Viral load is still the marker of choice for monitoring adherence to combined antiretroviral therapy (cART) and confirming the success of HIV treatment. Unfortunately it is difficult to access in many resource-poor settings. We aimed to measure the performance of caregiver reporting adherence for detecting virological failure in routine practice during the first 2 years after cART initiation in infants.PEDIACAM is an ongoing prospective cohort study including HIV1-infected infants diagnosed before 7 months of age between November 2007 and October 2011 in Cameroon. Adherence was assessed using a questionnaire administered every 3 months from cART initiation; the HIV-RNA viral load was determined at the same visits. Virological failure was defined as having a viral load ≥ 1000 cp/mL at 3 and 12 months after cART initiation or having a viral load ≥ 400 cp/mL at 24 months after cART initiation. The performance of each current missed and cumulative missed dose defined according to adherence as reported by caregiver was assessed using the viral load as the gold standard.p = 0.005, LR + =4.4 and LR− = 0.4). Whatever the adherence reporting criterion, the negative predictive value was high (NPV ≥ 75 %) 12 and 24 months after cART initiation, whereas the positive predictive value was low (PPV ≤ 50 %).cART was initiated at a median age of 4 months (IQR: 3–6) in the 167 infants included. The cumulative missed dose showed the best overall performance for detecting virological failure after 12 months of cART (AUC test, The adherence questionnaire administered by the health care provider to the infants’ caregivers is not reliable for detecting virological failure in routine practice: its positive predictive value is low. However, the cumulative missed dose measurement may be a reliable predictor of virological success, particularly after 12 months of cART, given its high negative predictive value. Mortality and morbidity due to HIV/AIDS in both adults and children has been dramatically reduced by combined antiretroviral therapy (cART) –3. The C. But it is difficult to access for routine use in many resource-limited settings. There are other indicators some of which have been used in biomedical research: direct measures, including assaying plasma for drugs or drug metabolites; and indirect evaluations including self-reports, electronic drug monitoring, pill counts and pharmacy refill records [Satisfactory virological and clinical responses require adherence to cART; adherence also limits the emergence of antiretroviral drug resistance, particularly important in resource-limited settings with restricted cART regimen options –11. Vira records –18. Elec records .Pediatric cART is increasingly widely used in low-income countries and therefore there is a corresponding need for reliable methods, easier to access than viral load determinations, for assessing infant adherence to cART in routine practice. We are unaware of any previous study in Sub-Saharan Africa using indirect evaluations specifically to evaluate the adherence to cART among infants.The main objective of this study was to assess the performance of caregiver adherence reporting questionnaires for detecting virological failure in routine practice during the first 2 years of cART in infants in Cameroon.PEDIACAM is a prospective cohort study of HIV-infected infants included between November 2007 and October 2011 in three referral hospitals in Cameroon: the Centre Mère et Enfant de la Fondation Chantal Biya (CME/FCB) and Centre Hospitalier d’Essos (CHE) both in Yaounde and Hôpital Laquintinie de Douala (HLD) in Douala. The inclusions in PEDIACAM were organized in two phases and are described elsewhere . In brieOverall, 210 HIV-infected infants were included in the second phase: cART was proposed systematically to these children as soon as the HIV status was confirmed. The initial cART regimen depended on PMTCT (Prevention of Mother-to-Child Transmission) history and followed the Cameroonian guidelines at the time of the study which recommended zidovudine (or abacavir or stavudine if anemic), lamivudine and lopinavir/r if there was any use of nevirapine for PMTCT (or nevirapine otherwise). The caregivers were asked to administer the drugs to the infants twice daily (every 12 h). A standardized questionnaire was administered every 3 months after initiation of cART by a health care provider  to assess infant adherence to cART during the period and to identify and help families with any difficulties. In total, eight questionnaires were planned from month 3 (M3) to month 24 (M24) of follow-up. The HIV viral load was measured at the same follow-up time points using RT PCR .The adherence questionnaire was similar to the questionnaire used in the PENTA trial, with some adaptations . It inclAmong the 210 HIV-infected infants included in phase 2 of the PEDIACAM study, 13 died before treatment started and five refused cART. For the 192 infants who initiated cART, 25 died during the first three months of treatment. Thus, 167 HIV-infected infants treated for at least three months were included in this analysis  or higher than 400 copies/mL (at M24). We chose a higher threshold for M3 and M12 because a previous African study of HIV-infected infants under 12 months of age reported very high viral loads at baseline and that treatment duration contributed to lower viral loads at M24 . HIV resThe outcome measures also included non-adherence at the various time points recorded as: “current missed doses” (criterion A) defined as the number of cART doses not administered to an infant at a particular time point  and “cumulative missed doses” (criterion B) which was the sum of cART doses reported as not administered every three months during the corresponding interval M3 to M12 or M3 to M24. We also used mixed criteria . When a quarterly visit was lacking, all the cART doses for this visit were considered to be missing. When a quarterly visit was performed but the adherence questionnaire not completed, we considered the previous number of missed doses for that particular visit.Many different thresholds  for current missed doses have been used in previous studies in the literature to define non-adherent HIV-infected patients , 19–21. We studied associations between missed dose A or B criteria and various other items in the adherence questionnaire: problems with giving medication , type of caregiver and health care provider, the time point of the visit. Univariate analysis was first performed, using Chi square or Fisher’s exact test, as appropriate. Adjusted odds ratios (aOR) were estimated in multivariate logistic regression models, with reported missed dose as the dependent variable. For all analyses, p-values less than 0.05 (two-sided) were considered statistically significant. All the statistical analyses were performed using STATA 12.0 .The ANRS-PEDIACAM study was granted ethical approval in Cameroon by the National Ethics Committee and in France by the Biomedical Research Committee of the Pasteur Institute of Paris. The Cameroon Ministry of Public Health gave administrative authorization to start the study. Caregivers signed written informed consent for participating in the study.. Three quarters of the infants were enrolled in Yaounde. Two years after cART initiation, 92.2 % (154/167) of infants were alive, and 144 attended the M24 visit. Thirteen infants died, including nine before M12, and four between M12 and M24 , pharmacists (31.5 %), psychosocial workers (19.8 %) and nurses (13.9 %), and the respondent was mostly the mother . According to respondents, cART medication was usually administered by the mother  although other caregivers may also have been involved in cART administration .For current missed doses (criterion A), the cutoff point which provided the best compromise between sensitivity and specificity, according ROC curves, was ≥1 missed dose at all visits Table . For cump = 0.005 for VL ≥ 1000 cp/mL) (Table The area under the curve (AUC) was not significantly different to 0.5 for any missed dose criterion, except for cumulative missed doses at M12  3 and 12 months after cART initiation were respectively: 47.8 % (61/146) and 23.8 %. Twenty-four months after cART initiation, the proportion of virological failure (defined as >400 copies/mL) was 27.9 % (22/79) (Table The best sensitivity was observed with the cumulative missed doses criterion as reported at M12 63.3 %) and M24 (72.7 %). But its specificity decreased with time . For all criteria used, the PPV was low (≤50 %) and NPV was high (≥75 %) at both M12 and M24. Cumulative missed doses at M12 gave the best likelihood ratios (LR + =2.0 and LR− = 0.5)  and 20 children as virological successes at M24 (VL < 400 cp/mL) were classified as non-adherent according to cumulative missed dose criteria but adherent according to current missed dose criteria.Missed dose reporting was not significantly associated with health care provider, visit time point or caregiver.This study is the first to evaluate the value of a face-to-face adherence reporting questionnaire administered to caregivers for detecting virological failure 3, 12 and 24 months after initiation of cART for HIV-infected infants less than 6 months of age in an African country. As described elsewhere , 19–21, The measure based on current missed doses had only low sensitivity; cumulative missed doses reported at M12 performed better for detecting virological failure . This means that the probability of classifying a child as “non-adherent” is 2 times higher if he/she is in virological failure than success. Conversely, the probability of being classified as “adherent” is 2 times higher in cases of virological success than failure.For all criteria considered, the negative predictive values were mostly higher than 75 % but positive predictive values were mostly below 50 %. Few relevant studies report estimates of likelihood ratios and predictive values. Predictive values are more helpful than specificity and sensitivity for clinical decision-making in routine practice, but depend on the prevalence of virological failure in the population studied. Here, prevalence of virological failure was 47.8 % at M3, 23.8 % at M12 and 27.9 % at M24, which explains the high NPV. A cohort study in Uganda reported that 28.8 % (17/59) of children had viral loads ≥1000 copies/mL at M12, and this is consistent with our findings .Some children were classified as non-adherentaccording to cumulative missed dose, because of poor adherence at the beginning or part way through cART although they subsequently improved and obtained virological success at M12 or M24. This led to false positive cases, erroneous detection of virological failure and consequently decreased specificity, LR+ and PPV, particularly at M24. The single measures of viral load at M12 and M24 did not allow us to examine short-term effects of adherence fluctuations on virological outcomes ., 7 had genotypic evidence of resistance to antiretrovirals in their regimens. The findings from this study strongly suggest that in the majority of cases, patients whose caregivers report high adherence and have virological failure have resistance mutations. The caregiver desire to please health care providers, as reported in previous studies [Other children were classified as “adherent” although they showed virological failure at M12 or M24, which would be considered false negatives, reducing the sensitivity, NPV and LR+ of caregiver adherence reporting as a test for virological failure. Among the 11 infants presenting with these “divergent” unexpected results studies –25, accoOther factors which were not assessed by our study may be involved, such as concomitant disease, insufficient doses of ARV at home, drug interactions, individual differences in drug absorption and metabolism .The sensitivity of the current missed dose measure for detecting virological failure was low and did not significantly vary over time during the first 2 years of cART. Reporting a non-null number of missed doses is likely to indicate that some of the doses was taken only in part, rather an exact number completely not consumed . This tyOur study has some limitations including sample size to be analyzed at M12 and in particular at M24 being smaller than the number of children initially eligible for the study. This may have reduced the statistical power (sensitivity) for detection of virological failure. Viral load measurements were not made for some children because of death, loss to follow-up and caregiver decision to leave the health facility before blood could be drawn at the lab for plasma HIV-1 RNA tests. The ANRS 12140-PEDIACAM initially planned to follow HIV-infected infants until 2 years of age; however, many of the children who reached age 2 years (65 of 144) had not completed 2 years of cART and their viral loads were not subsequently measured. There was no association found between absence of viral load measurement at M12 or M24 and baseline characteristics.In conclusion, the adherence questionnaire administered by the health care provider to the infant’s caregiver is not a useful tool for detecting virological failure. As described for older children, its performance is poor, and its positive predictive value is too low for use in routine practice. Therefore, viral load measurement remains the best approach in routine practice to managing children under antiretrovirals and to avoid the emergence of resistance. We nevertheless report that the adherence questionnaire has a relatively high negative predictive value; adherence questionnaires and caregiver reporting may be useful in similar resource-limited settings, not to detect virological failure, but to identify virological success or resistance to antiretrovirals . Indeed, two or fewer missed doses reported in all trimestral questionnaires during the 12 months following the initiation of treatment is reassuring for the continuation of the treatment unchanged if the clinical and immunological status is satisfactory. Discordance suggests that it may be valuable to interview the family carefully, or propose home visits to identify any difficulties experienced with administration of medications, or to conduct genotyping tests.


Staphylococcus aureus, in both human and animal hosts, is largely influenced by the acquisition of mobile genetic elements (MGEs). Most S. aureus strains carry a variety of MGEs, including three genomic islands  that are diverse in virulence gene content but conserved within strain lineages. Although the mobilization of pathogenicity islands, phages and plasmids has been well studied, the mobilization of genomic islands is poorly understood. We previously demonstrated the mobilization of νSaβ by the adjacent temperate bacteriophage ϕSaBov from strain RF122. In this study, we demonstrate that ϕSaBov mediates the mobilization of νSaα and νSaγ, which are located remotely from ϕSaBov, mostly to recipient strains belonging to ST151. Phage DNA sequence analysis revealed that chromosomal DNA excision events from RF122 were highly specific to MGEs, suggesting sequence-specific DNA excision and packaging events rather than generalized transduction by a temperate phage. Disruption of the int gene in ϕSaBov did not affect phage DNA excision, packaging, and integration events. However, disruption of the terL gene completely abolished phage DNA packing events, suggesting that the primary function of temperate phage in the transfer of genomic islands is to allow for phage DNA packaging by TerL and that transducing phage particles are the actual vehicle for transfer. These results extend our understanding of the important role of bacteriophage in the horizontal transfer and evolution of genomic islands in S. aureus.The virulence of  Genetic variation of bacteria can be achieved through mutations, rearrangements and horizontal gene transfers and recombinations. Increasing genome sequence data have demonstrated that, besides the core genes encoding house-keeping functions such as essential metabolic activities, information processing, and bacterial structural and regulatory components, a vast number of accessory genes encoding antimicrobial resistance, toxins, and enzymes that contribute to adaptation and survival under certain environmental conditions are acquired by horizontal gene transfer of mobile genetic elements (MGEs) , 2. MobiStaphylococcus aureus is a major pathogen that colonizes the skin and mucous membranes of humans and animals, causing diseases ranging from mild skin infections to severe invasive diseases such as necrotizing pneumonia, infective endocarditis, and osteomyelitis [S. aureus [myelitis –8. The pmyelitis . Three t. aureus . Each ty. aureus –12. Geno. aureus , 14. Whi. aureus , 16, dir. aureus . The ind. aureus . Here weStaphylococcus aureus strains were cultured in tryptic soy broth (TSB) or agar (TSA) plates (Difco) supplemented with tetracycline  as necessary. Escherichia coli were grown in Luria-Bertani (LB) broth and agar plates supplemented with ampicillin  as necessary. Twenty nine bovine mastitis isolates and 22 human isolates were kindly provided by QIA  and Patrick Schlievert (University of Iowa), respectively.All strains and plasmids used in this study are listed in Cultures were incubated at 37°C with 200 rpm until reaching mid-exponential phase, and then mitomycin C  was added. Cultures were incubated at 30°C with 80 rpm until clear lysis was observed. The lysates were sterilized with syringe filers . A phage spot test and the plaque-forming unit (PFU) were determined by soft agar (0.5%) overlay method.7 CFU/mL. A phage solution containing approximately 108 PFU/mL was added to the culture, and incubated for 30 min at 30°C for the phage absorption, followed by addition of sodium citrate solution . After centrifuging at 4,000 rpm at 4°C for 15min, the pellet was suspended in sodium citrate solution and plated on TSA supplemented with tetracycline (5 μg/mL).For transduction experiments, the recipient strains were cultured to mid-log phase and adjusted to approximately 2×10E. coli K-12 was added to the culture lysates induced by mitomycin C as a control for verification of DNase treatment previously described [Phage DNA purification was performed as previously described . Brieflyescribed . Excessiset gene), νSaβ (the lukE gene), and νSaγ (the hla gene), SaPIbov1 (the tst gene), SaPI122 (the mdr gene), and ϕSaBov (the int gene). To estimate the frequency of transducing phage particles harboring MGEs, the absolute copy number of each MGE per nanogram of phage DNA was determined using quantitative real time PCR. Briefly, PCR products specific to MGEs (above) were cloned into pCR™4-TOPO® TA Vector (Life Technologies). Plasmids were quantified using a Nanodrop (Thermo Scientific) and the number of DNA copies/ng was calculated as described previously [2) using Microcal OriginPro . Quantification of MGEs was calculated by interpolation the CT from the standard curve.All primer pairs used in PCR, outward PCR, and quantitative real time PCR are listed in eviously . QuantittetM temperature-sensitive shuttle vector system [E. coli DH5α, and then to strain RF122. RF122 harboring the constructed plasmid were cultured at 43°C (non-permissive temperature for the replication of pMAD) to promote the first homologous recombination, followed by culturing 37°C to promote the second recombination, resulting in allelic exchange.All PCR primer pairs used in allelic exchange constructs were listed in r system , respectde novo. Recombined regions among the RF122 donor (GenBank NC_007622), CTH96 recipient, and transductants were identified through local alignments.Phage capsid DNA was isolated as described above, and bacterial genomic DNA was isolated with a DNeasy kit according to the manufacturer’s instructions (Qiagen). The dsDNA was quantified with a Qubit HS Assay Kit (Invitrogen). Indexed, paired-end libraries were prepared with a Nextera XT DNA Sample Preparation Kit (Illumina). Libraries were cleaned with 1.2× AMPure XP beads (Agencourt) and sequenced using a 300 cycle MiSeq Reagent Kit v2 on an Illumina MiSeq instrument (Illumina). CLC Genomics Workbench v6 software was used to trim and filter reads for quality and to assemble reads E. coli chromosomal DNA was added to the phage-induced lysates and treated with an excessive amount of DNase I. A PCR targeting the waaQ gene of E. coliwas performed to confirm the removal of chromosomal DNA in the phage DNA preparation (set gene), νSaβ (the lukE gene), and νSaγ (the hla gene), SaPIbov1 (the tst gene), SaPI122 (the mdr gene), and ϕSaBov (the int gene) (hlgB and rnaIII) were not detected  . The gendetected . The SaPolecules .10 copies/ng phage DNA), followed by νSaβ (5.25 Log10 copies/ng phage DNA). The copy numbers of νSaα, νSaγ, SaPIbov1, and SaPI122 were lower than the ϕSaBov and νSaβ, and ranged between 1.64–3.00 Log10 copies/ng phage DNA . The copy number of the ϕSaBov was the highest  were assembled which clustered in MGEs including νSaα, νSaβ, SaPIbov1, SaPI122, and ϕSaBov, except for a single short contig at SAB1107-8 not known to be located on MGEs . The ϕSaS. aureus, the tetM gene, conferring tetracycline resistance, was introduced into the MGEs as depicted in tetM gene was confirmed by PCR (tetM gene into the non-MGE gene (hlgB) to test random excision and packaging of phage DNA as in true generalized transduction, but transduction was not observed even with the recipients belonging to the ST151 lineage (data not shown).Results of PCR and phage DNA sequencing demonstrated the presence of phage particles harboring MGEs in the RF122 strain. To test the transfer of MGEs by these transducing phage particles to other d by PCR . The trad by PCR . Other ttetM gene found in the CTH96 νSaα transductant, approximately 27,162 bp of νSaα was transferred from donor to recipient, ranging from SAB0378 to SAB0403 and including a part of the set gene locus, hsdM, hsdS, and the lip gene locus (pheT), excinuclease ABC subunit C (uvrC), recombination and DNA strand exchange inhibitor protein (mutS2), ribonuclease III (rnc), succinate dehydrogenase (sdh), extracellular fibrinogen binding protein (efb), and the hla gene  and transductants (CTH96 νSaα transductant and CTH96 νSaγ transductant) was determined. Based on single nucleotide polymorphisms (SNPs) and the ne locus . The seqhla gene .int) and terminase large subunit (terL) encoded by ϕSaBov in the transfer of these genomic islands, the int or terL gene was deleted in the RF122 strain. PCR analysis of phage DNA extracted from these strains showed that disruption of the terL gene completely abrogated phage DNA packaging, which could be restored by complementation of terL  recognizes and binds to the phage-specific packaging (pac) site typically located in or near to the terL gene that initiates a hetero-oligomer complex with TerL resulting in headful phage DNA packaging [pac sites in the MGE by TerS that induce the headful packaging mechanism in concert with TerL and other factors in the ϕSaBov.In ackaging , 20, 21.pac-type phage DNA excision event is highly specific to the direct repeat sequence recognized by the integrase, excisionase, and terminase small unit [A transfer of MGEs by temperate phages requires a series of successful sequential events including excision from the host genome, packaging into transducing phage particles, transfer to a recipient and integration into the recipient genome. To investigate phage DNA excision events, phage DNA analysis using PCR and sequencing was performed. These results revealed that phage DNA excision events by ϕSaBov are highly specific to chromosomal locations of MGEs, suggesting a mechanism of sequence-specific excision events. It has been shown elsewhere that a typical all unit , 21. ForAs determined by PCR and DNA sequence analysis of phage DNA, transduction events are specific to MGEs rather than the random events that are expected from generalized transduction. A recent study demonstrated that the TerS encoded by SaPIs induced sequence-specific excision and packaging of phage DNA at unlinked chromosomal segments . In our stl and str [A higher frequency of MGE transfer by ϕSaBov was observed with recipient strains belonging to ST151 than with other strain lineages. These results suggest that the host specificity of phage might be a primary barrier in the transfer of MGE by phage. The host specificity of phage is expected to be determined by interactions between the tail proteins of phage and the target molecules in recipient cells such as proteins and wall teichoic acid (WTA) which were generally conserved in the core genome of the same or related lineages of strain –25. As s and str , 28, migtetM gene among donors, recipients, and transductants. Based on these comparisons, it was hypothesized that linear phage DNA segments harboring parts of νSaα and νSaγ were transferred, rather than a transfer of a single contig harboring entire νSaα or νSaγ. This result supports findings that the overall structure of genomic islands can vary as a result of “plug and play” type of recombinational replacements [hla gene was largely thought to have evolved with the core chromosome of S. aureus, recombination events are evident in some strains [hla gene could be horizontally transferred among S. aureus strains.To confirm the transfer of MGEs by ϕSaBov, a draft genome sequence of the transductants for νSaα and νSaγ was determined. The extent of the transfer of MGEs was estimated by comparing single nucleotide polymorphisms and the presence of the acements . To our  strains , 31. Ourint gene encoded by the ϕSaBov is required for integration of phage DNA harboring ϕSaBov and νSaβ into recipient genome, but not for phage DNA excision events of ϕSaBov and νSaβ [terL encoded in the ϕSaBov is required for packaging phage DNA of ϕSaBov and νSaβ [int gene encoded by ϕSaBov is not required for phage DNA excision events in νSaα and νSaγ nor for the integration into recipient genome. However, the terL is essential for packaging into transducing phage particles. These results suggest the major role of temperate phage in transfer of νSaα and νSaγ is to package DNA and shuttle it via transducing phage particles.Our previous study demonstrated that the and νSaβ . Furtherand νSaβ . SimilarS. aureus.Although it has long been acknowledged that bacteriophages contribute the horizontal transfer of MGEs by generalized and specialized transduction, the causal link to the transfer of genomic islands by bacteriophages or other means has not been established. In this study, for the first time, we present experimental evidence demonstrating transfer of genomic islands by a temperate phage utilizing MGE-specific DNA excision events from the host genome and presumably recipient-derived factors for integration. These results represent the complexity of underlying mechanism of phage-bacterial interaction in horizontal gene transfer and extend our understanding of the important role of bacteriophage in the horizontal transfer and evolution of genomic islands in


The demonstrated infrared plasmonic metamaterial sensing platform offers great potential for improving the specificity and sensitivity of label-free, biochemical detection.Infrared vibrational spectroscopy is an effective technique which enables the direct probe of molecular fingerprints, and such detection can be further enhanced by the emerging engineered plasmonic metamaterials. Here we experimentally demonstrate ultrasensitive detection and characterization of polymer molecules based on an asymmetric infrared plasmonic metamaterial, and quantitatively analyze the molecule detection sensitivity and molecule-structure interactions. A sharp, non-radiative Fano resonance supported by the plasmonic metamaterial exhibits strongly enhanced near-field, and the resonance frequency is tailored to match the vibrational fingerprint of the target molecule. By utilizing the near-field nature of the plasmonic excitation, significantly enhanced absorption signal of molecules in the infrared spectroscopy are obtained, enabling ultrasensitive detection of only minute quantities of organic molecules. The enhancement of molecular absorption up to 10 Large spectral and spatial overlap between the strongly confined near-field of the plasmonic mode and molecular vibrational absorption dramatically boosts transduction of molecular structural properties into detectable infrared signals, enabling the detection of minute amount of molecules on the plasmonic platforms.Here we experimentally demonstrate the ultrasensitive detection and quantitative characterization of poly(methyl methacrylate) (PMMA) molecules utilizing specially designed asymmetric Babinet-inverted Fano-resonant plasmonic metamaterials (FRPMs). As a complementary structure of nanoantennas normally used for biodetection17383940y axis, in which the right one is end-connected to a perpendicular shorter slot antenna. By breaking the spatial inversion symmetries of the unit cell in the structure plane, Fano interference is enabled for the x-polarized incident light for the Babinet-inverted FRPM42x- and y-polarized incidence, as shown in x-polarized reflection spectrum (red solid), the broad symmetric Lorentzian resonance at 83 THz represents the slot dipole resonance (bright mode ωD), and the narrow asymmetric resonance around 53 THz corresponds to the Fano interferences between the slot dipole and two-slot quadrupole resonance (dark mode). The resonant frequency (ωQ) of the latter mode can be determined from y-polarized reflection spectrum (blue solid) where the dark mode is excited alone without the interference with the bright mode38x- and y-polarized excitations are shown in the top panel of quadrupole excitation at ωQ exhibits significant field concentration near the end and the gap of the slots with typical Fano interference field patternquadrupole mode are reflected as a narrowing of the far-field spectral response. No interference effects are observed, however, for the slot dipole resonance at ωD. On the other hand, the electric field distributions for both modes  are carried out to obtain the reflection spectra, electromagnetic field distributions and field enhancement spectra of the FRPMs. Here the permittivity of bulk gold in the mid-infrared is described by the Drude model  possess large intrinsic dipole moment, and uniform PMMA layers can be formed with accurately controlled thicknesses by varying the molecule concentrations in the anisole solvent. A series of FRPMs with uniformly scaled dimensions are designed and fabricated to have tunable Fano resonance frequencies sweeping across the stretching absorption band of PMMA. A good match of the plasmonic resonance to the molecular absorption featureR = Rfunc − Rbare) in 10nPMMA ≈ 1.5) aside from any molecule-FRPM interaction effect has been taken into account  (PMMA) molecules dissolved in anisole  is deposited by spin coating on the fabricated metamaterials. The carbonyl bonds in PMMA molecules  are shown in h is smaller than the near-field decay length of the plasmonic mode (detailed in h as h ≤ 40 nm) with almost identical slopes. By comparing the measured reflection spectra of the respective samples, we note that a distinct molecular absorption signature is observed around 52 THz when the frequency of Fano resonance is tailored to match the frequency of the carbonyl bond of PMMA molecules , corresponding to the strongest interaction between PMMA molecules and FRPMs, is observed for sample B which combines the strengths of narrow line-width of the Fano resonance (DR ≈ 0.12 (0.14) for sample A (C) at c = 2%) is detected due to the weak molecule-FRPM interaction at the off-resonance condition. As the solid content of molecules is progressively diminished from 2% to 0.25%, the signal strength observed for sample A and C decreases to less than 0.02 and approaches the noise level of our measurement. Contrastingly, signal strength larger than 0.04 is observed for sample B. Especially, when the solid content of PMMA molecules further decreases from 0.2% down to 0.125%, the detectable signal strength is only observable from sample B  in the case of 0.4% solid content is obtained from about 77400 molecules or 130 zeptomoles for the entire array (50 × 50 μm2). Absorption signals from further diluted PMMA solution (molecule amount down to tens of zeptomoles) have also been observed as shown in 3 or less molecules within each unit cell), but the signal strength subsequently appears to saturate at larger number of molecules when the film thickness is more than 40 nm. The observed saturation behavior of the measured signal strengths can be attributed to the evanescent plasmonic near-fields decaying away from the FRPMs surface, which was also observed in nanorod arrays loaded with varying protein film thicknessz axis to a value of 1/e2 of the maximum within about 30 nm, which validates the near-field nature of enhancement mechanism of the FRPM-based molecular detection. The demonstrated detection sensitivity (zeptomole level) is quite impressive as compared to previous works, and it is also important to note that the estimation of detectable amount of molecules is conservative because only the portion of molecules that experience the near-field interaction with the FRPM exhibit the enhanced infrared absorption and contribute to the measured enhanced signals. According to the near-field intensity distribution of the Fano resonance in The controllable PMMA film thickness ranging from hundreds of nanometers to a dozen nanometers provides an opportunity to investigate the ultimate detection limit of molecule numbers using the FRPMs. According to the X-ray reflectivity measurements, the thickness of a diluted PMMA layer with 0.4% solid content is 12.8 nm , correspDR ≈ 0.22 from sample B with solid content c = 1%) is compared to the signal strength from the same amount of molecules on a reference bare gold film (DR ≈ 0.007). Then an enhancement by the mirror-dipole effect (a factor of 2) from the gold filmns)39E|2) distribution of the Fano resonance is plotted in x and y directions with spatial extents of 60 nm (lx) and 90 nm (ly) respectively. The active area can be estimated by (lx + ly)·hAu, where hAu is the thickness of Au film. Taking into account all the above mentioned factors, the enhancement factor of the infrared absorption of molecules that interact with the FRPM is estimated to be DR(FRPM)/DR(Au)·2·(1 + ns)·A0/(lx + ly)·hAu ≈ 163,000, where A0 is the size of unit cell. Therefore, the demonstrated FRPM provides an enhancement factor of the molecular absorption signal larger than 105, which is remarkable compared to the reported SEIRA results17Furthermore, in order to provide more insights into the sensing mechanism, the absorption enhancement factor of the FRPMs is estimated from the experimental and numerical results. At first, the measured signal strength from PMMA molecules on the FRPM structures  mode is σdm (σqm). The motion equations of three oscillators can be written as:To examine the enhanced molecule-FRPM interaction strengths in the presence of the xd, xq and xm of the oscillators are harmonic with xd, q, m = cd, q, m·eiwt where the amplitudes cd, q, m can be calculated analytically. We obtain the reflection spectrum by summing the square of three oscillation amplitudes and subtract them from unity, which represents light scattering efficiency of the system. The reflection spectra of sample B obtained from the coupled-oscillator modeling are shown in γq = 7 THz, γm = 0.7 THz) compared to that of the dipole resonance (γd = 28 THz), which capture our observations in the experiment well. The extracted coupling coefficients from the measured three samples . The asymmetric Fano resonant plasmonic metamaterials (FRPMs) are fabricated via focused ion beams, carried out in a FEI Helios Nanolab 600 DualBeam microscope system with the focused beam of gallium ions of the current of 9.7 pA and the energy of 30 eV. Each sample has a 50 × 50 εdilute = f·εPMMA + (1−f)·εanisole in numerical simulations, where εPMMA and εanisole are permittivity of readily obtained PMMA (950-A2) and anisole and f is the filling ratio of the PMMA. The thicknesses of diluted polymers with different concentrations are determined through X-ray reflectivity (Philips X'Pert-MRD) measurement and the respective thicknesses of different concentrations are shown in Different concentrations of poly(methyl methacrylate) (PMMA) molecules, a commonly used positive electron-beam resist, are used as model analyte in our experiment. For the largest concentration, a thin layer of PMMA  is spin-coated on top of the metamaterials at 2000 rpm. PMMA is chosen in the present work due to the accurate control of the uniform thickness obtainable via control of the spin speed and molecule concentration used. Then molecule concentration is diluted progressively in anisole and the diluted polymer solution is spun onto nanostructures. The dielectric function of the polymer can be modeled as −1 and 512 scans. The measurements are normalized with respect to a silver coated mirror (THORLABS). An IR polarizer  is used to polarize the incident electromagnetic field perpendicular or parallel to the slot antennas. The reflectance difference spectra displayed in R = Rfunc − Rbare, where Rfunc and Rbare are the reflectance spectra of PMMA-coated FRPMs and that of frequency-shifted bare FRPMs, respectively. The frequency shift is performed via a transformation Rbare = R0 (ω0) → R0 (ω0 + ∆ω) to coincide the maximum of two spectra as illustrated in R0 (ω0) is the original reflectance of a bare structure and ∆ω is the frequency shift due to the presence of PMMA layers with refractive index nPMMA ≈ 1.5. The absorption signal strength is defined as difference between maximum and minimum of the ∆R spectra around the absorption line of carbonyl bond stretching.The reflection spectra of the FRPMs are recorded using a Fourier transform infrared (FTIR) spectrometer . Reflected signals are collected with a 0.4 NA Compensation objective and recorded by a liquid-nitrogen-cooled mercury cadmiumtelluride (MCT) detector. All the spectra are recorded with a resolution of 4 cmx and y axes to account for the periodic arrangement of the unit cells. Perfectly matched layers (PMLs) surrounded by scattering boundary condition faces are utilized along the propagation direction  to avoid multiple reflections due to geometry truncation. The permittivity of bulk gold in the mid-infrared is described by the Drude model ε∞ = 1, the plasma frequency ωp = 1.37 × 1016 rad/s and the damping constant γp = 4.08 × 1013 rad/s. Due to the surface scattering and grain boundary effects in thin films, the damping constant of gold film in our simulation is taken to be three times that of bulk gold in order to match the experimental results. Meanwhile, to take the molecular absorption effect into account, here the PMMA layer is modeled as a Lorentz oscillator material described by εb = 2.2 is the background relative permittivity of PMMA, fm is the reduced oscillator strength, ω0 = 3.269 × 1014 rad/s is the Lorentz resonance frequency of PMMA molecules and γ0 is the Lorentz damping rate. In our simulation, the values of f and γ0 are appropriately chosen to provide a close match with the experimental measurements. The experimentally observed reflection spectra with the molecule vibrational absorption signals can be numerically reproduced as shown in Finite element method (FEM) simulations are performed to obtain the optical reflection spectra and field distributions using the software (COMSOL Multiphysics). In the simulations, periodic boundary conditions are employed along the How to cite this article: Cheng, F. et al. Ultrasensitive detection and characterization of molecules with infrared plasmonic metamaterials. Sci. Rep.5, 14327; doi: 10.1038/srep14327 (2015).


In order to explore marine natural resources using remote robotic sensor or to enable rapid information exchange between ROV (remotely operated vehicles), AUV (autonomous underwater vehicle), divers, and ships, ultrasonic underwater communication systems are used. However, if the communication system is applied to rich living creature marine environment such as shallow sea, it suffers from generated Impulsive Noise so-called Shrimp Noise, which is randomly generated in time domain and seriously degrades communication performance in underwater acoustic network. With the purpose of supporting high performance underwater communication, a robust digital communication method for Impulsive Noise environments is necessary. In this paper, we propose OFDM ultrasonic communication system with diversity receiver. The main feature of the receiver is a newly proposed Frequency Domain Diversity Combined Impulsive Noise Canceller. The OFDM receiver utilizes 20–28 KHz ultrasonic channel and subcarrier spacing of 46.875 Hz (MODE3) and 93.750 Hz (MODE2) OFDM modulations. In addition, the paper shows Impulsive Noise distribution data measured at a fishing port in Okinawa and at a barge in Shizuoka prefectures and then proposed diversity OFDM transceivers architecture and experimental results are described. By the proposed Impulsive Noise Canceller, frame bit error rate has been decreased by 20–30%. Underwater wireless communication system  will offIn this paper, we have designed underwater acoustic Orthogonal Frequency Division Multiplexing (OFDM) communication system with four receiving transducers diversity –4. The pWe have performed ocean experiments at two sites. One is OJIMA located at the south of Okinawa Island in Okinawa Prefecture in Japan. The other is barge in UCHIURA-cove in Numazu city in Shizuoka, which is roughly 400 m offshore and the sea depth is 30 m. Background noise distribution data at OJIMA site is also shown in k) is the output of each MRC block:k)1–4 are 4 equalizer outputs, H(k)1–4 are Channel Transfer Functions (CTFs) of each underwater channel, index k corresponds to subcarrier number, and σn1−42 are estimated average noise power at each underwater channel. Highlight of the system design is frequency domain Impulsive Noise Canceller with two-stage MRC combiners, although the reference paper [Following the equalizer, there are two stages of 4 inputs Maximum Ratio Combiner (MRC) blocks. Equation  shows thce paper  shows a T is used.r(n) is nth time sample of received signal after preamplifier, Pavg is the average power of an OFDM symbol, and α is threshold parameter. Obviously, if the power of input signal r(n) exceeds the threshold of  α · Pavg, the signal is replaced by zero. This method is called “blanking” [α, cut and try experimentation was applied and finally α = 20 is determined. The system performance is not so sensitive to the parameter.Because of the presence of Impulsive Noise so-called Shrimp Noise, time and frequency domain Impulsive Noise Cancelling (time-IMP and freq.-IMP) methods , 5 are elanking” , 11. In Subcarriers, which should be silent (no data or pilots), are set to zero.Subcarriers, which are used as pilots, are replaced by known pilot values.Subcarriers, which are used for data transmission, are demapped to nearest position in constellation plot.In addition, a frequency domain Impulsive Noise Cancellation is applied. The detail of freq.-IMP  and is IFFTed. If the output of IFFT has a large peak, it can be considered as estimated Time Domain Impulsive Noise signal u(n) according to nth time sample of IFFT output, σ2 is the average power of the IFFT output, and β is threshold parameter. Obviously, if the power of input signal β · σ2, the signal is considered as an Impulsive Noise. Then the estimated Time Domain Impulsive Noise signal u(n) is converted to frequency domain by FFT block and division by Hi. Finally, the estimated frequency domain Impulsive Noise is withdrawn from equalized signal of branch i (i = 1 to 4) EQAi. Then the noise reduced outputs EQBi (i = 1 to 4) are obtained and then they are combined in the 2nd MRC block to generate further noise reduced constellation. The parameter β is determined by cut and try experimentations and β = 8 is finally applied to the system. The obtained noise cancel performance is not so sensitive to the parameter β.Then succeeding subtraction block computes frequency domain noise components. In the following blocks, the frequency domain noise components are converted to a Time Domain Impulsive Noise. The frequency domain noise is multiplied with Channel Transfer Function of branch Figures R = 1/3 is applied to this system, all error free communications (BER = 0) for QPSK modulation of all speeds and 16QAM stable case have been confirmed.Finally, Figures In this paper, an ultrasonic OFDM transceiver architecture with four diverse receivers supporting 2 MODEs is proposed with enhancement of Impulsive Noise insusceptibility. It utilizes 20–28 KHz ultrasonic channel and subcarrier spacing of 46.875 Hz (MODE3) and 93.750 Hz (MODE2) OFDM modulations.Two different environment ocean experiments were conducted to evaluate the statistics of Impulsive Noise so-called Shrimp Noise, which is generated by living creatures in marine environment. To deal with challenges posed by the Impulsive Noise interference, we have employed a Frequency Domain Diversity Combined Impulsive Noise Canceller.Measured data at a fishing port in Okinawa and at a barge in Shizuoka Prefecture are disclosed and in both environment randomly generated Time Domain Impulse Noise distribution is observed. Although more than 90 to 99% of background noise shows AWGN type distribution, less than 1 to 10% noise components show large amplitude such as Impulsive Noise.R = 1/3 forward error correction, all error free communications using QPSK and 16QAM with not moving case are also confirmed.By enabling the proposed MRC combined Frequency domain Impulsive Noise Canceller, 20–30% BER reduction has been successfully obtained. In addition, 71.5 m shallow fishing port horizontal underwater communication BER is also shown using QPSK and 16QAM modulations. By applying TURBO


Proxy records of temperature from the Atlantic clearly show that the Younger Dryas was an abrupt climate change event during the last deglaciation, but records of hydroclimate are underutilized in defining the event. Here we combine a new hydroclimate record from Palawan, Philippines, in the tropical Pacific, with previously published records to highlight a difference between hydroclimate and temperature responses to the Younger Dryas. Although the onset and termination are synchronous across the records, tropical hydroclimate changes are more gradual (>100 years) than the abrupt (10–100 years) temperature changes in the northern Atlantic Ocean. The abrupt recovery of Greenland temperatures likely reflects changes in regional sea ice extent. Proxy data and transient climate model simulations support the hypothesis that freshwater forced a reduction in the Atlantic meridional overturning circulation, thereby causing the Younger Dryas. However, changes in ocean overturning may not produce the same effects globally as in Greenland. The Younger Dryas cooling event has been identified in ice records in the Northern Hemisphere, but the effects of this cold snap on the tropics are poorly known. Here, the authors present a speleothem record and model simulations, showing that tropical hydroclimate recovered slower than temperatures in Greenland. CE, hereafter456The Younger Dryas (YD) was an abrupt climate change event that affected climate across much of the Earth approximately 12.5 kyr ago  in the layer counting of young ice (∼12 kyr ago)9Greenland ice core records, particularly δIn the present paper, a new hydroclimate record from the deep tropics is combined with previously published hydroclimate and temperature records to better understand how a climate change event such as the YD evolves. Our conclusions hold important implications for the occurrence and prediction of future climate events.The YD event is captured in select stalagmite records, including those from China151718192021222324252627293031323318Osw marine sediment records  from the South China Sea293020161519252627The hydroclimate response to the YD in the Western tropical Pacific and adjacent regions was spatially and seasonally variable : the nor18O record of hydroclimate variability from the Puerto Princesa Subterranean River National Park in Palawan, Philippines (10.2° N and 118.9° E), to better understand the precipitation response of the Western tropical Pacific during the YD. The rainy season today in Palawan (May–November) contributes ∼80% of the total annual rainfall of ∼2,000 mm, and the seasonal variation in average temperature is small (27–29 °C). The limited seasonal variation in temperature and the high humidity of tropical cave environments suggest that changes in rainfall δ18O values dominate the stalagmite δ18O signal in Palawan18O signal most likely records changes in wet season rainfall amount via the ‘amount effect,' which is an empirical relationship between rainfall amount and rainfall δ18O values in the tropics4018O composition: a change of ∼1,000 mm of rainfall per year results in a 1‰ change in rainfall  δ18O values on suborbital timescales (decades to centuries) when ocean basin water transfer is minimalHere we present a stalagmite δ18O record consists of 12 U–Th dates with an average precision of ±78 years , occurs at 12.92±0.08 kyr ago  or abrupt (duration of 10–100 years to reach and recover from full YD conditions). These timings are based on the adaptation time of human and natural systems to drastic changes in climate5118O values to rainfall amount.We compare the proxy records with the deglacial climate transition simulated by two climate models of different complexities  and western Pacific  records . The main passage of the cave system is an 8.2-km long underground river that empties into the South China Sea. The stalagmite was collected in a chamber (40 × 20 m) located ∼250 m into a side passage. The entrance to the side passage begins ∼200 m from the cave entrance to the sea.18O analyses were performed using a Thermo MAT253 isotope ratio mass spectrometer or a Thermo Delta V Plus isotope ratio mass spectrometer with a Kiel IV Carbonate Device, both at the Analytical Laboratory for Paleoclimate Studies at the Jackson School of Geosciences, University of Texas at Austin . All stalagmite stable isotope values are reported relative to Vienna Pee Dee Belemnite (VPDB) in standard delta notation.Stable isotopic samples were drilled at 1 μm per subsample for the upper 2 cm and 0.5 μm for the lower 16.5 cm of the stalagmite with a computer-assisted micro-milling drilling system . A totalσ). A Monte Carlo approach simulated age model construction  based on a spline fitting using the 2σ error of the U–Th dates, allowing for quantification of the temporal error for each δ18O measurement  and the DGNS experiment with the earth system model of intermediate complexity (LOVECLIM)We compared the proxy records with the deglacial climate transition simulated by two climate models of different complexity: the TraCE-21ka experiment with the full physics climate model developed at NCAR (CCSM3)We compared the tropical Indo-Pacific rainfall simulated in the CCSM3 TraCE-21ka experiment with instrumental data and the Palawan record. The climatological seasonal cycle before the YD in CCSM3 agreed well with instrumental data . However230Th age-depth models and δ18O data from the Palawan stalagmite are archived at the NOAA World Data Center for Paleoclimatology at https://www.ncdc.noaa.gov/data-access/paleoclimatology-data/datasets.The How to cite this article: Partin, J. W. et al. Gradual onset and recovery of the Younger Dryas abrupt climate event in the tropics. Nat. Commun. 6:8061 doi: 10.1038/ncomms9061 (2015).Supplementary Figures 1-8, Supplementary Tables 1-2 and Supplementary References


The evolved ‘idealized’ network was then analyzed to gain insight into: (1) optimal network connectivity among different kinds of neurons—excitatory as well as inhibitory pacemakers, non-pacemakers and tonic neurons—within the preBötC, and (2) possible functional roles of inhibitory neurons within the preBötC in rhythm generation. Obtained results indicate that (1) synaptic distribution within excitatory subnetwork of the evolved model network illustrates skewed/heavy-tailed degree distribution, and (2) inhibitory subnetwork influences excitatory subnetwork primarily through non-tonic pacemaker inhibitory neurons. Further, since small-world (SW) network is generally associated with network synchronization phenomena and is suggested as a possible network structure within the preBötC, we compared the performance of SW network with that of the evolved model network. Results show that evolved network is better than SW network at exhibiting synchronous bursts.We hypothesize that the network topology within the pre-Bötzinger Complex (preBötC), the mammalian respiratory rhythm generating kernel, is not random, but is optimized in the course of ontogeny/phylogeny so that the network produces respiratory rhythm efficiently and robustly. In the present study, we attempted to identify topology of synaptic connections among constituent neurons of the preBötC based on this hypothesis. To do this, we first developed an effective evolutionary algorithm for optimizing network topology of a neuronal network to exhibit a ‘desired characteristic’. Using this evolutionary algorithm, we iteratively evolved an  In thigLeak, gNaP) for which a neuron possesses pacemaking property i.e. for low enough value of gtonic neurons in this region remain silent, and as the gtonic value is progressively increased, they exhibit ‘bursting’ behavior for some range of gtonic before transiting to ‘tonic’ behavior at higher values of gtonic  of CC is subdivided into. To quantify the closeness of the network with respect to Objective-2, the following measure was usedβ is a scaling factor, and IBIobjective = 7s. Thus, for evolving the excitatory network towards dual objectives EN1 and EN2, Total Cost = (Cost1 + Cost2) was minimized. We empirically set scaling factors α = 50 and β = 1.0. The value of all sub-intervals Δxj while computing Cost1 using The degree of synchronization of a network is quantitatively obtained by computing the cumulative curves (CC) as illustrated in gtonic for both excitatory and inhibitory neurons, in comparison to 0.10nS in gtonic (SynFrac = 0.06), with excitatory and inhibitory SynStrength values set to 0.6nS and 0.36nS, respectively. Using the simulated annealing algorithm, as described in section 2.3, we evolved a randomly initialized network towards the following three objectives:We constructed a neuronal network, comprising of 160 neurons—80 excitatory and 80 inhibitory neurons. The neuronal properties of both 80 excitatory and 80 inhibitory neurons were allocated as depicted in n gtonic . The netObjective-CN1: Evolved network produces better synchronized population bursts. Criteria used: A network in which higher fraction of excitatory neurons participate in each population burst is a better synchronized network.Objective-CN2: Identical with objective-EN2 stated in section 2.5.Objective-CN3: Sporadic bursts of excitatory neurons is minimum. This is perhaps effected by inhibitory neurons by providing IPSP to excitatory neurons.The motivation behind choice of objectives is discussed in section 4.1. Note that the contribution of inhibitory neurons to network synchronization (i.e. to Objective-CN1) is assumed to be indirect; they may potentially influence the activity of excitatory neurons by providing IPSPs, which may help match the onset of bursting of excitatory neurons and, thus, to exhibit synchronized population burst.The costs associated with first two objectives CN1 andCN2) are quantified using Eqs . For evolving both ‘excitatory network’ and ‘composite network’, during each iteration the newly mutated networks were simulated for 180,000ms  with a time step size of 0.05ms using forth order Runge-Kutta method in C++. To discount the initial transients, first 40s of real neuronal time were discarded. Data saved for the time range 40–180s (with a bin-width of 160ms) were used to evaluate performance of networks against the set objectives described in previous two sections. Note that the chosen bin-width of 160ms is larger than the largest inter-spike interval belonging to the same neuronal burst and smaller than typical interburst interval; thus it is adequate to identify the beginning and end-timings of individual neuronal bursts. The data thus saved is adequate for computations of Cost1, Cost2 and Cost3 besides keeping the saved data to manageable size.In real time, simulation duration for evolving ‘excitatory network’ amounts to ≈3.6min per iteration. Since ‘composite network’ consists of twice as many neurons as in ‘excitatory network’, simulation duration for its evolution is ≈7.2min per iteration.MM1 and CCobjective reflects a limit to the maximum optimization that may be achieved by ‘constrained’ mutation method MM1; ‘constrained’ in the sense that MM1 attempts to optimize network’s performance by keeping SynFrac value fixed. This is corroborated by further evolution of network using MM3  network is one of the most common network studied in context of network synchronization phenomena , and is First we constructed a SW network with 10% of all-to-all connectivity (moderately sparse) using Watts and Strogatz model . SpecifiSynStrength for the comparison shown in SynStrength at which the network evolution in SynStrength = 0.48nS, SW network fail to exhibit synchronized population burst; none of the 21 initialized SW networks exhibit synchronized population burst as opposed to 5–8 of 21 randomly initialized network.The performance of 19 different SW networks against those of 19 different randomly initialized network  is depicted in MM4 is farther away from CCobjective, as compared to CCMM3, it may be concluded that even the best evolved SW network provides suboptimal synchronization of neuronal network. Additional result supporting this conclusion is provided in Following this, the random hill climbing algorithm using MM4 as mutation method was used to evolve a SW network with the objective of improving the network synchronicity. Note that MM4 preserves the initial SW network topology intact; performance of neuronal network improves as a consequence of neurons swapping positions within the network. SynFrac value in the evolved network is ≈0.12, which is greater than SynFrac = 0.06 with which the network was randomly initialized. SynFrac = 0.06, while the ‘Evolved’ network has a SynFrac = 0.12. Thus, to compare its performance, a random network with SynFrac = 0.12 was constructed and simulated; its performance is depicted in rightmost column of SynFrac) when measured against the objectives-CN1, CN2 and CN3 stated above. Elimination of IPSPs in the evolved network 1) decreased IBI of network from 7.1s to 6.64s, 2) increased sporadic bursting of excitatory neurons, and 3) activity of inhibitory neurons got better synchronized with each population burst . These neurons tend to exhibit tonic behavior even when synaptically connected, within the neuronal networks.readily to EPSPs they receive from other neurons to exhibit bursting.‘Core region’ primarily comprises non-tonic pacemakers neurons i.e. pacemaker neurons which do not exhibit tonic behavior in synaptically isolated state. Within neuronal network, they respond ‘Activated region’ neurons are intrinsically silent non-pacemaking neurons which become active in the network as a consequence of EPSPs they receive from other neurons. The level of EPSPs required to make these neurons active is greater than that necessary for exciting ‘core region’ neurons.The intrinsic excitability of neurons is highest in the ‘tonic region’, intermediate for ‘core region’ and lowest in the ‘activated region’. However, since the excitability of neurons within these regions varies gradually, the region boundaries are not strict, but slightly overlapping. Note that both excitatory and inhibitory group of neurons of composite network may be subclassified into ‘tonic region’, ‘core region’ and ‘activated region’ neurons. Thus, composite network neurons may be categorized into ‘excitatory tonic region’ neuron, ‘inhibitory core region’ neuron, and so forth.Referring to SynStrength values are set to 0.6nS and 0.36nS, respectively. To check whether results are robust against variation in SynStrength values, additional simulations were carried out by varying excitatory and inhibitory SynStrength values in the range 0.2–0.6ns (incrementing in steps of 0.2nS) and 0.12–0.48nS (incrementing in steps of 0.12nS), respectively—9 cases in all. The reduction in Synstrength values lead to increase in total synapse count within the evolved network (see section 4.5) and vice versa; however, synaptic connectivity pattern within these additional evolved composite networks were found to be consistent with the pattern summarized in All 12 evolved composite networks for which the results are summarised in Objective-CN1: Objective-CN1 for evolving random networks used in the present study is an idealization towards improved synchronicity. Such behavior is typically not experimentally observed in preBötC slices. However, it serves as an idealization of the functional purpose of excitatory network within the preBötC. According to current understanding [standing , 24, 25,Objective-CN2: Initially, attempts were made to evolve composite networks towards Objectives-CN1 and CN3 alone. However, during the evolution process, the evolving networks illustrated a tendency to minimize Cost3, given by Objective-CN3: The relationship between intrinsically bursting and rest of the neurons within the ‘excitatory network’ may be analogous to that of ‘leader-follower relation’ found in social communities, with the former set of neurons acting as leaders and the later set of neurons as followers. Objective-1 was devised with the idea that for robust rhythm generation, it is desirable that this leader-follower relation between intrinsically bursting and rest of the neurons in the excitatory network is strong. Extending the sociological analogy further, it was presumed that inhibitory neurons act analogous to (constructive) social critiques—they criticise (inhibit) those activity of individual neurons which tend to disrupt the desirable harmony of ‘leader-follower relation’ between intrinsically bursting and rest of the neurons in the excitatory network. It is this idea that motivated us to formulate Objective-3.Scaling factors for Costs: The scaling factors—50 for Cost1 [non-trivial costs—Cost1 (measuring network synchronicity) and Cost3 (measuring sporadic bursting of excitatory neurons); Cost2  is considered trivial cost here. This may be noted from reduction history of costs depicted in or Cost1 , 1.0 foror Cost1  and 100 or Cost1 —were fixObjectives EN1 and EN2: Objectives EN1 and EN2 used for evolving random excitatory networks are equivalent forms of objectives CN1 and CN2; simplified to a form so that they are applicable to excitatory networks. Since excitatory networks do not comprise any inhibitory neurons, no equivalent of Objective CN3 is used for evolving excitatory networks.Evolved excitatory network: The common feature of the evolved network is to have ‘collector’ and ‘spreader’ neurons within network. Interestingly, neurons rich with out-going synapses are mostly pacemaker neurons in the core region while less readily excitable non-pacemaker cells in the activated region are rich in in-coming synapses. That is, the evolved network is rich with synapses having pacemaker and non-pacemaker neurons at pre- and post-synaptic ends, respectively.The skewed/heavy tailed degree distribution of incoming synapses in the evolved network may be related to inhomogeneous cellular properties. Neurons in the ‘activated region’ require increasingly high degree of EPSPs provided by incoming synapses to make them exhibit synchronized bursting with rest of the network neurons, which results in the elongated tail towards the right in model neurons act as nodes, which have varied intrinsic properties depending on the gLeak and gNaP values that are randomly allocated to them. Consequently, network burst synchronization depends on both network topology and intrinsic property of neurons at specific nodal positions. In a random network, a few intrinsically bursting neuron may by chance end up occupying nodal positions in network with above average out-going synapses. Such neurons end up making greater contribution in synchronizing the network by providing EPSPs to greater number of network neurons. However, a SW network is constrained by its characteristic that all nodes necessarily have  identical number of neighbours; equivalently, equal number of outgoing synapses. This constraint limits the extent to which individual neurons may contribute towards network synchronization. It is probably this difference that renders SW network less effective at synchronizing the network when compared to the random network in Our results suggest that random networks show better performance than SW networks in terms of synchronization . In convEvolved composite network: As in case of ‘evolved excitatory network’, it was common to find several ‘collector’ and ‘spreader’ neurons, of both excitatory and inhibitory types, within evolved composite networks. The chief features of evolved composite network is concisely summarized in Similar to ‘evolved excitatory network’, evolved composite network also possesses rich projections from excitatory neurons in the core region to excitatory non-pacemaker neurons in the activated region. Excitatory neurons in the core region also project synapses to inhibitory neurons belonging to both core and activated regions. Thus, in essence, excitatory neurons in the core region may be thought of as a subpopulation of evolved composite network dictating the network’s rhythmic synchronous bursts through its phasic excitatory drive.Inhibitory ‘core region’ neurons provide synaptic projection to excitatory ‘tonic region’ and ‘core region’ neurons. With the objectives employed for evolving the network, this may be rationalized as follows: (1) inhibitory synaptic inputs provided to excitatory tonic neurons help to minimize sporadic bursts of excitatory network, and (2) inhibitory synaptic inputs provided to excitatory ‘core region’ neurons most probably assists the excitatory network to exhibit network bursts with (desired) inter-burst interval of 7s; in the absence of this inhibitory inputs, the inter-burst interval of network is lower (see section 3.2). It is interesting to note that inhibitory neurons have little influence on excitatory ‘activated region’ neurons, primarily because unlike ‘tonic region’ and ‘core region’ neurons they do not participate in sporadic bursting.As is discussed in section 4.1, the three objectives used for network evolution are (hypothesized) ‘idealizations’ of functional purpose of excitatory and inhibitory networks within the preBötC. Consequently, the connectivity characteristics of evolved composite network may be thought to reflect characteristics of an ‘idealization’ of preBötC network, referred to as ‘idealized network’ henceforth.in situ imaging technique, they have shown that the network within the preBötC has a modular structure consisting of clusters having transverse  structure. They found that the mean number of cells within a cluster are 6.7±0.6 with each cell having 4.4±0.5 connections to other cells. For cells outside the clusters, the number of connections was considerably sparse, 2.1±0.4. Further, Carroll and Ramirez [SynFrac ≈ 0.10–0.12 that we employed for evolving the excitatory and composite networks is probably a reasonable one. Recently Wang et al. [SynFrac ≈ 0.13 generates respiratory-like cycle periods. However, they used Rubin-Hayes preBötC neuron model [Rekling et al. , based o Ramirez , based og et al. , throughreal network whose topology is bound to the neuroanatomical structure [Gaiteri and Rubin  comparedtructure .no better than random networks as far as synchronization of neuronal network is concerned. The evolved excitatory network, which exhibits highest degree of network synchronization, possesses skewed/heavy-tailed degree distribution with rich synaptic projections extending from pacemaker neurons to non-pacemaker neurons. Further, our work shows that evolved ‘composite networks’ also have ‘spreader’ neurons in both excitatory and inhibitory sub-populations, whose cellular properties are in the ‘core region’.Gaiteri and Rubin  tested sCarroll and Ramirez  investig2+-activated non-selective cationic current (ICAN), motivated and supported by experimental studies [2+ accumulation by the activation of ionotropic and metabotropic glutamate receptors leads to the activation of ICAN, resulting in shaping the driving potential of the burst phase of neuronal and network activity. More recently, models of rhythmogenesis in preBötC combining mechanisms associated with INaP and ICAN [In the present study, we used a mathematical model described by Purvis et al.  . Consequently, the best evolved network achievable through evolutionary process is a function of these initially fixed parameters. The product (SynStrength × SynFrac), is one of the most important parameter that is required to be set correctly during network initialization so as to ensure its proper evolution. This is relatively straight forward to comprehend for the case of evolution of ‘excitatory network’ towards complete synchronicity (section 3.1): when the initialized value of product (SynStrength × SynFrac) is low, the network fails to evolve completely to exhibit full synchronization; a fraction of neurons belonging to the ‘activated region’ remain silent and fail to participate in population burst. On the other hand, when the initialized value of product (SynStrength × SynFrac) is high, the network does evolve to exhibit full synchronization but possess several redundant synapses in it. Both cases hinder identification of essential features of network connectivity.The fact that the random hill climbing algorithm, using MM1 or MM2 as mutation method, is intrinsically ‘constrained’ is already explained in section 2.3. While network initialization, SynFrac to vary, we let the value of product SynStrength × SynFrac evolve along with the evolving network. This extra degree of freedom introduced in the simulated annealing algorithm, allows the network to evolve to full synchronization even when initialized value of product SynStrength × SynFrac is low or high. Moreover, since the simulated annealing algorithm preferentially selects networks with lower synapse count in every iteration of evolution, the redundant synapse count in the evolved network is minimal. These features of the simulated annealing algorithm are advantageous when it is used to evolve ‘composite networks’.However, in the simulated annealing algorithm used for the present study, by allowing and random addition of equal number of synapses—must simultaneously combine in a ‘positive way’. The frequency of occurrence of such a favorable mutation is substantial when the network is composed of only excitatory neurons. However, when the network is composed of both excitatory and inhibitory neurons, the frequency of occurrence of such a favorable mutation decreases considerably. To a lesser degree, the increased size of composite network reduces the probability of finding favorable mutations as well. In contrast to this, success of simulated annealing algorithm in producing a favorable network mutation depends only on one operation—addition (or deletion) of synapses—and hence it produces favorable network mutations with greater frequency.Another appealing aspect of simulated annealing algorithm is that it is more prolific in producing favorable network mutations when compared with random hill climbing. Random hill climbing algorithm utilizes mutation methods which preserve the total synapse count. Consequently, for a favorable network mutation to occur two operations—the random deletion of synapses It is basically for the reasons specified in preceding two paragraphs that we directly employed simulated annealing algorithm for evolving the composite network. Note however that MM2, which is a total synapse preserving mutation method, is ‘active’ in parallel see  all the not on the topology of the network but on the behavior of the network, i.e., the synchronicity of the network activity  on network behavior on account of their differing intrinsic characteristic is essential here). Such addition and elimination of nodes and connections—simulated annealing—may be viewed as partially analogous to addition and elimination of neurons and synapses associated with the development of brain structures during ontogeny [gLeak/gNaP values) of constituting neurons as well, which is not incorporated in the present work.Scale-free network, which has extreme skewed/heavy-tailed degree distribution, emerges when nodes and connections are added with a higher probability to connect with a highly connected cells  or when ontogeny . SimulatIn summary, we conclude the following:An effective evolutionary algorithm is developed to evolve the synaptic connectivity within a neuronal network towards desired objective function. The developed algorithm is sufficiently generic—it can handle neuronal networks comprising both excitatory and inhibitory neurons.skewed/heavy-tailed degree distribution among network neurons. Network is rich with synapses projecting from tonic/core region neurons to non-pacemaking neurons in the activated region.superior network synchronization when compared to a small-world network with identical number of synapses.Evolved excitatory network, which is an ‘idealization’ of excitatory subnetwork of preBötC, possesses the following features:excitatory subnetwork retains the overall salient features of evolved excitatory network mentioned above.excitatory ‘core region’ neurons dictate the network’s rhythmic synchronous bursts through its phasic excitatory drive.inhibitory subnetwork influences excitatory subnetwork primarily through ‘core region’ neurons; their inhibitory synaptic inputs (1) minimize sporadic bursting of excitatory tonic neurons, and (2) assists excitatory ‘core region’ neurons to exhibit network bursts with desirable inter-burst interval.Evolved composite network, which is an ‘idealization’ of preBötC network, possesses following features:Four different network mutation methods were devised.Mutation Method-1 (MM1): Network mutation is exercised by deleting a set of synapses followed by adding an equivalent number of synapses to the network. To determine synapses to be deleted, two neurons, say n1 and n2, are randomly chosen from the neuronal group and then, their respective neighborhoods N1 and N2 were defined. To define neighborhoods N1 and N2, we define an Euclidean metricgLeak/gNaP-parametric space depicted in ni and nj. Consequently, neighborhood N1 is composed of all neurons ni ≡  corresponding to δ(N1) is the radius of neighborhood N1 with neuron n1 ≡  at its center; δ(N1) is randomly specified during each mutation process. Neighborhood N2 is similarly computed. A random fraction of synapses directed from N1 to N2 is deleted during each mutation step. An equal number of synapses directed from neighborhood N3 to neighborhood N4, is subsequently added to the network; where N3 and N4 are neighborhoods of two additional randomly selecting neurons n3 and n4 from the neuronal network.gLeak and gNaP, and thus occupying same local neighborhood on gLeak/gNaP-parametric space depicted in gLeak/gNaP-parametric space also serve similar purpose within the neuronal network, and thus may be added or deleted together. This method of mutating the network ensures that all the added/deleted synapses effect favorable or unfavorable change to the network unanimously (more or less) and thus is more effective than simply randomly adding or deleting a fraction of network’s total synapse.MM1 is devised on the assumption that neurons with similar values of N1, N2, N3 and N4 are of the same kind .Additional remark: while employing MM1 to mutate neuronal network composed of both excitatory and inhibitory network (composite network), caution is exercised such that all neurons within neighbourhoods Mutation Method-2 (MM2): This method is comprised of two distinct mutations depicted in n1 and n2 are chosen at random and a fraction of incoming (or outgoing) synapses of n1 are rewired to n2. These changes are effected by appropriately changing the elements of connectivity matrix . Note that here it is possible that excitatory synapses from E-E part of the adjacency matrix may get reallocated to E-I part .Mutation Method-3 (MM3): This mutation method has following two subtypes:MM3(SynAdd) This adds new synapses to the network. Method: A neuron in the network is randomly chosen and a small fraction of (maximum possible) incoming or outgoing synapses are added to it. MM3(SynDel) This deletes existing synapses from the network. Method: A neuron in the network is randomly chosen and a small fraction of all incoming or outgoing synapses to it are deleted. Both subtypes may be implemented in conjunction (i.e. some new networks may be obtained by employing MM3(SynAdd) and others by using MM3(SynDel)) or in isolation. To prevent sudden large change in network topology in one mutation step, maximum number of synapses that may be added by MM3(SynAdd) or deleted by MM3(SynDel), during mutation is restricted to 5 synapses.While employing MM3 to mutate neuronal network composed of both excitatory and inhibitory network, caution is exercised that all synapses added or deleted from the network are of same kind .Mutation Method-4 (MM4):homeomorphic to each other.Except for MM3, all other three mutation methods described above, preserve the total number of synaptic connections within the neuronal group. All three methods simply keep rearranging the synaptic connections within neuronal network so as to help it exhibit desired population behavior.It is remarked that all four mutation methods (MM1-4) are custom-made algorithm, devised for the sole purpose of serving the application at hand . However, the key ideas employed in these mutation methods, particularly MM2-4, are similar to those found in ‘swap mutation’ and ‘scramble mutation’ of Genetic Algorithm .The procedure for computing ‘total sporadic burst count’ and ‘total sporadic burst count possible’ required for determination of Cost3 using Since, pre-inspiratory (pre-I) bursting of a subset of network neurons is an essential precursor to help the entire network exhibit a synchronized population burst subsequently, short pre-I activity of neurons—for example, pre-I bursting of neurons-3 and 4 in Similarly, short post-I activity of neurons—for example, post-I bursting of neurons-2 and 4 in Taking the above conventions into account, ‘total sporadic burst count possible’ for a given neuron is the sum-total of all the time-bins during which neuron’s activity would potentially be considered as sporadic bursting. Initially, several explorations were done to evolve the composite network using simulated annealing ‘manually’. Details of one such ‘manual’ attempt is provided in Referring to Iterations 0–1000: All mutation methods—MM2, MM3(SynAdd) and MM3(SynDel)—are active. As the network continues to evolve, the Cost (Cost1+Cost2+Cost3) continues to diminish and reaches a stage where rate of decrease of Cost per iteration becomes relatively small. This initial phase of network evolution is accompanied with increase in synapse count in the network.Iterations 1000–3400: Mutation methods MM3(SynDel) is periodically deactivated and activated for a period of 100 and 200 iterations, respectively. During the period when MM3(SynDel) is inactive, networks evolves under the influence of mutation methods MM2 and MM3(SynAdd); network’s synapse count tends to increase. During the period when MM3(SynDel) is active, networks evolves under the influence of all three mutation methods—MM2, MM3(SynAdd) and MM3(SynDel); this helps network ‘relax’. It also ensures that only Cost-minimizing synapses are added to the network by effecting deletion of redundant synapses present in the network.Iterations 3400–6400: Mutation methods MM3(SynAdd) is periodically deactivated and activated for a period of 100 and 200 iterations, respectively. During the period when MM3(SynAdd) is inactive, networks evolves under the influence of mutation methods MM2 and MM3(SynDel); network’s synapse count tends to decrease.The evolved composite network obtained at the end of this iterative random mutation and selection process is expected to have captured essential features of desirable network connectivity pattern.Note that the utilized evolutionary process comprises of only one cycle of increase and decrease in network’s synapse count. Conventional paradigm of simulated annealing algorithm suggests execution of more than one such cycle. However, since care has been exercised to periodically ‘relax’ the network during its evolution process so that the effect of random drift is minimal, one cycle was considered sufficient. The experience gained from preliminary explorations described in section S1 FigN1, N2, N3 and N4 are randomly selected on neuron property plane. A fraction of synaptic connections directed from N1 to N2 is deleted, and an equal number of synaptic connections directed from N3 to N4 is added.Four different neighborhoods (PDF)Click here for additional data file.S2 Fign1 and n2 are chosen at random. Then (A) a fraction  of incoming synapse to neuron n1 (indicated in gray color) are removed and allocated to neuron n2, and (B) a fraction of outgoing synapse from neuron n1 are removed and allocated to neuron n2. These changes are effected by appropriately changing the elements of connectivity matrix.Two different neurons (PDF)Click here for additional data file.S3 Figsmall fraction  of ‘maximum possible incoming synapse that can be added to the selected neuron’ is added (indicated in gray color); in the case presented, ‘maximum possible’ incoming synapse that may be added to selected neuron is 3, (B) a small fraction of ‘maximum possible outgoing synapse that can be added to the selected neuron’ is added, (C) a small fraction of all incoming synapse to the selected neuron is deleted, and (D) a small fraction of all outgoing synapse from the selected neuron is deleted. (A) and (B) together constitute MM3(SynAdd), while (C) and (D) together constitute MM3(SynDel); see A neuron within the neuronal group is randomly selected, then (A) a (PDF)Click here for additional data file.S4 Fign1 and n2 are chosen at random and their position within the network is interchanged. This change is effected by exchanging the rows and columns of connectivity matrix corresponding to neurons n1 and n2, respectively.Two different neurons (PDF)Click here for additional data file.S5 Figgtonic (their interburst period is zero). Neurons depicted by hollow-tiny circles remain silent at the set value of gtonic (they have no interburst period).A & B: Intrinsic interburt periods of 80 neurons depicted in (PDF)Click here for additional data file.S6 Fig(Refer (PDF)Click here for additional data file.S7 FigCost1 and Cost2 are given by Eqs ((PDF)Click here for additional data file.S8 Figobjective: CC corresponding to the desired case when every neuron in the network bursts with every population burst. CCinitial, CCMM1 and CCMM3 are CC corresponding to network activities depicted in 1, CC2 and CC3 are representative intermediate CC obtained during the evolution process.CC(PDF)Click here for additional data file.S9 FigIllustration for computing ‘Total sporadic burst count’ and ‘Total sporadic burst count possible’ required for computing Cost3 using (PDF)Click here for additional data file.S10 Figobjective: CC corresponding to the desired case when every neuron in the network bursts with every population burst. CCMM4 is CC corresponding to network activity depicted in (A)‘Evolved NET1 using MM4’. CC0, CC1, CC2 and CC3 are representative intermediate CC obtained during the evolution process. CCMM3 is CC corresponding to evolved excitatory network by method described in section 2.5  Network activity of NET1, initially and after evolving it using MM4. Objective of evolution is to minimize (Cost1+Cost2) as defined by Eqs ((PDF)Click here for additional data file.S11 FigEffect of elimination of IPSPs within the evolved composite network (same evolved network as depicted in (PDF)Click here for additional data file.S12 Fig(PDF)Click here for additional data file.S13 FigSynStrength and SynFrac values as in SynFrac = 0.06, which is lower than that of evolved network (SynFrac ≈ 0.10) and hence is not shown.Synaptic distribution among neurons of evolved network for a different evolutionary run but with identical (PDF)Click here for additional data file.S14 Fig(A) Depiction of evolution of network in terms of ‘Cost’ minimization. Cost1, Cost2 and Cost3 are given by Eqs ((PDF)Click here for additional data file.S15 Fig(A) Depiction of evolution of network in terms of ‘Cost’ minimization. Cost1, Cost2 and Cost3 are given by Eqs ((PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S3 Text(PDF)Click here for additional data file.


There was no effect on PS II function at low or high light intensity. We propose a significant physiological function for the chloroplast NDH at low light intensities commonly experienced during the reproductive and ripening stages of rice cultivation that have adverse effects crop yield.Cyclic electron transport around photosystem I (PS I) was discovered more than a half-century ago and two pathways have been identified in angiosperms. Although substantial progress has been made in understanding the structure of the chloroplast NADH dehydrogenase-like (NDH) complex, which mediates one route of the cyclic electron transport pathways, its physiological function is not well understood. Most studies focused on the role of the NDH-dependent PS I cyclic electron transport in alleviation of oxidative damage in strong light. In contrast, here it is shown that impairment of NDH-dependent cyclic electron flow in rice specifically causes a reduction in the electron transport rate through PS I (ETR I) at low light intensity with a concomitant reduction in CO Regulation of photosynthetic electron transport in the thylakoid membrane of chloroplasts is fundamental for the maximum photosynthetic yield and plant growth. The light reactions in photosynthesis convert light energy into chemical energy in the forms of ATP and NADPH. The reactions involve two types of electron flow in the thylakoid membrane. While linear electron transport generates both ATP and NADPH, cyclic electron transport around photosystem I (PS I) is exclusively involved in ATP synthesis without the accumulation of NADPH. ATP and NADPH generated by light reactions are utilized primarily in the Calvin cycle and photorespiratory cycle.pgr5 single mutant showed the sensitivity to high light2PGR5 knocked-down lines showed a mild decline in CO2 assimilation and biomass productionThe role of cyclic electron transport around PS I is proposed to be essential for balancing the ATP/NADPH production ratio and/or for protecting both photosystems from the damage via stromal over-reductionin vivo and on isolated thylakoids1316On the other hand, NDH-deficient mutants show a sensitivity to various severe stresses, including strong lightMarchantia polymorphaAbiotic stresses often limit crop productivity and play a major role in determining the geographical distribution of plant species. Thus, understanding the physiological processes underlying stress responses and the mechanisms of tolerance is of immense importance for us. Many studies have focused on the role of PS I cyclic electron transport in alleviation of oxidative damage. However, it has been recently reported that the defect in chloroplast NDH led to a reduction in electron transport at low light intensity in ricecrr6 gene in rice also led to the lack of intact chloroplast NDHcrr6 plant provided by the National Institute of Agrobiological Sciences of Japan, plants with the homozygous Tos17 insertion allele  and plants with the homozygous wild-type (WT) CRR6 allele  were identified by PCR analysis and used in the present experiment. Immunoblot analysis showed that the crr6 mutant did not accumulate the CRR6 protein, resulting in the absence NdhK, a subunit of subcomplex A of the chloroplast NDH complex  and plants with the heterozygous Tos17 insertion (+/–) showed the similar dry weight to the WT plants. In contrast, F2 plants with the homozygous Tos17 insertion (–/–) showed the significant reduction in dry weight than the other genotypes  was also lower in the crr6 mutant than in the control plants, when plants were grown at low light.The effect of the nditions . When pl2 assimilation rate at CO2 concentration of 390 μmol mol−1 (A390) above the growth light intensity of 800 μmol photons m−2 s−1 were all similar between crr6 mutant and the control plants, whereas ETR I and A390 below 800 μmol photons m−2 s−1 were significantly lower in crr6 mutant than in the control plants  and dark respiration rate (Rd) were also greater at high light intensity than those at low light intensity  and a low transthylakoid pH gradient (ΔpH) (low NPQ)  . The red0 and gs . The Rd crr6 mutation on the Fv/Fm level was measured after exposure to strong light at 2,000 μmol photons m−2 s−1 for 90 min in plants grown at two different light intensities   , and plaA390)  at high A390) , leading0) (A390  and cons0)  during the reproductive and ripening stages has an adverse effect on potential rice yield since the photosynthetic activity is decreased with lowering light intensity30OsCRR6 gene (Os08g0167500) by the Tos17 retrotransposon insertion and its wild type (Oryza sativa ssp. japonica cv. Hitomebore) were used. The plants were grown hydroponically in an environmentally controlled growth chamber as described2 concentration was 390 μmol mol−1. Plants were grown under two different light intensities, 200 μmol photons m−2 s−1 (low light) or 800 μmol photons m−2 s−1 (high light).The rice mutant defective in the 2 concentration of 390 μmol mol−1 at various light intensities, after leaves were irradiated for over 30 min to obtain steady-state photosynthesis. The electron transport rate (ETR) was calculated as ETR I (or ETR II) = 0.5 × 0.84 × ϕ PS I (or ϕ PS II), where 0.5 is the fraction of absorbed light reaching PS I or PS II, and 0.84 is the leaf absorptance.Measurements of gas exchange, chlorophyll fluorescence and P700 redox state were performed simultaneously with a GFS-3000 and a Dual-PAM-100 measuring system  in uppermost, fully expanded new leaves of 60- to 80-days-old plants as described−1 CO2 concentration and 60% relative humidity in a portable gas exchange system . The leaves were exposed to strong light at 2000 μmol photons m−2 s−1 by a cool light source  for 90 min. The fraction of active PS II (Fv/Fm) was measured in the strong-light treated leaves after dark incubation for 30 min with a chlorophyll fluorescence measuring device .The leaves were placed in a temperature-controlled chamber at 390 μmol molf of the cytochrome b6/f complex and NdhK, a subcomplex A subunit of the NDH complex were determined by the immunoblot analysis with each antibodyImmediately after the measurements of gas exchange, leaf samples were taken, immersed in liquid nitrogen and stored at −80 °C. The frozen leaf samples were ground in liquid nitrogen and homogenized in an extraction buffer. Contents of leaf nitrogen, chlorophyll and Rubisco were quantified−1. After incubation on ice for 10 min and centrifugation at 12,000 × g for 10 min, the supernatants were supplemented with 1/10 volume of BN sample buffer ). Equal amounts of chlorophyll were loaded in each lane of a gel.BN-PAGE was performed as describedHow to cite this article: Yamori, W. et al. Photosystem I cyclic electron flow via chloroplast NADH dehydrogenase-like complex performs a physiological role for photosynthesis at low light. Sci. Rep.5, 13908; doi: 10.1038/srep13908 (2015).