In total, 229 homologs of Bf Hyds were identified in complete genome sequences, of which 95% were from the genomes of anaerobes, 4% were from the genomes of facultative anaerobes, and 1% were from the genomes of putative aerobes that belong to the phylum Spirochaetes  ; the ability of T. parva and S. pacifica to use O2 has not been robustly tested. The genomes that coded for homologs of Bf Hyds belonged exclusively to the bacterial domain, which is consistent with previous studies  homologs detected in complete genome sequences, 24% were multimeric  and hence can potentially bifurcate electrons ( studies .2) , arsenate reduction , nitrate reduction , sulfur reduction , and sulfate reduction , although it is not necessarily clear if these homologs are active under these physiological conditions. Moreover, homologs of the Bf Hyd were identified in the genomes of organisms that inhabit a range of environments based on where they were isolated from, including those that are of moderate  to high temperature .Hyd is one of three evolutionarily distinct hydrogenases that catalyze the reversible reduction of protons to hydrogen (H2) . Bf HydsFigure 3A). Mvh homologs were identified in the genomes of obligate anaerobes  and were largely confined to members of the archaeal domain  with the remaining 26% of the genomes that encode Mvh being from members of the bacterial domain  in complete genomes were predicted to bifurcate electrons based on those genomes also coding for MvhADG and HdrABC and the presence of unique sequence motifs in MvhA , halophilic bacteria , ammonifying bacteria , and sulfate-reducing archaea . This suggests that the reversible reduction of Fd+ and CoM-CoB with H2 may be involved in a diversity of metabolisms , (ii) a [FeFe]-hydrogenase-like complex (termed Hyl), and (iii) a NADP(H)-dependent [FeFe]-hydrogenase complex (termed Hyt). Among the total 2,516 homologs of formate dehydrogenase that were detected in available complete genomes, 2% were predicted to form a complex that would allow for bifurcation of electrons ; the remaining formate dehydrogenase homologs are unlikely to bifurcate electrons and likely function as canonical formate dehydrogenases which reversibly oxidize formate to CO2  that were primarily within the archaeal phylum Euryarchaeota (Supplementary Table Formate dehydrogenase can form a Bf complex with (i) a Hdr complex  are homologous to HydABC of the Bf [FeFe]-hydrogenase complex (Hyd). However, Hyt appears to be specific for NADP(H) whereas Hyd utilizes NAD(H) . The roles of HytE1, HytE2, and HytD in the functioning of Hyt are not presently known, however, they harbor conserved cysteine residues that may serve as ligands for FeS clusters. This may suggest that they are involved in electron transfer within the enzyme complex.In total, the genomes of five acetogenic and obligately anaerobic bacteria that belong to the genus  complex . Howevers NAD(H) . Nonethe to HydA , Fd bind to HydA , and NAD to HydA  . Of the 397 archaeal and bacterial genomes that encoded Nfn, 72 were from Archaea and 325 were from Bacteria. The majority (82%) of the archaeal and bacterial genomes that coded for Nfn belonged to the bacterial phyla Firmicutes (n = 138 genomes), Proteobacteria (n = 53 genomes), Bacteroidetes (n = 44 genomes), Thermotogae (n = 24 genomes), and the archaeal phylum Euryarchaeota (n = 66 genomes) (Supplementary Table Candidatus Koribacter versatilis Ellin345 (ABF41796), which has been characterized as an aerobe , and NfnIII, which are all proposed to bifurcate electrons . HoweverPyrococcus furiosus shows that flavin is indeed the site of bifurcation , acetogenesis , elemental sulfur reduction , dinitrogen reduction , heterotrophy , and phototrophy , among others . Unlike other Bf enzymes that are largely confined to anaerobic or facultatively anaerobic taxa, the majority  of the homologs of Fix were identified in the genomes of aerobes . Of the 178 genomes that coded for homologs of Fix, 25% were from Archaea within the phylum Crenarchaeota (n = 36) (Supplementary Table n = 85 genomes) and Firmicutes (n = 33)  are termed Fix , iron reducing anaerobic archaea , anaerobic acetogenic bacteria , anaerobic sulfate-reducing bacteria , and anaerobic fermentative thermophilic bacteria   and hence can potentially bifurcate electrons . Ninety-eight percent of Bf-Bcd encoding genomes were from anaerobic taxa . Bf-Bcd homologs were confined to Bacteria where the enzyme was detected in the phyla Firmicutes (n = 56 genomes), Fusobacteria (n = 15 genomes), and Bacteroidetes (n = 12 genomes) (Supplementary Table Butyryl-CoA dehydrogenase (Bcd) can form a complex with EtfAB and bifurcate electrons . In tota+ and crotonoyl-CoA to the oxidation of NADH during clostridial fermentation , Firmicutes , and Fusobacteria  encoded Bf-Bcd. In addition, homologs of Bf-Bcd were detected in the genomes of metal reducers , acetogens , and thermophilic heterotrophs  , and thus are predicted to bifurcate electrons . The Car complexes were identified in the genomes of anaerobic bacteria , primarily within the phylum Firmicutes  or in complex with Etfs  . In totaA. woodii where it was shown to couple the simultaneous and reversible reduction of Fd+ and caffeyl-CoA with the oxidation of NADH , a fermentative halophile , and in several fermentative thermophiles within the Thermotogales , among others  and these homologs were detected only in the genomes of anaerobes  within the phylum Firmicutes , metal reducers , arsenite oxidizers , acetogens , sulfate-reducers , and thermophilic heterotrophs   and bifurcate electrons . The Bf-Ldh homologs identified belonged to the bacterial domain and were from either obligate anaerobes or facultative anaerobes .Lactate dehydrogenase (Ldh) can form a complex with EtfAB  and these included metal reducers , acetogens , sulfate-reducers , and anaerobic phototrophs  . In total, 63% (n = 226 homologs) of the HdrABC homologs identified were identified in genomes that encoded copies of HdrABC attributable to Fdh, Mvh, or Met complexes. The extra copies of HdrABC in these genomes were thus designated as Hdr2 . Hdr2 homologs were primarily identified in genomes from the phyla Euryarchaeota (n = 66 genomes), Proteobacteria (n = 44 genomes) and Firmicutes (n = 25 genomes) .A total of 770 homologs of heterodisulfide reductase (HdrABC) were identified, of which 46% were predicted to form a complex with either FdhAB, MvhAGD, or MetFV (described below) or to function alone , as previously noted (M. thermoacetica), sulfate-reducers , and anaerobic phototrophs  suggesting that it is widely distributed among organisms with diverse metabolic backgrounds  of these were from genomes that also encoded MetFV, MvhD, and HdrABC. Thus, these homologs can putatively bifurcate  . Homologs of Met were primarily confined to the phyla Firmicutes (n = 12 genomes) and Proteobacteria (n = 8 genomes) and all were from obligate anaerobes .A total of 1,017 methylene-H4F to the oxidation of NADH and a yet to be identified electron acceptor that is thought to be Fd+ , acetogens , sulfate-reducers , and nitrate reducers   different Bf enzymes were detected in a single genome and this was particularly true for organisms belonging to the phyla Firmicutes, Thermotogae, and Euryarchaeota , positively co-occurred . This is consistent with biochemical data that indicates both [NiFe]-hydrogenase (MvhAG) and formate dehydrogenase (FdhAB) compete to bind MvhD that is associated with HdrABC , consistent with previous reports . Importantly, since these five enzymes share common substrates , several authors have proposed that the reductant generated from one Bf enzyme can be utilized by another Bf enzyme  are generated and caffeyl-CoA is kept from building up, thereby allowing for metabolism to continue  and Hyd (46% of the Bf-Bcd encoding genomes) .Twenty eight of the 41 phyla represented in our complete genome sequence dataset coded for at least one homolog of a Bf enzyme  of the 12 Bf enzymes were identified in genomes from both bacterial and archaeal domains indicating that they may have been a property of the LUCA. To more precisely assess this possibility, we examined (i) the extent that subunits comprising a complex co-evolved and (ii) the phylogeny of the oxidoreductase subunit for each of the Bf enzymes relative to paralogs as described below; the phylogeny of Hdr2 was not assessed . Moreover, the oxidoreductase phylogenies were used to assess if subunits that allow for bifurcation (flavoproteins) were recruited or lost (or both through multiple events) during the evolution of the oxidoreductases.Hyd, Hyl, and Hyt were only detected in bacterial genomes, suggesting that these three enzymes evolved after the divergence of Archaea and Bacteria from the LUCA. This conclusion agrees with a previous analysis of the evolution of Hyd , but is T. maritima (NP_229226) and T. africanus (ACJ75249)] and Dictyoglomi . These lineages were well-supported, suggesting that Hyd likely evolved and diversified in an ancestor of Thermotogae or Dictyoglomi (Table 2). Importantly, the taxa comprising the earliest evolving clade are thermophilic anaerobes, suggesting that Bf Hyd may have evolved in an anoxic, high temperature environment. Numerous clades of Bf HydA homologs formed interspersed lineages among monomeric non-Bf HydA lineages. The positive, strong relationships observed in the pairwise distances of HydABC  reveals positive correlations . HylA homologs formed a single monophyletic clade suggesting that Hyl evolved once during its evolution ] belonging to the phylum Firmicutes (100% bootstrap support). The next branching lineage also comprised homologs from thermophilic Firmicutes. This suggests an origin for Hyl among an anaerobic ancestor of the Firmicutes in an anoxic high temperature environment (Table 2).The earliest evolving Hyl is from an anaerobic thermophile -hydrogenase isoform.Previous phylogenetic reconstructions of the large subunit of Mvh  suggests that it is derived from a non-Bf [NiFe]-hydrogenases through recruitment of the Hdr complex  and other subunits  , an asseD. autotrophicum (ACN14546)] with high bootstrap support, suggesting that Bf Fdh likely emerged in a bacterium in an anoxic environment. Bf archaeal FdhA lineages are nested among, and are paraphyletic to, bacterial FdhA lineages. This suggests that FdhB/HdrABC may have been recruited to an ancestor of proteobacterial FdhA. Moreover, these observations suggest that once FdhB/HdrABC were recruited to function with FdhA as a Bf Fdh complex, the Fdh complex was then laterally transferred to Archaea. This interpretation is supported by strong positive correlations between the pairwise distances of the subunits of Fdh complex  , the lack of monophyly among archaeal and bacterial Bf FdhA homologs, and the observation that euryarchaeote homologs are nested among bacterial FdhA homologs to indicate that Bf Fdh was unlikely to be a property of the LUCA.Bf Fdh homologs exhibit a narrow taxonomic distribution but were identified in both archaeal (Euryarchaeota) and bacterial (Proteobacteria) genomes. Phylogenetic reconstruction of the oxidoreductase subunit of Fdh  suggests that Bf Fdh postdates non-Bf formate dehydrogenases  belonging to the phylum Actinobacteria . Paraphyly among bacterial and archaeal Fix lineages, combined with data indicating that Fix is primarily encoded in the genomes of aerobes and facultative anaerobes , suggests that Fix was not a property of the LUCA. Rather, these features suggest a more recent origin for this Bf complex and that this likely occurred after the advent of oxygenic photosynthesis and the increase of atmospheric O2 ∼ 2.4 Ga , suggesting that they emerged after the divergence of Archaea and Bacteria from the LUCA. Phylogenetically, the catalytic subunits of Bf-Bcd and Car ] each form coherent lineages that are interspersed with putatively non-Bf lineages  within the phylum Firmicutes (100% bootstrap support), which is followed by several lineages comprising additional homologs from Firmicutes. These lineages are well-supported, suggesting that Bf-Bcd likely evolved first in an anaerobic member of the Firmicutes. Likewise, the earliest evolving putatively Bf CarC  homologs were identified in the genome of Natranaerobius thermophilus (ACB85453) within the phylum Firmicutes (100% bootstrap support)  lineage is from Figure 5) suggesting that Bf-Ldh also evolved after the divergence of Archaea and Bacteria from the LUCA  were only detected in bacterial taxa (Thermovirga lienii (AER67312) within the phylum Synergistetes (100% bootstrap support). The next branching lineage comprised homologs from genomes affiliated with the Thermotogae. Collectively, these observations suggest that Bf-Ldh evolved in an anaerobic ancestor of the Synergistetes or Thermotogae after the divergence of Archaea and Bacteria from the LUCA , we can evaluate the evolutionary history of HdrA to determine whether HdrABC were likely to be a property of the LUCA.Desulfobacca acetoxidans) and Firmicutes   and bacterial (Firmicutes) taxa. Phylogenetic reconstruction of homologs of MetF suggests that Bf Met postdates non-Bf methylene-HD. acetoxidans (AEB08698) that belongs to the phylum Proteobacteria, with high bootstrap support. This suggests that Met likely evolved in an anaerobic ancestor of Proteobacteria. Only one archaeal Met homolog was identified, and this was encoded in the genome of the methanogen M. intestinalis. Phylogenetic reconstruction of MetF from this putative Bf complex reveals that it is nested among bacterial MetF homologs suggesting that it was likely acquired via horizontal gene transfer from Bacteria. Collectively, our findings suggest that Met was not a property of the LUCA but rather emerged in a bacterial anaerobe and was laterally transferred to Archaea.The earliest evolving lineage of Met comprised only one homolog from the anaerobe Results from our screening and analysis of complete genome sequences reveal that homologs of Bf enzymes are enriched in anaerobes and strongly suggest that each enzyme system has an independent origin in anaerobic taxa, with the potential exception being Fix. We therefore hypothesized that microbial communities inhabiting anoxic environments would (i) be enriched in homologs of Bf enzymes relative to oxic environments and (ii) host the earliest evolving homologs of enzymes involved in FBEB. Specifically, we hypothesized that subsurface environments, which are characteristically limited in redox gradients and feature a low flux of oxidants and other nutrients , would sTo test these hypotheses, we compiled 3,136 available metagenomic sequences and classified them as being from ‘surface’ or ‘subsurface’ environments based on a previously described classification scheme (Supplementary Table R2 = 0.18) (data not shown). This suggests that the lack of detection of homologs with more subunits was not due simply to the lower probability of assembling contigs with the minimal number of specified genes in near synteny.Screening of the 3,136 metagenomes revealed that 893 metagenomes coded for a homolog of at least one Bf enzyme  when compared to metagenomic datasets. For example, several taxonomic groups of methanogens or anaerobic alkane degraders have only recently been discovered or characterized via environmental genomics surveys , suggestFigure 6), with most being derived from surface soils  and surface waters  that included lakes, freshwater streams, and oceans  , but were surprisingly not enriched in subsurface sediment microbiomes relative to those from surface environments.Of the 893 metagenomes that encode Bf enzyme homologs, 275 were classified as being derived from subsurface-like environments, and 618 were classified as being derived from surface-associated environments . Most of the Bf complexes were identified in at least seven of the eight metagenome subcategories, with the exception of Hyt (not detected), Hdr2 (not included) and Car, the latter of which was only identified in one deep subsurface and one soil metagenome. Homologs of Bf enzyme complexes were generally overrepresented in metagenomes associated with deep subsurface habitats, such as subsurface rock fracture fluids and were overrepresented in environments that share characteristics with subsurface environments, such as hydrothermal springs/vents . This was particularly true for Nfn, Hyd, Fix, and Bf-Bcd. Like deep subsurface environments, hydrothermal springs/vents often exhibit highly reducing conditions and minimal energetic gradients  of the Bf complexes featured deep-branching lineages comprising homologs from either deep subsurface or hydrothermal springs/vents metagenomes . Many surface environments considered to be broadly oxidized  harbor anoxic microcompartments (2) penetrates only millimeters to centimeters  where phylogenetic data potentially suggest an origin in a surface like environment are nonetheless found almost exclusively in the genomes of anaerobes (artments  that maytimeters . Thus itTable 2). Specifically, both analyses indicate that several of the Bf enzymes are likely to have originated in thermophiles, sulfate reducers, and members of the Firmicutes. Thermophiles were well-represented in the earliest branching homologs of Bf hydrogenases (Hyd and Mvh), Nfn, Fix, and Hyl. Origins for Bf enzymes in thermophiles is consistent with the generalized lack of O2 in high temperature environments due to the inverse relationship between O2 solubility and temperature . It is possible that the function of these Bf homologs may differ from those that have been biochemically characterized. For example, the deepest-branching [NiFe]-hydrogenase Mvh homolog was identified in a deep sea hydrothermal vent community and is distantly related to Mvh from Acetomicrobium thermoterreneum (Table 2). To date, Bf Mvh complexes have only been characterized in Archaea . The lack of close representation in existing genomic databases or culture collections for many of these early evolving homologs from metagenomic datasets suggests the presence of a greater diversity of Bf enzyme complexes in natural systems than is currently available in genome databases from cultivars. To assess the diversity of Bf enzyme homologs in genome and metagenomic datasets and to determine the extent that the diversity present in metagenomic datasets is captured by genomic datasets, we calculated the pairwise sequence identities of each pair of homologous sequences within each of the ten Bf enzymes that were detected/considered in our metagenomic sequence analysis. Specifically, we conducted pairwise sequence comparisons of (1) metagenome versus complete genome homologs and (2) complete genome versus complete genome homologs. We quantified an E-value  as a proxy for protein identity distance, where higher values indicate that average pairwise identities among sequences are higher and that they are thus less diverse on average.With few exceptions, the amino acid sequence identities of earliest branching homologs for the ten Bf enzymes identified/considered in our metagenomic sequence dataset to the nearest characterized cultivar in our genome dataset were low . In particular, the significantly lower median E-values for Bcd, FdhA, HylA, Ldh, and MetF homologs in metagenomic sequences when compared to those from complete genome sequences indicates that a greater diversity of Bf enzymes exists in natural environments than currently exists in genomic databases from cultivars. Although the median E-values for the remaining Bf enzymes were not significantly different between the datasets, they were all comparatively lower in the metagenomes than in the complete genomes and many metagenomes comprised unique outlier homologs , suggesting the presence of novel protein sequences. Collectively, these results suggest that there is a far greater diversity of uncharacterized homologs of Bf enzymes in natural environments that may harbor unique functional properties that are distinct from those homologs that have been biochemically characterized.Calculation of E-values for homologs indicates that those identified in complete genomes have a relatively narrow distribution of amino acid identities when compared to the homologs identified in the metagenomes , and inferred origins of homologs among different taxonomic domains suggests that the ability to bifurcate electrons evolved after the divergence of Archaea and Bacteria from the LUCA and that these enzyme complexes evolved independently multiple times. This suggests an ecological advantage to integrate FBEB into the energy metabolism of functionally distinct and phylogenetically diverse taxa, specifically those that are anaerobic. Importantly, interspersed lineages of Bf and non-Bf homologs in phylogenetic reconstructions and strong evidence for co-evolution of protein subunits indicates that loss of flavoproteins and other subunits was common during the evolution of the oxidoreductase subunits.Interestingly, our conclusion that homologs of the 12 currently known Bf enzymes were not likely a property of the LUCA indicates that extant Bf enzyme complexes are not primordial and contrasts with conclusions from previous reports on the natural history of FBEB  and the Our analysis of the distribution and composition of Bf enzyme homologs in metagenomic sequences revealed enrichment of homologs in subsurface environments relative to surface environments. This is particularly true for deep subsurface rock fracture fluids and thermal spring/hydrothermal vent environments, where homologs of Bf enzymes were shown to be highly enriched. Moreover, phylogenetic reconstructions of the catalytic subunits of Bf enzyme complexes identified in metagenomic sequences revealed that the earliest evolving lineages were often  from deep subsurface rock fracture fluid and thermal spring/hydrothermal vent communities. This suggests that the characteristics of these environments, that at one time promoted the origin of these Bf complexes, may also help to explain the prevalence of Bf enzyme homologs in these environments today. The specific characteristics of these environments that may have promoted the origins of several of the Bf enzyme classes, and that promote the establishment of contemporary communities enriched in these protein complements, includes minimal redox gradients, highly reducing conditions, and low nutrient fluxes . Many of2 concentrations were low (2 to the cells or the sensitivity of the enzymes themselves to O2. In the case of Fix, it appears that aerobic diazotrophic cells specifically acquired and maintained this enzyme complex as an adaptation to generate Fd- for use in supporting nitrogenase activity (The observations made here suggest that FBEB is a widely distributed strategy in organisms inhabiting anoxic environments where the energy yield of redox reactions is generally low and where increased efficiency of energy capture would be highly selectable ,b. The pwere low  and enerwere low . Phylogeactivity , which eactivity .Significantly more work needs to be completed to identify the specific triggers that led to the emergence and extinction of Bf systems as biology diversified to occupy the breadth of niche space as it does today. Moreover, additional work needs to be conducted to identify the unique environmental characteristics that selected for the emergence of Bf systems. Such studies will shed new light on the evolutionary history of FBEB and provide new perspective on the physiological and ecological role of Bf enzymes in these cells and the natural environment. The insights and perspectives obtained from conductance of these evolutionary ecology studies, framed within a robust understanding of the physiology and biochemistry of these enzymes, will provide a plethora of exciting new questions to guide future thinking and research into the nature of microbial life on early Earth and the role of FBEB in the physiology and ecology of that life. Insights from such studies will ultimately be required to evaluate the merits of placing the process of FBEB as a property of the LUCA, as has been suggested elsewhere , and theSP and EB designed the study and wrote the paper. SP conducted informatics analyses. SP, ED, ML, MA, EF, DC, and EB downloaded the metagenomes, provided feedback, and contributed to the writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dear Editor,1. Although customized DNA-binding proteins such as zinc-finger proteins and transcription activator-like effectors (TALEs) have been used as tools for sequence specific DNA targeting and gene regulation, the complexity of the design limits their applications2. Recently, due to its much convenience and high efficiency, the CRISPR/Cas9 system has been widely applicated in both genome editing and programmable regulation of gene transcription in many organisms4. Besides of Cas9, the class 2 type V-A CRISPR-Cas12a offers additional capabilities with better performances including shorter CRISPR RNAs (crRNAs) and the RNase activity for pre-crRNA maturation, facilitating multiplex gene editing6. Moreover, the DNase-dead Cas12a (namely ddCas12a) can also function as a platform for programming diverse types of transcriptional regulation or epigenetic manipulation of the genome, without changing the genome sequence7. However, to accurately control the transcription of a target gene with the CRISPR systems, one may need to either test different targeting sites or choose an inducible CRISPR system8, bringing much inconvenience. To solve these problems, we here employed the CRISPR/ddCas12a system that was combined with crRNAs harboring mutated direct repeats (DR) to conveniently regulate gene transcription in a programmable and accurate way.The ability to regulate gene expression may facilitate the study of gene functions underlying complex biological processes and has great potential in both basic biological research and clinical applications9, indicating the importance of the crRNA DR sequences in Cas12a recognition and binding. To further demonstrate this relationship between the DR sequences and the ddCas12a-based transcriptional regulation, we first constructed a reporter system using the gfp gene as the reporter. We designed two crRNAs targeting the promoter region and four crRNAs targeting the coding region of gfp  and the reduction of the gfp fluorescence signal proved the effectiveness of the above crRNAs. However, crRNAs targeting to the promoter region and to the template DNA strand were much more effective in gene silencing   Fig. , the fluEscherichia coli and sorted the cells by the fluorescence signal intensity with a flow cytometer  showed a similar secondary structure of DR with that of the wild type  to analyze the binding affinities of ddCas12a against mutated crRNAs and target dsDNA, employing either labeled crRNAs Fig.  or label11. Furthermore, this accurate strategy in gene regulation can be combined with the RNase activity of ddCas12a, which allows the generation of multiple mature crRNAs from a crRNA array, facilitating quantitative regulation of multiple genes8.Taken together, we demonstrate here that mutations in the crRNA DR sequences could allow both controllable and quantitative regulation of gene transcription, which may become useful for metabolic engineering of cell factories. For example, the metabolic burden can be minimized by controllable regulation of the corresponding pathways to maximize the yield of target productsSupplementary Information


The lymphatic vasculature has traditionally been thought to play a passive role in the regulation of immune responses by transporting antigen presenting cells and soluble antigens to regional lymph nodes. However, more recent studies have shown that lymphatic endothelial cells regulate immune responses more directly by modulating entry of immune cells into lymphatic capillaries, presenting antigens on major histocompatibility complex proteins, and modulating antigen presenting cells. Secondary lymphedema is a disease that develops when the lymphatic system is injured during surgical treatment of cancers or is damaged by infections. We have used mouse models of lymphedema in order to understand the effects of chronic lymphatic injury on immune responses and have shown that lymphedema results in a mixed T helper cell and T regulatory cell (Treg) inflammatory response. Prolonged T helper 2 biased immune responses in lymphedema regulate the pathology of this disease by promoting tissue fibrosis, inhibiting formation of collateral lymphatics, decreasing lymphatic vessel pumping capacity, and increasing lymphatic leakiness. Treg infiltration following lymphatic injury results from proliferation of natural Tregs and suppresses innate and adaptive immune responses. These studies have broad clinical relevance since understanding how lymphatic injury in lymphedema can modulate immune responses may provide a template with which we can study more subtle forms of lymphatic injury that may occur in physiologic conditions such as aging, obesity, metabolic tumors, and in the tumor microenvironment. The lymphatic system is comprised of a series of blind ended, single cell thick initial lymphatic vessels that drain progressively into successively larger vessels and eventually return interstitial fluid back to the systemic circulation. Lymphatic flow is regulated by coordinated pumping of smooth muscle cells that partially envelop collecting lymphatics and compressive forces from surrounding skeletal muscles. One-way valves in collecting lymphatics ensure forward flow of interstitial fluid and prevent reflux when a segment of the collecting vessel located between two valves contracts .In addition to draining interstitial fluid, the lymphatics system is responsible for lipid and fatty acid absorption and is an important regulator of cholesterol metabolism . The lymGiven the important role of the lymphatic system in a wide range of physiologic processes, it is not surprising therefore that abnormalities in lymphatic function have been implicated in inflammatory disorders , 13, immLymphatic function is highly variable clinically and modulated by numerous factors including chronic inflammation, tumors, external stimuli such as radiation, age, obesity, and metabolic dysfunction. For example, reports published in the late 1990s showed that aging results in structural changes in the lymphatic system including loss of elasticity, reduced smooth muscle coverage, decreased number of mesenteric collecting vessels, and decreased mesenteric lymphatic flow , 23. MorVariability in lymphatic function resulting from aging, obesity, or metabolic syndrome may play a key role in immune responses to solid tumors and provide a rationale for the fact that these comorbid conditions increase the risk of tumor development and metastasis. Solid tumors such as melanoma and breast cancer are surrounded by abnormal, leaky lymphatics with impaired lymphatic transport function. Tumor, draining lymph node lymphangiogenesis and increased VEGF-C expression by inflammatory cells increase tumor growth and metastasis. Lymphatic vessel density and VEGF-C expression correlates with cytotoxic T cell infiltration and expression of immunosuppressive factors  in patients with melanoma indicating a possibility of LECs playing a dual role in promoting and hindering anti-tumor responses . These cLymph node dissection for cancer treatment is the most common cause of lymphedema development in Western Countries. Because lymphedema in this scenario develops secondary to surgical injury, this type of lymphedema is referred to as secondary lymphedema. Patients with secondary lymphedema develop progressive fibroadipose deposition in the affected limb and have an increased risk of developing infections and secondary malignancies. These pathologic changes cause significant morbidity and decrease quality of life . It is eHistorically, development of lymphedema has been thought to be secondary to impaired development of collateral lymphatics that bypass the zone of injury. Indeed, this concept led to the multiple preclinical studies reporting on the use of exogenous lymphangiogenic growth factors as a therapeutic treatment for lymphedema –40. HoweRecent studies from our lab and others have shown that lymphatic injury results in chronic inflammatory changes in the skin distal to the zone of injury and that this response, in turn regulates development of lymphedema by causing lymphatic leakiness, decreasing lymphatic pumping, increasing tissue fibrosis, and impairing development of collateral lymphatics. These inflammatory changes illustrate the important coordination of immune responses by the lymphatic system. The changes in inflammatory responses after significant lymphatic injury during surgery enable us to study the effects of more subtle forms of lymphatic injury as may occur in aging, obesity, metabolic dysfunction, or the tumor microenvironment. Thus, studying lymphedema is broadly relevant and may provide important insight into the role of the lymphatic system in regulating immune responses in other physiologic or pathologic events.in vivo and in vitro (Danger-associated molecular patterns (DAMPs) are the endogenous cellular products released by stressed, damaged or cells undergoing necrosis that alarm and activate the innate immune system components. By activating innate immune system, DAMPs create a pro-inflammatory state in the damaged tissues with an intention of host defense. However, in excess DAMPs can be harmful due to continuous activation of innate immune reactions , 46. Earin vitro , 49. In DAMPs initiate innate immune responses by interacting with pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). To understand the role of DAMPs interaction with PRRs in lymphedema development, we previously studied lymphedema development in different TLR knockout mice  using a mouse tail model of the disease . Consist+ cells and aggravates fibrosis  whose ligands [CCL21 (C-C motif ligand 21) and CCL19] are expressed by LECs. Gradients of CCL21 guide DCs to initial lymphatics  or mice lacking CD4+ cells (CD4KO) do not develop lymphedema after skin and lymphatic excision. Similarly, depletion of T cells using antibodies or topical treatment with tacrolimus, a medication that prevents T cell proliferation/differentiation prevents development of lymphedema in preclinical models and can be used to treat the disease once it has developed. In contrast, depletion of other inflammatory cell types such as cytotoxic T cells, B cells, and macrophages either has no significant effect or worsens the severity of lymphedema   . In thes stimuli . Identif stimuli .+ cells that co-express interferon gamma  and CD4+ cells that co-express interleukin 4 (IL4) or IL13 (putative Th2 cells). T cells in lymphedematous tissues tend to cluster around initial lymphatics and lymphatic collectors  and Th2 (CD45+/CD4+/CCR4+/CCR8+) cells. More importantly, we found that release of activated T cells from the lymph node via sphingosine 1 phosphate (S1P) signaling into the systemic circulation is necessary for the development of lymphedema. Treatment with an S1P inhibitor (FTY720) prevented release of activated T cells from the lymph node and prevented development of lymphedema in a mouse tail model of the disease.Lymphatic injury results in a mixed T cell inflammatory reaction consisting of T helper 1 (Th1), T helper 2 (Th2), and Tregs . Lymphedllectors , 70. Usi+ cell migration to the skin in other inflammatory conditions is regulated by cell surface expression of chemokine receptors including cutaneous leukocyte antigen (CLA), cc chemokine receptor 4 (CCR4), CCR8, and CCR10 . In contrast, we found no changes in the number of induced Tregs in the skin and no changes in any Treg population in the blood or the spleen suggesting that Treg activation and proliferation was localized to the forelimb skin rather than systemic changes. Depletion of Tregs using diphtheria toxin treatment in Fox-P3-diphtheria toxin receptor (FoxP3-DTR) transgenic mice significantly increased the number of Th1 and Th2 cells in the forelimb tissues. In addition, this treatment increased the number of infiltrating macrophages (CD11b+F4/80+), neutrophils (Ly-6G+), and activated DCs (CD11c+MHCII+CD86+). Consistent with the immunosuppressive effects of Tregs in general, we found that Treg depletion improved T cell and B cell mediated immune responses after sensitization of the forelimb skin distal to the zone of lymphatic injury. Moreover, we found that Treg depletion increased bacterial phagocytosis and removal as compared with control mice after injection of heat inactivated bacterial particles in the forelimb skin. Other recent studies have shown that depletion of Tregs in mouse models of lymphedema results in increased severity of lymphedema while adoptive transfer of Tregs ameliorates the phenotype (In addition to a mixed Th1/Th2 immune response, our lab and others have shown that lymphedema results in the accumulation of Tregs in lymphedematous tissues , 95, 96.The lymphatic system, acting via direct and indirect mechanisms, is an important regulator of immune responses . LymphatBM, RK, JB, HP, and IW did the literature search, complied, and wrote the manuscript. SR and JS proof read the manuscript. RK and JB prepared the figures and tables.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 socio-economic burden of allergic respiratory conditions on continental Europe is even higher than that of mainstream diseases, such as diabetes and cardiovascular disease, as allergic rhinitis alone accounts for billions of Euros in healthcare expenses across Europe. House dust mites (HDM) are one of the most common triggers behind allergic rhinitis and asthma. The role of probiotics in the treatment and prevention of some allergic conditions, such as atopic dermatitis, is already well recognized, whereas evidence about their efficacy in patients with respiratory allergies—while increasing—is still limited. Here the current evidence for the use of probiotics in patients with allergic rhinitis and/or asthma is discussed. Allergies and asthma are health conditions that have become highly prevalent across the globe, triggering respiratory symptoms—such as runny nose and sneezing, shortness of breath, and wheezing—in affected individuals. One of the most common triggers behind such allergic reactions are the allergens of house dust mites (HDMs). HDMs, which thrive in warm and humid household conditions, are classified as Arachnids and form a very diverse class in the Animalia kingdom. While mites are associated with a wide range of pathological and parasitic conditions in plants and animals, HDMs are rather unique as they can trigger a type I hypersensitivity reaction in predisposed persons, which can lead to severe allergic manifestations . The orgThe geographical distribution of different subspecies of HDMs is truly multi continental, as numerous research studies have indicated their prevalence across all continents except for Antarctica . In regaDermatophagoides pteronyssinus in 1967 [Dermatophagoides in samples of house dust during her research. At that time, however, the organism had no research significance regarding its role as an allergen in humans [HDMs belong to the taxonomic group called Astigmata and were established as a prominent source of indoor allergens with the discovery of the house mite species n humans .It can be estimated that approximately 1–2% of the general population worldwide has allergic sensitization towards HDMs with significant variability depending on the cohort being studied . In deveThis result demonstrates that substantial diversity in HDM allergic sensitization prevalence exists and depends on factors such as ethnicity, socio-economic conditions, geographical variations and even the diagnostic paradigm that is adopted. For this reason, a wide range of confounding factors need to be considered to understand the epidemiology of HDM-mediated allergies .A very common manifestation of HDM allergic sensitization is rhinitis, the symptoms of which include sneezing, and stuffy and running nose. These symptoms may not appear to be very serious and thus people often ignore them until the conditions become unbearable. However, a recent research article stated that the socio-economic burden of allergic rhinitis on continental Europe is even higher than that of mainstream health conditions, such as diabetes and cardiovascular disease . When scDue to the rising prevalence of different allergic conditions in recent decades, there has been a massive surge in research endeavours towards understanding their underlying pathogenic mechanisms and clinical implications . ResearcA study by Calderon et al.  describeLactobacillus bacteria affect Treg cells by generating semi-mature dendritic cells and increasing the expression of CD40; they are capable of inhibiting Interleukin (IL)-4 and IL-5 while inducing specific regulatory cytokines, such as transforming growth factor (TGF-β) and IL-10 [Considering the massive socio-economic burden of different allergic conditions on global healthcare resources, there is a considerable research impetus towards finding pioneering therapeutic and preventive interventions. In this regard, the role of the gut microbiota in shaping the immune response against conditions such as HDM allergy was anticipated in a previous section. Following up, there are new findings that suggest that the dynamics of gut colonization in infants play a critical role in shaping the immune system, and there is a possibility of enriching the gut microflora using probiotics that could contribute towards the prevention of allergic conditions in later life , 13. Thend IL-10 . Probiotnd IL-10 , and thend IL-10 .Lactobacillus rhamnosus was given for 2–4 weeks prenatally and 6 months postnatally; these results were confirmed in two subsequent follow-up studies in which a minor incidence of atopic disease was confirmed after 4 and after 7 years, respectively [One of the first observations about the role of probiotic supplementation for the prevention of atopic eczema dates to 2001, when Kalliomaki et al.  found a ectively , 19.A recent systematic review and meta-analysis of the literature that documented the use of probiotic agents for the prevention of allergic conditions in early life was carried out by Zuccotti et al. . The autIn regard to the role of probiotic supplementation in patients with allergic rhinitis, Vilà-Nadal et al. , recentlThe authors of this review conclude that probiotics appear to have beneficial therapeutic effects in allergic response as improvements in symptom scores and quality of life have been observed in these patients. In contrast, no positive impact was shown for the prevention of asthma and respiratory diseases .In another systematic review on the effect of probiotics in AR, Zajac et al.  considerIt has to be underlined that probiotics effects must be considered strain-specific; therefore, future studies are required to investigate which strain(s) can be considered the most suitable in order to achieve the best immunomodulatory effect in patients affected by allergic respiratory diseases .Scientific evidence assessing the role of probiotics in the treatment of HDM-related respiratory allergies, while limited, is increasing.Lactobacillus plantarum NCIMB8826 to reduce the allergic response in HDM allergic patients was evaluated in vivo and in vitro. In this study [L. plantarum and Der p 1 induced a Th1-shift in the immune response with upregulation of IgG and Th1-cytokine (IFN-γ and IL-12) production, moderate stimulation of IL-10, reduction of bronchoalveolar lavage eosinophilia after allergen challenge and suppression of allergen-specific IgE-response.The potential capacity of is study , it was In one study on 29 patients affected by allergic asthma and HDM sensitization, a 4-week treatment with synbiotics significantly reduced the systemic production of Th2-cytokines  after allergen challenge and improved peak expiratory flow, despite a lack of any observed effects on bronchial inflammation markers .Another recent and interesting approach consists of probiotic-impregnated bedding material. In the first pilot RCT in which 20 patients with AR and HDM sensitization used such materials for 8 weeks, no differences were found in HDM allergen levels compared to the placebo, whereas a significant improvement was reported by patients in terms of rhinitis-related symptom reduction and quality of life .Lactobacillus paracasei supplementation significantly improved rhinitis-related quality of life scores in a group of 90 children with HDM sensitization.Of interest, it has been shown that even heat-killed lactic acid bacteria can be of help in the treatment of patients with respiratory allergy and HDM sensitization. Peng et al.  showed tLactobacillus acidophilus NCFM, Bifidobacterium lactis BL-04 and fructo-oligosaccharide (FOS); Lactobacillus plantarum LP01, Lactobacillus paracasei LPC00 and FOS) in the management of seasonal allergic rhinitis (SAR) and perennial allergic rhinitis (PAR) [Lactobacillus plantarum and Lactobacillus paracasei in patients with PAR, whereas the symbiotic combination against SAR showed a statistically significant improvement at only the first timepoint. However, a concomitant decrease in the consumption of symptomatic medications, such as antihistamine, and corticosteroids drugs significantly and progressively decreased after 2 and 4 months of treatment.The therapeutic potential of probiotic strains against allergic conditions was also explored by an observational study published in 2014. This study focused on the efficacy of two different symbiotic formulations (is (PAR) . Aspectsis (PAR)  for AR sThe observed decrease in the utilization of co-administered oral antihistamine drugs and oral corticosteroid drugs during the study period with this symbiotic multistrain combination may represent additional evidence of the beneficial effect of probiotics in the management of allergic rhinitis .As it has already been mentioned before, identification of the most suitable probiotic strains in order to achieve a significant immunomodulatory effect in patients with HDM-related allergic conditions, will be one main challenge in this research field in the near future. Moreover, more research and effort are needed to improve the formulation of probiotic products  to assure higher efficacy.Allergen-specific immunotherapy (AIT) is a well-established treatment option for allergic rhinitis, including HDM-triggered AR . AIT triAdjuvants have been shown to amplify the effect of AIT by modulating the immune response to this therapy, thus increasing both its efficacy and safety .In recent times, it has been hypothesized that probiotics, which stimulate innate immunity via toll-like receptor activation, could be used as adjuvants in AIT.The effectiveness of probiotics as potential adjuvants in sublingual AIT (SLIT) has been studied in animal models; multiple lactic acid bacteria strains were found to act as Th1/possibly Treg inducers, and therefore—in the authors’ opinion—they could represent valid candidates for adjuvants in SLIT .E. coli Nissle 1917) prevented the development of allergic symptoms after an airway challenge with mite extracts; moreover, it was observed that it contributed to the upregulation of allergen-specific IgG2a production, down-regulation of specific IgE production and induction of a strong reduction of IL-5 secretion by allergen-restimulated splenocytes [In a murine model of mite allergy, immunotherapy with a recombinant Der p 1 allergen plus a probiotic strain  significantly enhanced SLIT efficacy .Lactobacillus rhamnosus GG) to SLIT improved symptom scores and the induction of T regulatory cells after 5 months of treatment relative to children treated with SLIT alone or placebo.In a study by Jerzynska et al.  on childLactobacillus acidophilus and Bifidobacterium lactis contributed to improved asthma control test (ACT) scores in children with allergic asthma undergoing AIT for HDM; the effect was additive relative to the sole effect of AIT [In one study, t of AIT .Clostridium butyricum coadministration with AIT has shown to improve the efficacy of AIT in patients with AR and asthma [d asthma , 39; thiLactobacillus rhamnosus, Bifidobacterium lactis and FOS) during AIT for AR may act synergistically to improve the clinical efficacy of sublingual allergen immunotherapy. After 2 and 4 months of treatment, patients who were administered AIT plus probiotics showed a trend towards the reduction of symptoms scores together with a significant improvement in medication scores, number of “well days”; and no relevant local or systemic adverse reactions [Recently, in an observational real-life study, Rossi et al.  showed teactions .The use of probiotics in the treatment of allergic diseases is very promising, thanks to increasing evidence of their immunomodulatory effects. Their role in the treatment and prevention of some allergic conditions, such as atopic dermatitis, is already well recognized, whereas evidence about their efficacy in patients with respiratory allergies—while increasing—is still limited.The prospective of employing probiotics as novel adjuvants for AIT is intriguing and already supported by a number of interesting studies.9 colony forming units generally considered) exists.Future research is needed to improve our understanding of the use of probiotics in the treatment of allergic conditions and as an adjuvant in immunotherapy; in particular, we need to better understand which probiotic strains are the most suitable for these purposes and which immunologic mechanisms are involved. Moreover, evidence is still lacking regarding which formulations will guarantee the best results, what are the best timing and duration for probiotic administration in order to prevent/modulate HDM-related allergic conditions, and if a critical dose (beside the 10


A radicular cyst (RC) in deciduous dentition is relatively rare. This clinical report presents a case of RC that condition derived from a primary molar undergone an endodontic treatment with gutta-percha approximately one year ago. In addition, we also considered whether intracanal medicaments and gutta-percha filling material related to the formation and development of the cyst or not. Key words:Primary tooth, radicular cyst, pulp therapy, gutta-percha filling material, intracanal medicament. The radicular cyst (RC) is known as a common type of odontogenic cyst arising from the proliferation of the Hertwig’s epithelial root sheath . The RC is stimulated by a condition of pulpal necrosis. Nevertheless, the radicular cyst in deciduous dentition is an extremely rare condition with just under 4% among 1300 cases which were diagnosed as those cysts in both primary and permanent teeth , displacement of the adjacent teeth and malocclusion. 23 in 28 cases on Lustmann’s research were observed and authors informed that: 1) the age of patients varied between 4 and 12 except one case at 19 years old, 2) the proportion of male to female was 1.6, 3) the lower molars were most common involved, 4) all cases caused by caries, 5) and all cases presented cystic lumen which was covered with non-keratinized stratified squamous epithelium in histological examination 6).In this case, we decided the final diagnosis as a RC related to a primary tooth because of the following pieces of evidences: 1) the existence of a painless swelling and a bony expansion associated with a primary molar had endodontic treatment, 2) the existence of a well-defined radiolucent lesion surrounding the roots of a primary molar but not involving in the successive premolar, 3) histopathological features of a cystic lumen lined with non-keratinized stratified squamous epithelium, and 4) no relation between the cystic sheath and the successive permanent tooth during surgery. Marsupialization following extraction of the associated primary tooth aims to eliminate the growth of the cyst and preserve the permanent tooth. After 6-month follow up, the eruption of the permanent successor could be clearly seen through the cystic opening.et al.  tend to promote the growth of the cysts , various endodontic sealers for primary teeth have been recommended, for instance, Zinc oxide-eugenol paste, Iodoform and Calcium hydroxide. However, no material has been known as an ideal root canal sealer yet. Gutta-percha has been introduced to dentistry since the mid-19th century and known as the most common root canal filling for permanent teeth because it was obtained the Grossman’s standards of an optimal root canal filling material, which should be bacteriostatic without irritating to the periapical tissue. In contrast, gutta-percha has been contraindicated to primary teeth, except when there is no successive tooth. Ansari G and Mirkarimi  followedTogether, several intracanal medicaments  used for pulp therapy were considered as antigenic stimuli to the development of the radicular cyst and still no evidence indicated the relation between gutta-percha material filling and these lesions. In our case, as the primary molar was treated in a private dental clinic, no available evidence was obtainable about the material dressed in root canals except clear evidence of gutta-percha filling in the radiographic image and after surgery.The radicular cysts occurring in deciduous dentition are relatively rare and it is extremely important to early diagnose and appropriately treatment aiming to conserve the successive permanent teeth. In addition, it is necessary to have routine clinical and radiographic check-up for primary teeth had pulp treated.


Estimation and comparison of results after incisional hernia repair (IHR) modo onlay or sublay with abdominoplasty in patients who lost the weight following Roux-en-Y Gastric Bypass (RYGB). Analysis and comparison of changes in quality of life (QL) of these patients prior to RYGB, before and after simultaneous IHR and abdominoplasty.Clinical analysis involved 40 patients with abdominal disfigurement (following RYGB and massive weight loss) after one-time IHR sublay method with abdominoplasty—group 1 or IHR onlay method with abdominoplasty—group 2. We evaluated postoperative results and long-term QL changes .We noted abnormal wound healing (2), pneumonia (3) and dysesthesia (3) in patients from group 1, and abnormal wound healing (2), seroma (2), pneumonia (2), and dysesthesia (4) in group 2. Quality of life was improved in the functional, esthetic and psychological aspects.One stage incisional hernia repair by onlay as well as sublay method with abdominoplasty are safe surgical methods improving the functioning of patients after major weight loss following RYGB. Sublay hernia repair and abdominoplasty was connected with longer time of the: operation, drainage, analgesic agents use, time to mobilization and to full oral diet than the onlay method. Significant improvement of the quality of life was noted after every subsequent step of surgical treatment in both groups. Reduction of the risk of BMI re-growth after bariatric surgery is related to the need for constant, specialized care for these patients at every stage of follow-up after bariatric surgery. Bariatric surgery is a commonly performed highly effective treatment in achieving long-term weight loss and resolution of obesity-related comorbidities. However, in the majority of these patients body contour irregularities and postoperative hernias (in 10–50%) form after open bariatric procedures  (± 3) days, similar to Berry’s report—9.0 ± 1.3 days . DuratioSome literature systematic reviews reported outcomes comprising different sections of improved QL as well as patient’s satisfaction and at the same time application of varied scales following body contouring surgery in patients after massive weight loss –27. In oWeight regain altogether was confirmed by 65% of patients (55% of patients from the onlay group and 75% from the sublay group) between the third and fifth year after RYGB, and resulted in their opinion from improper self-control, mainly eating habits, and periodically lack of physical activity, which corresponds with our earlier and other authors’ observations , 4. ThisPresented here two methods of IHR and abdominoplasty are varied in respect of polypropylene mesh insertion. Onlay IHR, as less technically demanding, is connected with shorter operative time and shorter recovery. Sublay mesh placement poses smaller risk of wound and mesh complications or hernia recurrence. Both simultaneous techniques of IHR and abdominoplasty provide an opportunity to improve QL for patients after major weight loss, following open bariatric operations. Qualification for surgery and the choice of method should be individual in any case, depending on the general patient’s status and size of abdominal wall distortion, as well as patient’s expectations. Based on our experience, we recommend onlay IHR in larger of incisional hernias and abdominal wall distortions as less burdensome method for the patients especially with concomitant systemic  disorders, which can be also exasperated with age. Sublay method is safe in younger individuals, with less extensive incisional hernia and smaller abdominal integument requiring removal.One stage incisional hernia repair by onlay as well as sublay method with abdominoplasty are safe surgical methods improving the functioning of patients after major weight loss following RYGB. Sublay hernia repair and abdominoplasty was connected with longer time of the: operation, drainage, analgesic agents use, time to mobilization and to full oral diet than the onlay method. Significant improvement of the quality of life was noted after every subsequent step of surgical treatment in both groups. Reduction of the risk of BMI re-growth after bariatric surgery is related to the need for constant, specialized care for these patients at every stage of follow-up after bariatric surgery.


Applying theories of lifelong psycho-social growth and life course principles of human development, older couples generally possess rich and valuable resources from diverse aspects of life. However, empirical evidence, on whether these supports are fully transferred to benefit themselves as they age, remains uncertain due to inevitable physiological decline and increasing caregiving strains. The present study explicated the underlying processes of the older Chinese couples in forming a dyadic resilience model as they age with chronic illnesses. Using in depth interviews, 15 pairs of older Chinese couples were interviewed twice through home visits. The conversations were transcribed and analyzed using thematic analysis. Eight themes related to resilience and challenges of spousal caregiving were identified: 1) insights and acceptance of the chronicity of illnesses, 2) a sense of purpose and commitment as being the spouse in providing care for each other, 3) mastering skills and resources to handle the duties, 4) mixed feelings when the spouse was functioning changes, 5) realizing the importance of self-care and life-management, 6) cultivating positive meanings amidst difficult circumstances, 7) accepting things that cannot change, and cherishing on those aspects which are still functioning well, 8) working closely with other formal and informal support for common good. Findings have both practice and policy implications to health and social care practitioners in understanding different patterns of resilience as experienced by older Chinese couples in mitigating the developmental challenges from individual, relational and societal levels, and research implications in examining actor-partner interdependence practice in addressing caregiving challenges.


Accumulating evidence has highlighted the accumulation of mast cells (MCs) in tumors. However, their impact on tumor development remained controversial. Indeed, cumulative data indicate an enigmatic role for MCs in cancer, whereby depending on the circumstances, which still need to be resolved, MCs function to promote or restrict tumor growth. By responding to multiple stimuli MCs release multiple inflammatory mediators, that contribute to the resolution of infection and resistance to envenomation, but also have the potency to promote or inhibit malignancy. Thus, MCs seem to possess the power to define tumor projections. Given this remarkable plasticity of MC responsiveness, there is an urgent need of understanding how MCs are activated in the tumor microenvironment (TME). We have recently reported on the direct activation of MCs upon contact with cancer cells by a mechanism involving an autocrine formation of adenosine and signaling by the A3 adenosine receptor. Here we summarized the evidence on the role of adenosine signaling in cancer, in MC mediated inflammation and in the MC-cancer crosstalk. Though best known for their involvement in allergic disorders, accumulating evidence highlights mast cell (MC) involvement in multiple inflammatory disorders, including cancer ,2. MCs iAdenosine is a nucleoside consisting of adenine (the purine base) in glycosidic linkage with the sugar ribose. Serving as a precursor and metabolite of adenine nucleotides, adenosine plays a central role in energy transfer and metabolism in living organisms. Adenosine is also generated and released from cells, specifically by either nucleoside transporters  or by miAdenosine functions are mediated by four G-protein coupled receptors (GPCRs), designated A1 adenosine receptor (A1R), A2a adenosine receptor (A2aR), A2b adenosine receptor (A2bR), and the A3 adenosine receptor (A3R) . The recAdenosine concentrations in the TME are significantly higher than in normal tissue alluding to the role of extracellular adenosine in controlling tumor progression ,44,45,46−/− MCs [Adenosine has long been incriminated in the pathogenesis of bronchoconstriction of MC-dependent allergic asthma. Inhaled adenosine provokes bronchoconstriction in atopic and asthmatic individuals, but not in normal subjects, implicating adenosine in the induction of airway hyperresponsiveness (AHR), a hallmark of asthma . Post-mo−/− MCs . However−/− MCs ,67. This−/− MCs .Analysis of the crosstalk between MCs and hepatocarcinoma (HCC) using transplantable H22 HCC tumors demonstrated that MCs promote infiltration of myeloid derived suppressor cells (MDSCs) and production of IL-17 by MDSCs . IL-17 tWe have also extended our studies to include two additional cancer types and found that similarly to the pancreatic cancer cell derived membranes, also membranes derived from two non-small cell lung carcinoma cell lines , as well as membranes derived from a leiomyosarcoma cell line  could activate the MCs and also in both cases, activation was sensitive to inhibition by APCP and MRS1220, demonstrating their dependence on autocrine formation adenosine and signaling by the A3R [Therefore, taken together, the results of others and us identify autocrine signaling of adenosine as a central factor in the crosstalk between MCs and tumor cells in the TME. By activating the MCs, autocrine/paracrine signaling of adenosine is likely to mediate the upregulation of angiogenesis and tissue remodeling genes , as wellThe finding that adenosine is involved in MC-cancer crosstalk, both directly and via complex interactions with other cells of the immune system may explain the controversary concerning the role of MCs in the TME. In view of the fact that adenosine signals through four distinct receptors that differ in their affinity to adenosine, as well as in their functions, it is anticipated that changes in the concentration of adenosine could alter the functional impact of adenosine. For example, while at low concentrations only the high affinity receptors would signal, an increase in adenosine concentration, brought about by the extracellular metabolism of ATP by the CD39/CD73 ecto enzymes, would allow the low affinity adenosine receptors to signal as well. In a similar manner, changes in the relative expression of the receptors may influence the biological outcome. In this regard, it is important to note that we have shown that adenosine itself affects the expression level of the A3R . Therefo


Currently, BrS is estimated to accountfor 12% of all sudden cardiac deaths and up to 20% of the sudden cardiac deaths inindividuals with no structural heart disease.5The usual clinical manifestation of BrS is arrhythmic syncope, nocturnal agonalrespiration or sudden death secondary to polymorphic ventricular tachycardia (VT) orventricular fibrillation (VF). The symptoms usually occur during sleep, at rest duringthe day or in situations with increased vagal tone, such as binge drinking or heavymeals. In addition, fever is a common trigger, mainly among children.5 Although threeECG patterns have been described, the diagnosis of BrS can only be established inpatients with type 1 ECG pattern (coved-type), characterized by a concave ST-segmentelevation ≥ 2 mm in at least two right precordial leads  positioned onthe 2nd, 3rd and 4th intercostal spaces, occurring spontaneously or after provocativedrug test with the intravenous administration of class I antiarrhythmic drugs . The other ECG patterns (types 2 and 3) do not establish thediagnosis of BrS.5The diagnosis and risk stratification of BrS are mainly based on clinical history and ECGpattern, which can generate controversy because of the incomplete penetrance of thechannelopathy and the dynamic pattern of ECG manifestations.8 Thus, in those patients, ICD placement should be avoidedbecause of the risk of complications, such as inappropriate shocks.5In BrS risk stratification and treatment, individuals with history of resuscitatedcardiac arrest (class I indication) and those with history of syncope and type 1 ECGpattern (class IIa indication) are at high risk for sudden death and have indication forimplantable cardioverter-defibrillator (ICD) placement for secondary prevention. Severalstudies have reported that asymptomatic patients with type 1 ECG pattern only afterinfusion of class I antiarrhythmic drugs are at low risk for arrhythmic events duringclinical follow-up.10 other studies have questioned thosefindings.7 It is worth noting the results of a recent systematicreview of eight observational prospective studies, including 1,312 patients with BrS andno history of cardiac arrest, in which the induction of polymorphic VT or VF during PVScould predict an increased risk for arrhythmic events (cardiac arrest or ICD shocks)during clinical follow-up, with an increased risk for events when VT/VF inductionoccurred with only one or two extra-stimuli. However, those authors have reported thatthe lack of VT/VF induction could not predict a lower risk of arrhythmic events,especially in the subgroup of patients with type 1 ECG pattern and history ofsyncope.8 Thus, the most recentexpert consensus recommend caution when indicating ICD placement in asymptomaticpatients with BrS when ventricular tachyarrhythmias were induced by PVS, emphasizing theneed to consider an individualized approach for ICD implantation (class IIb indication)in those patients.5The role of programmed ventricular stimulation (PVS) during invasive electrophysiologicalstudy for risk stratification and management of asymptomatic patients with BrS iscontroversial. While some studies have shown that the induction of ventriculartachyarrhythmias (polymorphic VT or VF) during PVS is an independent predictor ofarrhythmic events during clinical follow-up, emphasizing their negative predictivevalue,1 its genetic changes,arrhythmogenic mechanisms and clinical management continue to be debated. This isattributed to the continuous report of new information on BrS and its constantlyevolving understanding, driven by new clinical and basic research findings.10It is worth noting that, despite the description of BrS by the Brugada brothers more than25 years ago,Arquivos Brasileiros de Cardiologia issue, WarpechowskiNeto et al.11 report the clinicalcharacteristics, management and follow-up of patients with an ECG pattern suggestive ofBrS, who had been referred to a tertiary center for risk stratification by use ofinvasive electrophysiological study. In the study, aligned with those previouslyreported in the literature, most patients were of the male gender, adults and hadspontaneous type 1 ECG pattern. The study provides a timely and updated overview of thecomplexity of the BrS clinical management, discussing the controversies of using PVS forrisk stratification of asymptomatic patients, as well as ICD placement to treat those athigher risk for fatal arrhythmias in the long-term clinical follow-up.In the present


Guard cells are pairs of epidermal cells that control gas diffusion by regulating the opening and closure of stomatal pores. Guard cells, like other types of plant cells, are surrounded by a three-dimensional, extracellular network of polysaccharide-based wall polymers. In contrast to the walls of diffusely growing cells, guard cell walls have been hypothesized to be uniquely strong and elastic to meet the functional requirements of withstanding high turgor and allowing for reversible stomatal movements. Although the walls of guard cells were long underexplored as compared to extensive studies of stomatal development and guard cell signaling, recent research has provided new genetic, cytological, and physiological data demonstrating that guard cell walls function centrally in stomatal development and dynamics. In this review, we highlight and discuss the latest evidence for how wall polysaccharides are synthesized, deposited, reorganized, modified, and degraded in guard cells, and how these processes influence stomatal form and function. We also raise open questions and provide a perspective on experimental approaches that could be used in the future to shed light on the composition and architecture of guard cell walls. Physcomitrella patens and Funaria hygrometrica, guard mother cells undergo incomplete cytokinesis, which results in a single guard cell encasing a stomatal pore , and non-commelinoid monocots, possess a Type I cell wall, with xyloglucan being the predominant hemicellulose and pectins composing 20–35% dry weight of the wall; in contrast, Type II cell walls are typical in commelinoid monocots such as grasses, and contain xylans and mixed-linkage glucans as the major hemicelluloses and much less pectin than Type I cell walls   or pectate lyases (PLs), resulting in wall loosening , rhamnogalacturonan-I (RG-I), rhamnogalacturonan-II (RG-II), xylogalacturonan, and apiogalacturonan Mohnen, . HG is tWe have gained our knowledge of the primary wall predominantly from studies in tissue types that undergo irreversible expansion, such as roots  encodes a putative cell wall glycoprotein that is required for the formation of the stomatal outer cuticular ledge  are not highly expressed in stomatal lineage cells  mutant, cesa3je5, that is deficient in cellulose  and XXT2, xxt1 xxt2, which is deficient in xyloglucan   is downregulated in a loss-of-function mutant of SCAP1, a transcription factor that is essential for maintaining the proper shape of guard cells and controlling stomatal conductance  is expressed in Arabidopsis guard cells, and its overexpression accelerates light-induced stomatal opening by reducing the volumetric elastic modulus of the guard cell, which likely reflects the effect of EXPA1 on the mechanical properties of the cell wall , and have a 25% reduction in arabinan content in leaves . Exogenous treatment with arabinanase in epidermal strips prevents stomatal opening or closure in species such as POLYGALACTURONASE INVOLVED IN EXPANSION3 (PGX3) in Arabidopsis stomata. In cotyledons, GFP-tagged PGX3 is enriched at sites of stomatal pore initiation and PGX3 expression is associated with pore dimensions, suggesting that pectin degradation by PGX3 contributes to the controlled cell separation between sister guard cells during stomatal pore formation  leads to malfunctioning stomata, possibly due to the presence of smaller pectins and/or holes at one or both ends of stomata in transgenic leaves  has been used to visualize the pattern and movement of cellulose microfibrils on the nanoscale in onion epidermis   Is cell wall degradation an essential molecular mechanism underlying stomatal pore formation? (2) How is the enrichment of pectin-degrading enzymes and pectins at stomatal pore initiation sites determined and established during stomatal development? (3) What is the composition and architecture of the guard cell wall? (4) Is there any difference in wall composition between guard cells and neighboring cells? (5) Is there any difference in wall composition and/or architecture between kidney-shaped guard cells and dumbbell-shaped guard cells? (6) What are the turgor pressure values in guard cells at different developmental stages, at different opening/closed states, and in different plant species? (7) During leaf senescence, mesophyll cells undergo a reduction in chloroplast number and degradation of cortical MTs (Zeiger and Schwartz, Future studies on how the amazingly strong and elastic cell walls of stomatal guard cells are constructed and how their dynamics are spatiotemporally regulated will help elucidate structure-function relationships during reversible cell expansion in plants, knowledge of which can be translated into applications such as creating elastic biomimetic materials (Li and Wang, YR and CA generated Movie 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.