Flowering time is an important trait for determining the adaptability of a crop cultivar. A novel heading date regulator, OsHAPL1, interacts with DTH8-Hd1 to negatively regulate flowering time in rice. Oryza sativa), and a complicated, preliminary regulatory genetic network has also already been established, but the protein regulatory network is poorly understood. We have identified a novel heading date regulator, Heme Activator Protein like 1 (OsHAPL1), which inhibits flowering under long-day conditions. OsHAPL1 is a nuclear-localized protein that is highly expressed in leaves in a rhythmic manner. OsHAPL1 can physically interact with Days To Heading on chromosome 8 (DTH8), which physically interacts with Heading date 1 (Hd1) both in vitro and in vivo. OsHAPL1 forms a complex with DTH8 and Hd1 in Escherichia coli. OsHAPL1, DTH8, and Hd1 physically interact with the HAP complex, and also with general transcription factors in yeast (Saccharomyces cerevisiae). Further studies showed that OsHAPL1 represses the expression of the florigen genes and FLOWERING LOCUS T 1 (RFT1) and Hd3a through Early heading date 1 (Ehd1). We propose that OsHAPL1 functions as a transcriptional regulator and, together with DTH8, Hd1, the HAP complex, and general transcription factors, regulates the expression of target genes and then affects heading date by influencing the expression of Hd3a and RFT1 through Ehd1.Heading date is an important agronomic trait related to crop yield. Many genes related to heading date have already been identified in rice ( Flowering  involves the transition from vegetative to reproductive growth in plants. The correct flowering time is not only important for reproductive success, but also affects crop quality and grain yield . MultiplArabidopsis and rice (Oryza sativa). In Arabidopsis (a long-day (LD) plant), the CONSTANS (CO) gene is regulated by GIGANTEA    is regulated by OsGI (the GI homologue in rice) (Hd1 promotes flowering by activating Hd3a expression under SD conditions and delays flowering by repressing Hd3a expression under LD conditions (Early heading date 1 (Ehd1) pathway that is independent of the Hd1 pathway  and Hd3a , a LD crop, promotes flowering under LD conditions when expressed in rice, suggesting functional conservation in flowering time control between the two diverse crops , natural short days , controlled long days , and controlled short days . ehd1, hd3a, and rft1 mutants in Nipponbare were created using the CRISPR-Cas9 genome editing system.Transgenic plants were produced in the ProUbi::OsHAPL1-VP64 plasmid was produced using the OsHAPL1 coding sequence (CDS) driven by the maize ubiquitin (Ubi) promoter fused to the binary vector LP042 nVP64-hyg-asRED via the Gateway cloning system. The ProUbi::OsHAPL1-Flag plasmid was produced using the OsHAPL1 CDS driven by the maize Ubi promoter fused to the binary vector pCAMBIA1390 via an In-Fusion Advantage PCR Cloning Kit (Clontech). The ProOsHAPL1::GenomeOsHAPL1-GFP plasmid was produced from the genomic DNA sequence and driven by the OsHAPL1 promoter fused to the binary vector pCAMBIA1390 using the In-Fusion Advantage PCR Cloning Kit. The oshapl1 mutant was obtained using the CRISPR-Cas9 system. The Pro35S::OsHAPL1-GFP plasmid was produced using the OsHAPL1 CDS driven by the cauliflower mosaic virus (CaMV 35S) promoter fused to the pAN580 vector. Vectors for yeast two-hybrid assays were constructed by CDS of relevant genes fused to pGBK T7 or pGAD T7 (Clontech). Vectors for pull-down assays were obtained by fusing gene CDS to the vectors pGEX-2T(GST), pET-28a(+)(His), and pMAL-c2X(MBP). Vectors for bimolecular fluorescence complementation (BiFC) were constructed by fusing the gene CDS to pSPYNE173 or pSPYCE(M) (JXB online.The SPYCE(M) . All priTotal RNA was extracted using an RNA Prep Pure Kit . A reverse transcription (RT) kit (Qiagen) was used in RT reactions. Quantitative RT-PCR (qRT-PCR) was performed using SYBR Premix Ex Taq Kit  in an ABI PRISM 7900HT (Applied Biosystems), and ubiquitin was used as the internal control. Standard errors were calculated from three biological replicates.Pro35S::OsHAPL1-GFP and Pro35S::OsMADS51-mCherry were co-transformed to rice protoplasts by polyethylene glycol-mediated transformation  AH109 cells. Positive interactions were confirmed by the ability of AH109 cells to grow on Synthetic Dropout/-Trp-Leu-His-Ade medium in a 30°C incubator.Yeast two-hybrid assays were carried out to detect the interaction of relevant proteins using a procedure provided by Clontech. Baits consisted of proteins fused with the GAL4 DNA binding domain in pGBKT7 (Clontech). Preys were constructed by fusing proteins with the GAL4 activation domain from pGADT7. Bait and prey were co-transformed into yeast , 150 mM NaCl, and 1 mM DTT. The sample liquid was then sonicated to destroy the cell . If the resulting bacterial liquid was not clear, sonication was repeated. Equal amounts of the two protein solutions were mixed and centrifuged, and the supernatant was incubated with glutathione agarose beads  overnight at 4°C. After spinning , the beads were collected and washed five times with buffer containing 25 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1 mM DTT, 1% Triton X100, and 0.1% SDS. Finally, the proteins bound to the beads were separated from the beads by boiling in 2×SDS loading buffer in a 95–100°C water bath. The proteins were separated in SDS-PAGE gels and detected by western blot analysis using anti-GST  and anti-MBP antibodies .Plasmids containing GST-, HIS-, and MBP-labelled proteins were transformed into Agrobacterium tumefaciens (strain GV3101) and infiltrated into tobacco (Nicotiana benthamiana) according to the protocol described by The interacting proteins  referred to in this paper were separately fused with YFPC and YFPN from the expression plasmids pSPYCE(M) and pSPYNE173. The plasmids were introduced into OsHAPL1 was inserted into the pB42AD vector. The promoter region of Ehd1 were amplified and inserted into the pLacZi vector. These constructs were transformed into the yeast strain EGY48. The assay was performed as previously described (The coding region of ChIP assay was performed as previously described (UBI) promoter . Phylogenetic tree analysis showed that this protein is homologous to HAP2 subunit members  related to flowering  transgenic plants under NLD conditions . Thus, OsHAPL1 has a role in controlling flowering time under LD.To further confirm the function of OsHAPL1, an obtained . These pobtained , D but nobtained , E. We aProOsHAPL1::GenomicOsHAPL1-GFP transgenic rice (see JXB online).To investigate the subcellular localization of OsHAPLl, the OsHAPLl-GFP fusion protein was introduced into rice protoplasts. GFP fluorescence signals were detected in the nucleus, and the signals merged with known nuclear-localized signals . GFP sigOsHAPL1. OsHAPL1 was generally expressed in all tissues, but more highly expressed in the uppermost and second uppermost leaves   . High exOsHAPL1 exhibited the same diurnal rhythmic expression patterns under SD and LD conditions. It accumulated from the beginning of darkness, reached a peak at the start of the light period, and then rapidly decreased  as bait, we identified an amino acid sequence corresponding to residues 3–125 of DTH8-Ghd8-OsHAP3H (LOC-Os08g07740.1) -OsHAPLl and pSPYNE173-DTH8 . Together, these results suggest a possible interaction in planta between OsHAPLl and DTH8.To confirm the results from library screening, the interaction between the full-length amino acid sequences of OsHAPLl and DTH8 was tested on selective media plates . An in v OsHAPLl . To inve173-DTH8 , whereas173-DTH8 . We alsoTo identify the OsHAPLl domain involved in the interaction with DTH8, we performed yeast two-hybrid assays using either OsHAPLl with the HAP domain only (OsHAPLl-H) or OsHAPLl with the HAP domain deleted (OsHAPLl-∆H) and DTH8 . OsHAPLlin vitro pull-down and BiFC assays  depends on short domains adjacent to their histone fold motifs . Becauseoshapl1 mutant plants grown in LD and SD conditions using qRT-PCR. The expression levels of Ehd1 were upregulated in oshapl1 mutant plants relative to WT in LD conditions. The florigen genes Hd3a and RFT1 in oshapl1 mutant plants were also upregulated in LD conditions . These results suggest that OsHAPL1 regulates heading date by controlling the expression of Ehd1, Hd3a, and RFT1 in LD conditions.To study downstream genes regulated by OsHAPL1, we analysed the expression of key genes related to the heading date in 30-day-old WT and nditions . The expironment . These dOsHAPL1, which is a novel repressor of heading date. Our studies showed that OsHAPL1 can form a complex with DTH8 and Hd1 in E. coli. In addition, OsHAPL1, DTH8, and Hd1 also interact with some members of the HAP subfamily groups and with GTFs. The data strongly indicate that the OsHAPL1-DTH8-Hd1 complex acts as a transcriptional regulator of heading date by interacting with the HAP complex and GTFs , indicating they are not homologues. It seems that OsHAPL1 may function like other HAP family members. In addition, OsHAPL1 also interacts with the GTF TFIID, suggesting that it functions as a transcription regulator in the heading pathway in rice. The CCT domain of CO in Arabidopsis is similar to HAP2 in yeast, and CO may replace AtHAP2 in the HAP complex to form a trimeric CO-AtHAP3-AtHAP5 complex to regulate flowering-related gene expression , HAP3 (NF-YB or CBF-A), and HAP5 (NF-YC or CBF-C) . In yeasE. coli. OsHAPL1 interacts with the GTF TFIID, DTH8 interacts with TFIIA and TFIID, and Hd1 interacts with TFIIH. GTFs and RNA polymerase II play important roles in transcription in eukaryotes. Six GTFs and RNA polymerase II assemble into a stable pre-initiation complex responsible for RNA synthesis  in SD conditions, whereas it delays flowering by downregulating expression of Hd3a under LD conditions through unknown mechanisms . This is a consistent yield feature between oshapl1 and dth8. Altogether, OsHAPL1, DTH8, and Hd1 function in the same pathway and consistently behave as repressors of heading in LD conditions while having no influence on heading date in SD conditions. The Ehd1 pathway is a unique, Hd1-independent flowering pathway. Ehd1 encodes a B-type response regulator that is highly conserved in cultivated rice, but has no homologue in Arabidopsis (Hd3a and the florigen RFT1 (Ehd1, and then inhibits flowering by downregulating expression of RFT1 and Hd3a. Recent studies have shown that Hd1 interacts with Ghd7 to form a complex that can bind to the promoter of Ehd1 and repress its expression (Ehd1. OsHAPL1 may regulate other target genes to control heading date via Ehd1. Nevertheless, our results have demonstrated the possibility of using OsHAPL1 to increase the adaptability of rice varieties. Combinational manipulation of this gene, together with other trait genes such as GS3 for grain size, Gn1a for grain number, and AFD1 for plant height (Heading in rice is regulated by a complicated genetic network involving many genes. Rice is a SD plant that flowers rapidly under SD conditions, but is late flowering under LD conditions . We founJXB online.Supplementary data are available at Fig. S1. Protein structure and phylogenetic tree of OsHAPL1.oshapl1 mutants in Nipponbare background.Fig. S2. The phenotype of Fig. S3. Subcellular localization of OsHAPL1.DTH8.Fig. S4. Rhythmic expression analysis of OsHAPL1 between Nipponbare and ehd1, hd3a, and rft1 mutants, under CLD and CSD conditions.Fig. S5. Expression analysis of Fig. S6. Blast analysis of the amino acid sequences of OsHAPL1 and ScHAP4.Table S1. Primers used in this study.oshapl1 under NLD conditions.Table S2. Comparison of major agricultural traits between Kia-ake and supplementary_figures_S1_S6_tables_S1_S2Click here for additional data file.


In low resource settings, visual inspection with acetic acid (VIA) by allied health workers, has been suggested as an alternative for cervical cancer screening. However, there are concerns about the objectivity and time to diagnostic concordance with specialists. We evaluated the secular trend in interobserver agreement between nurse providers and a gynecologist/colposcopist over a five-year period.Nurses provided VIA screening with digital cervivography to 4,961 participants in five screening clinics from October 2010 to May 2014 in Nigeria in this observational study. Cervigraphs were reviewed at meetings where a gynaecologist/colposcopist made an assessment from the cervigraphs. We used weighted kappa statistics to calculate agreement in diagnosis between nurse providers and the gynecologist/colposcopist; linear regression models to examine overall trend and investigate potential clinic characteristics that may influence agreement; and time series models to characterize month to month variations.Mean age of participants was 37±8 years. Overall agreement was 0.89 at Site D, 0.78 and 0.73 at Sites A and C respectively, 0.50 for Site E and 0.34 for Site C. The number of trainings attended by nurse providers, high level of engagement by site gynecologists were associated with increased agreement; while increasing distance from the coordinating site was associated with decreased agreement. There were no associations between number of years screening clinics were operational, cumulative experience of nurse providers and agreement. There were no significant increases in weighted kappa statistics over time for all sites considered. Monthly variations were significant for only one of two sites considered in time series models .Our results showed a lack of objectivity, persistent variation and lack of convergence of diagnostic capabilities of nurse led VIA cervical cancer screening with the diagnostic capabilities of a specialist in a cervical cancer screening program in Nigeria. Cervical cancer is a major cause of morbidity and mortality, especially in low and middle-income countries (LMIC) ,2. In 20Barriers to the successful implementation of cytology or HPV DNA test based cervical cancer screening programs in LMIC include the need for well-trained laboratory technicians; adequate clinical and laboratory infrastructure to support sample collection and processing; and requirements for multiple clinic visits for screening and treatment. In order to address these limitations, screen and treat strategies which are based on visual inspection with acetic acid (VIA) followed by same day ablative treatment implemented by nurses and allied health workers, have been promoted as cost-effective cervical cancer screening methods in LMIC . The WorResults from studies in India, Latin America and Africa suggest that the sensitivity of VIA is comparable to cytology-based screening strategies , 8. HoweIn this study, we evaluate the secular trend in interobserver agreement between diagnosis made by trained nurse providers and a gynecologist/colposcopist over a 44-month period (October 2010 –May 2014) in a VIA and digital cervicography based cervical cancer screening program in Nigeria.Between October 2010 and May 2014, 4,961 women who were 18 years or older, participated in VIA based screen and treat cervical cancer screening program in five health facilities–National Hospital, Abuja (NHA); University of Abuja Teaching Hospital (UATH); Garki Hospital Abuja (GHA); Federal Medical Center, Keffi (FMCK) and Mother and Child Hospital, Ondo (MCHO) in Nigeria. Screening activities at these clinical sites were coordinated from a central office in Abuja, Nigeria. Three of the sites were located close to the coordinating office  while the other were located in the North Central (FMCK) and South Western (MCHO) regions of the country respectively. The distance from these sites to the coordinating office were 70 kilometers (km) for FMCK and 560km for MCHO.We trained nurses to perform a physical examination including a detailed abdomino-pelvic examination. This was followed by VIA and digital cervicography. Results of VIA examinations were recorded as either positive, negative, uncertain or suspicious of cancer according to the International Agency for Research Against Cancer (IARC) guidelines . Women wWe trained seventeen nurses and six gynaecologists in the conduct of VIA screening. The training module included practical and theoretical sessions on the female reproductive anatomy; cervical cancer aetiology and prevention; informed consent process; communication and counselling skills; performing VIA and digital cervicography; use of data collection tools; data entry; and treatment with thermocoagulation. The training modules were based on cervical cancer recommendations from the WHO and VIA practical manuals from IARC ,12. DuriWe implemented monthly Quality Assurance (QA) meetings to monitor provider performance and improve skills of providers except for a ten-month period between February 2012 and November 2012 when meetings could not be held for logistic reasons. These meetings were held at the central coordinating office at Abuja and were attended by all nurse providers. Nurse providers from sites close to the coordinating office attended in person while nurse providers from remote sites attended via video conferencing.At each QA meeting, all images of the cervix (cervigraphs) taken before and after application of acetic acid during VIA screening were reviewed by a consultant gynecologist/colposcopist (YO). One nurse from each site would present the cervigraphs from that site, noting the nurse providers’ diagnosis and its rationale. Attendees would ask questions and finally the consultant gynecologist/colposcopist would announce his diagnosis for the cervigraph. Discordant diagnoses were highlighted and discussed, and clients with such diagnoses were recalled for appropriate management.In addition to the monthly QA meetings, we also utilized a web-based application for real time consultation. In cases, where nurses wanted a second opinion, the nurses uploaded cervigraphs unto the web-based platform which immediately alerted collaborating gynecologists via SMS to provide a real-time diagnosis for the uploaded cervigraphs. Collaborating gynecologists working in each of the hospitals also provided on-site supervision.Nurse providers collected data on demographics; documented findings from physical and abdominopelvic examinations; recorded findings from VIA assessments, details of treatment if provided and follow up plans. The nurse providers performed all VIA procedures and the gynecologist/colposcopists only made an assessment at the review meeting after cervigraphs had been discussed.For these analyses, we de-identified the screening clinics and referred to them as Sites A, B, C, D, and E. We were able to retrieve the quality assurance meeting reports for 31 months for Site A; 30 months for Site B; 7 months for Site C; 6 months for Sites D and E. All the nurses working at a given screening site were grouped together and analyses were conducted per site.We calculated weighted kappa statistics to estimate the interobserver agreement for interpretations of cervigraphs between nurses and the consultant gynecologist/colposcopist by month and by site. We defined weights based on the following rationale: diagnosis of VIA positive and suspicious of cancer were reasonably similar; that VIA negative and uncertain are reasonably similar; but the VIA positive/suspicious of cancer group was not in any way similar to the VIA negative/uncertain group. We used Landis and Koch’s guidelines for the interpretation of kappa estimates where values below 0.0 are considered poor; 0.00–0.20 slight; 0.21–0.40 fair; 0.41–0.60 moderate; 0.61–0.80 substantial; 0.81–1.00 almost perfect . In someWe explored the temporal trends in agreement between nurse providers and the consultant gynecologist/colposcopist using two components—the overall trend in weighted kappa statistics over the study period and the monthly cyclical variation in weighted kappa statistics. We estimated the overall trend during the study period using linear regression models with time as the explanatory variable and weighted kappa statistic as the outcome variable for each site. We performed sensitivity analysis to investigate the trend in weighted kappa statistics for Sites A and B before and after the QA interruption by using linear piecewise regression models with splines at months 16 and 27, the period during which QA meetings were suspended.p, d, and q [d) for each site by performing Dicky Fuller tests [d = 0 was used in our ARIMA models. Next, we evaluated autocorrelation functions (ACF) and partial autocorrelation functions (PACF) to identify the autoregressive (AR) component  and moving average (MA) component . We fitted several ARIMA models and performed model diagnostics, including residual plots and autocorrelation functions. We compared models using the Akaike Information Criteria (AIC) and selected the model with the lowest AIC value. Time series analysis were not performed for sites C, D, and E because of limited time series data. All statistical analyses were performed in Stata (Stata version 15).We modelled monthly cyclical variation using autoregressive integrated moving (ARIMA) time series models. First, we used a time series filter, the compound, nonlinear, resistant, Hanning smoother to visually inspect the monthly cyclical patterns of the weighted kappa estimates over time. ARIMA model parameters were estimated using the Box Jenkins methodology, defined as d, and q . In modeer tests . As the This study was reviewed and approved by the National Health Research Committee of Nigeria . All participants provided a written informed consent.By May 2014, our VIA cervical cancer screening program was operational in five clinics around the country. Of these clinics, two were activated in 2010; two in 2012 and one in 2013 . Sites ABaseline characteristics of clients seen in the VIA screening program are provided in Of the 4,961 clients seen in the VIA screening program, cervigraphs of 4,602 clients were reviewed by the gynecologist/colposcopist at the monthly QA meetings. Details of the distribution of the reviewed cervigraphs by site are provided in Weighted kappa estimates for the duration under study were almost perfect for site D (0.89); substantial for sites A and B (0.78 and 0.73 respectively); moderate for site E (0.50); and fair for Site C (0.34) .In univariate linear regression models, site characteristics that were significantly associated with improved weighted kappa statistics were attendance at more formal VIA training sessions ; high level of engagement by onsite gynecologist  . Remote For every additional month of site being fully operational, the weighted kappa statistic, comparing nurse providers at the site and the consultant gynecologist/colposcopist, increased by 0.21  for Site B and 1.03  for Site E; and decreased for three other sites . These changes were only significant for Site A .In sensitivity analysis, weighted kappa statistics declined with each additional increase in month of being operational in the periods before  and after  QA interruption for Site A . However1 = -0.11, p = 0.54) indicating that the data in this series were random and independent. Therefore, further ARIMA models were not considered for Site A.Monthly kappa estimates for site A for the period under study were not serially correlated . The lagged change in weighted kappa estimate by one month (t -1) was a significant predictor of expected kappa estimate for the current month (t) . As the coefficient of the AR1 term was negative, monthly kappa estimates were highly variable and oscillatory such that an estimate above the mean in month t-1 was followed by an estimate below the mean in month t. For Site B, our data displayed a significant autoregressive behavior for the first order ARIMA model . The weighted kappa statistics per month at this site were slightly autocorrelated Click here for additional data file.


Subtractive inhibition can narrow the range of inputs that evoke spiking activity by eliminating responses to non-preferred inputs. Divisive inhibition is a form of gain control: it modifies firing rates while preserving the range of inputs that evoke firing activity. Since these two “modes” of inhibition have distinct impacts on neural coding, it is important to understand the biophysical mechanisms that distinguish these response profiles. In this study, we use simulations and mathematical analysis of a neuron model to find the specific conditions (parameter sets) for which inhibitory inputs have subtractive or divisive effects. Significantly, we identify a novel role for the A-type Potassium current (IA). In our model, this fast-activating, slowly-inactivating outward current acts as a switch between subtractive and divisive inhibition. In particular, if IA is strong  and fast , then inhibition has a subtractive effect on neural firing. In contrast, if IA is weak or insufficiently fast-activating, then inhibition has a divisive effect on neural firing. We explain these findings using dynamical systems methods  to define how a spike threshold condition depends on synaptic inputs and IA. Our findings suggest that neurons can “self-regulate” the gain control effects of inhibition via combinations of synaptic plasticity and/or modulation of the conductance and kinetics of A-type Potassium channels. This novel role for IA would add flexibility to neurons and networks, and may relate to recent observations of divisive inhibitory effects on neurons in the nucleus of the solitary tract.Neurons process and convey information by transforming barrages of synaptic inputs into spiking activity. Synaptic inhibition typically suppresses the output firing activity of a neuron, and is commonly classified as having a  Neurons process information by generating spikes in response to two types of synaptic inputs. Excitatory inputs increase spike rates and inhibitory inputs decrease spike rates . The interaction between these two input types and the transformation of these inputs into spike outputs is not, however, a simple matter of addition and subtraction. Inhibitory inputs can suppress outputs in a variety of ways. For instance, in some cases, inhibition adjusts the rate of spiking activity while preserving the range of inputs that evoke spiking activity; an important computational principle known as gain control. We use simulations and mathematical analysis of a neuron model to identify properties of a neuron that determine how inhibitory inputs affect spiking activity. Specifically, we demonstrate how the gain control effects of inhibition depend on the A-type Potassium current. This novel role for the A-type Potassium current provides a way for neurons to flexibly regulate how they process synaptic inputs and transmit signals to other areas of the brain. The activity of a neuron is driven by barrages of synaptic inputs. Synaptic inputs are classified as either excitatory (those that promote spike generation) and inhibitory (those that impede spike generation). The interplay between these two “opposing” inputs influences how neurons process and transmit information in the brain.subtractive effect on neural firing, versus inhibition that has a divisive effect , so there exists a fixed-point of this map [nav as the solution of the fixed point fixed point problem n = N ∘ Θ(n), and then set θ = Θ(nav).We have now defined the two maps, this map . We use Slope of output rates at onset of firing in the divisive case. By computing the firing threshold at rE = 0, we can study the behavior of the model near the onset of firing. In particular, if gA is sufficiently small or gSyn,E is sufficiently large, then inhibition is divisive and the neuron is able to fire in response to arbitrarily low firing rates , i.e. it is the value of sI so that Nlk = 0.This is done by simply differentiating the firing rate approximation in rE = 0 computed from both the theoretical prediction  was denoted as gA at which the effect of inhibition switches from divisive to subtractive.In ediction  and simuApproximation of output rates at arbitrary input rates. To more completely characterize output firing rates, we seek to extend our approximations to cases of higher output rates. In other words, we consider the firing rate equation  and performed a nonlinear curve-fit using R ≈ 10 ms (with some dependence on ga) provides accurate approximations to the firing rate functions in the absence of inhibition.equation  for rE >gA . When we include inhibition in these simulations, we find that the approximated firing rate (using the firing rate threshold computation outlined above) tends to overestimate simulated firing rates (colors). Nevertheless, approximations do capture the qualitative differences in firing rate curves for divisive inhibition  and subtractive inhibition .We compare our theoretical approximation to the firing rate of the model neuron to simulated firing rates in θ, the theoretical value of sI for which the neuron cannot produce a spike if an incoming excitatory event arrives at a time when sI ≥ θ. We plot values of θ as colored lines in the lower row of θ to simulations, we recorded the values of sI at the time of every excitatory event that triggered a spike. Then, for each input rate (x-axis), we found the maximum value of sI for these spike-triggering excitatory events. These maximum sI values qualitatively align with our approximations of θ, further supporting the heuristic concept of a firing threshold.The firing threshold calculation defines We point out that, following from Eqs a can be set to its voltage-dependent steady state value; that is, we set a = a∞(V). One effect of this change from a evolving dynamically with τa = 2 ms (the full model), to a evolving instantaneously as a∞(V) (the reduced model), is that excitation must be much stronger in the reduced model to observe subtractive inhibition. Typical values of gSyn,E in the full model are around 0.5  plays a role in switching the effect of inhibition from divisive to subtractive.A key assumption in the formulation and analysis of the reduced model is that the A-current activates sufficiently fast so that the dynamical variable  0.5 see , and typgA is sufficiently strong because the A-type channel activates “instantaneously” and can prevent spike initiation. In the one-compartment model with “non-instantaneous” a variable, large gA could switch the effect of inhibition to subtractive, but only if excitatory input strength was also sufficiently small , there is a delay of roughly 2 ms before the rapid upstroke of V at the onset of the action potential. During this “pause”, voltage is slowly ramping up and, simultaneously, recruiting additional A-current as the a variable activates. The amount of IA available to suppress spike initiation depends, therefore, on A-channel maximal conductance (gA) and also the time-constant of IA activation (τA).For inhibition to have a subtractive effect, responses to infrequent excitatory inputs must be suppressed. In the reduced model, this occurs when l recall . The impIA can act to switch the effect of inhibition from divisive to subtractive, but only if it activates rapidly enough relative to the dynamics of spike initiation. To illustrate our point, we simulated the model with three values of A-channel activation time constant , using gSyn,E = 0.5, gSyn,I = 1, and rI = 50 Hz. As shown in τA = 1, 2) and subtractive for faster activation kinetics (τA = 0.5). In gSyn,E, gA) parameter plane. There is a strong effect of τA. Faster activation kinetics  shift the critical point at which inhibition switches from divisive to subtractive to lower values values of gA.From this observation, we draw the following conclusion: “non-instantaneous” Our prior observation, that delaying spike initiation allows inhibition to have a subtractive effect for “non-instantaneous” A-channel activation, led us to investigate other cellular mechanisms that could have a similar effect. To this end, we considered a multi-compartment neuron model that describes a soma and passive dendrite. Inhibition and voltage-gated currents are restricted to the soma compartment, and excitation targets a location somewhere on the dendrite. Passive cable theory tells us that the amplitudes of excitatory post-synaptic potentials attenuate and their rising slopes become less steep as signals spread along the cable . By varygSyn,E = 2 in these simulations). Inputs that arrive proximal to the soma are large and fast-rising relative to responses to more distal inputs, and thus evoke action potentials with shorter latencies. The parameter cptin identifies the compartment that receives synaptic excitation. It takes values from 1  to 9 .Examples of action potentials, recorded in the soma compartment, in response to inputs at different locations along the dendrite are shown in We included synaptic inhibition in the model targeting the soma, and used simulations to characterize the effect of inhibition as either subtractive or divisive. In gA, gSyn,E) parameter plane and identified the boundary separating regions in which inhibition has a divisive effect on firing rates and regions in which inhibition has a subtractive effect  parameter plane over which inhibition has a divisive effect is smaller when inputs are more distant from the soma.We explored the  can act to switch the effect of inhibition on output firing from divisive to subtractive. This provides a clear demonstration of how the internal dynamics of a neuron can control the functional impact of inhibition.IA conductance (gA) at which the effect of inhibition switched from divisive  to subtractive   and slow variables . In fact, the inactivation variable b was sufficiently slow that it could be treated as a constant with a value that depended on the input rate  . In the ullcline , we idenc inputs . Key in put rate . This siput rate  as well put rate .IA activation, a, evolved instantaneously to its voltage-dependent equilibrium value , we found that, in conditions of slow spike initiation, IA could “ramp up” during slowly-developing spikes and suppress spike initiation. Weak excitatory inputs, or excitatory inputs targeting more distal regions in a model that included a spatially-extended dendritic process . We alsitiation . For our process  producedgA, inhibition can have a divisive effect on the input/ouput properties of a spiking neuron responding to a mixture of random excitatory inputs and periodic inhibitory inputs. The amount of gA altered the slope (gain) of the output firing rate, and thereby tunes the gain control in this system. This result is consistent with the results of a recent in vitro study of neurons in the rostral nucleus of the solitary tract [IA, as compared to neurons in the same nucleus that did not have IA. Thus, the presence of IA enhanced the divisive effect of inhibition. Previous modeling work has identified similar gain control effects by IA [Divisive inhibition is a mechanism of neural gain control and has been the subject of numerous studies; see  for reviry tract . In thatts by IA .gA, the A-type current can switch the effect of inhibition from divisive to subtractive. This demonstrates a novel example of how the internal dynamics of a neuron interact with synaptic inhibition to change the neuron’s computational properties (input/output relation). Previous studies have explored the multi-faceted ways in which IA current can alter neural dynamics. Connor and Stevens established IA current as a mechanism to prolong interspike intervals of repetitively-firing neurons to arbitrary lengths (“type I” firing dynamics) [IA include prolonging first spike latency [IA function.We observed, additionally, that at higher levels of ynamics) . Other i latency , produci latency , filteri latency , and aff latency . Our conIA can lead to subtractive inhibition. The strength and kinetics of the A-type Potassium channels can be modulated in a variety of ways [We have identified routes to subtractive inhibition that depends only on mechanisms that could be readily adjusted by processes of plasticity and neuromodulation. In particular, we have shown that strong and fast  of ways –36. For  of ways –39 and g of ways .IA can add flexibility to neural systems by allowing neurons to “self-regulate” whether inhibition acts in a subtractive or divisive manner.We also found that weak excitatory inputs or more distally-located excitatory inputs led to subtractive inhibition by slowing the onset of action potentials. Synaptic plasticity and modulation of the electrical properties of dendrites can adjust the strength and propagation of excitatory inputs , and plaS1 FigA, B: Output firing rates as a function of excitatory input rate, computed from simulations without inhibition  and with inhibition . Excitatory synaptic strength is gSyn,E = 0.5. In A: Divisive rescaling of the input/output relation with gA = 20. In B: Subtractive shifting of the input/output relation with gA = 40. C: Data from A and B are replotted with output firing rates in the absence of inhibition on the ordinate and output firing rates in the presence of inhibition on the abscissa. Threshold-linear functions are fit to simulation data (black lines). Rightward shift of threshold-linear function for gA = 40 is characteristic of subtractive inhibition.(TIF)Click here for additional data file.S2 FigA, B: For each parameter set, we fit threshold-linear functions to characterize the relationship between output firing rates in the presence and absence of inhibition. Dots in each panel identify the smallest value of gA (for a given parameter set) at which inhibition is subtractive. In A: We vary inhibition strength  and keep inhibition rate fixed at 50 Hz. In B: We vary inhibition rate  and keep inhibition strength fixed at gSyn,I = 1. The values of gA that define the boundary between subtractive and divisive inhibition decrease with increases in either inhibition parameter .(TIF)Click here for additional data file.S3 FiggA (compare slopes for gA = 0 and gA = 20), and shows both divisive and subtractive features for higher values of gA . We classify as subtractive any response for which the input/output curve is shifted rightward. Thus, subtractive responses (in our classification) also include “mixed” responses such as those shown here.Inpout/output firing rate relations for varying A-channel conductance. Inhibition is divisive for lower (TIF)Click here for additional data file.S4 FigA: Threshold-linear relations between output firing rates in simulations of the multi-compartment model with and without inhibition for varying inhibition input location and gA = 20. For simulations with inhibition: gSyn,I = 1 and rI = 50. Inhibition is divisive for inhibitory inputs that target the soma (cptinhib = 0), an intermediate position on the dendrite , and a distal location on the dendrite (cptinhib = 8). B: Threshold-linear relations for varying inhibition input location and gA = 30. For simulations with inhibition: gSyn,I = 1 and rI = 50. Inhibition is subtractive for inhibitory inputs that target the soma or positions on the dendrite.(TIF)Click here for additional data file.S1 DatasetExcel data set containing data used to produce figures.(XLSX)Click here for additional data file.


Nanoconfinement dramatically modifies the behaviour of solvent within protein crystals, allowing biophysical measurements in the presence of liquid solvent at temperatures down to ∼200 K. When internal ice forms it is stacking-disordered, consistent with nucleation within deeply supercooled solvent, and ice does not form in solvent within ∼5 Å of the protein surface. h) forms in external solvent, but internal crystal solvent forms stacking-disordered ice (Isd) with a near-random stacking of cubic and hexagonal planes. Analysis of powder diffraction from internal ice and single-crystal diffraction from the host protein structure shows that the maximum crystallizable solvent fraction decreases with decreasing crystal solvent-cavity size, and that an ∼6 Å thick layer of solvent adjacent to the protein surface cannot crystallize. These results establish protein crystals as excellent model systems for the study of nanoconfined solvent. By combining fast cooling, intense X-ray beams and fast X-ray detectors, complete structural data sets for high-value targets, including membrane proteins and large complexes, may be collected at ∼220–240 K that have much lower mosaicities and comparable B factors, and that may allow more confident identification of ligand binding than in current cryocrystallographic practice.Ice formation within protein crystals is a major obstacle to the cryocrystallographic study of protein structure, and has limited studies of how the structural ensemble of a protein evolves with temperature in the biophysically interesting range from ∼260 K to the protein–solvent glass transition near 200 K. Using protein crystals with solvent cavities as large as ∼70 Å, time-resolved X-ray diffraction was used to study the response of protein and internal solvent during rapid cooling. Solvent nanoconfinement suppresses freezing temperatures and ice-nucleation rates so that ice-free, low-mosaicity diffraction data can be reliably collected down to 200 K without the use of cryoprotectants. Hexagonal ice (I In its early days, crystallography was performed using protein crystals at or near room temperature. Once synchrotron X-ray sources became widely available in the 1990s, data collection shifted to the near-exclusive use of cryogenically cooled crystals. Cryocooling to ∼100 K reduces the rate at which diffraction properties degrade with X-ray dose by a factor of ∼50, increasing the amount of data that can be collected per crystal, and reduces thermal motions, often increasing the resolution   or more for high-solvent-content [>80%(v/v)] crystals. For data collection between the homogeneous nucleation temperature of bulk water, Th ≃ 235 K, and 180 K, crystals have been soaked in 75%(v/v) methanol . All crystals were grown by the hanging-drop vapour-diffusion method in 24-well plates.−1 in 0.1 M sodium acetate buffer pH 6.5 and 2 µl of a well solution consisting of 2%(w/v) CdSO4 and 15%(w/v) (NH4)2SO4 in the same buffer. Cubic crystals in space group F432 grew to dimensions of 300–500 µm within a week [Supplementary Fig. S1(a)].Crystals of equine spleen apoferritin  were grown in hanging drops consisting of 2 µl of protein at 10 mg ml−1 in 0.1 M sodium acetate buffer pH 6.5 and a well solution consisting of 14%(w/v) potassium/sodium tartrate in the same buffer. Tetragonal crystals in space group P41212 grew to dimensions of 200–300 µm within one week [Supplementary Fig. S1(b)].Crystals of thaumatin  were grown in hanging drops comprised of equal volumes of protein at 40 mg ml−1 in 0.1 M sodium acetate buffer pH 5.2 and a well solution consisting of 2.5%(w/v) NaCl in the same buffer. Tetragonal crystals in space group P432121 grew to dimensions of 300–800 µm. Crystals appeared within one week and stopped growing within four weeks.Crystals of lysozyme  were grown in hanging drops comprised of equal volumes of protein at 80 mg mlv/v), which were obtained by adding glycerol to a solution with the same composition as the previously mentioned well solutions. Each crystal was transferred to a separate drop of NVH oil (Cargille) and manipulated until all external solvent was removed from its surface  using a PILATUS 6M detector (Supporting Information Section S2). A cold nitrogen-gas stream programmed to the desired final sample temperature was directed at the crystal, but was initially blocked using a shutter. Each crystal was placed in the X-ray beam at room temperature, ten frames totalling 5° in rotation were collected to assess the crystal for damage or dehydration, and the crystal was then rotated back to its initial orientation. The gas stream was then unblocked and the collection of frames with 0.5° rotation and 0.1–0.2 s exposure per frame commenced .2.2.XDS in segments of five frames (Supporting Information Section S3). Structural models were derived from frames starting after the unit cells reached a stable equilibrium and until the end of data collection. Molecular replacement and model refinement were performed using PHENIX, and the results were checked using Coot (Supporting Information Section S4). Protein and solvent volumes were then evaluated using the final refined models (Supporting Information Section S5). Refinement statistics for the 45 apoferritin and 53 thaumatin structures used in the analysis are given in the Supporting Information.Diffraction frames were indexed, integrated and scaled using 2.3.DIFFaX  and cubic (Ic) ice. These fits were compared with those obtained assuming a simple mixture of cubic and hexagonal crystallites (Supporting Information Section S7).Diffraction frames from the detector were processed using Python scripts to remove protein Bragg scattering and background, and were azimuthally integrated (Supporting Information Section S6). The resulting intensity versus resolution plots were analysed by embedding the program 2.4.Structure factors calculated from models of protein structure and ice diffraction were used to normalize protein structure and ice diffraction data collected from the same crystal in the same X-ray beam using the same detector, yielding the ratio of ice to protein crystal volume (Supporting Information Section S8). The ice volume was then compared with the solvent-cavity volume of the protein crystal at the ice observation temperature, with corrections applied based on estimates of the solvent fraction that exited the unit cell on cooling.3.3.1.c) and 1d) and Supplementary Fig. S4], contributing to the difficulty in obtaining high-quality structural data sets from these crystals. For pure water in nanoporous silica and alumina, the effects of nanoconfinement on freezing and melting temperatures become pronounced for cavity sizes below ∼10 nm  and maximum solvent-cavity sizes of 68, 25 and 13 Å, respectively, as indicated in Figs. 1We studied cubic apoferritin, tetragonal thaumatin and (with limited measurements) tetragonal lysozyme crystals, with Matthews-coefficient-derived solvent contents of 63, 59 and 42%(v/v) glycerol or harvested as is [0%(v/v)] and then abruptly cooled (in <1 s) to temperatures between 180 and 260 K (Supporting Information Section S2). Fig. 3a)] and 20 s [Fig. 3b)], times that are sufficient to collect complete structural data sets on high-brilliance synchrotron beamlines, after their unit cell reached its steady state or minimum value. No apoferritin crystals, regardless of the concentration of glycerol, showed internal ice formation at temperatures above 240 K. Ice eventually appeared below 240 K in crystals with lower glycerol concentrations. However, at all temperatures internal solvent within at least a substantial minority of these crystals could be maintained in a supercooled state for at least a few seconds. Ice first became detectable up to ∼20 s after the crystals reached their steady-state temperature. Similar results were obtained using thaumatin crystals . The salt concentrations present within the crystallization solutions and internal crystal solvent suppress bulk freezing temperatures by only a few degrees. When bulk solutions containing these salt concentrations are cooled below the freezing temperature, ice forms in ∼10 ms unless the temperature drops below the solvent glass-transition temperature Tg first (Supporting Information Section S11). Consequently, the suppression of ice formation in protein crystals with solvent cavities of up to ∼70 Å must be owing to nanoconfinement.Time-resolved X-ray diffraction measurements were performed on apoferritin and thaumatin crystals soaked in solutions containing 10, 20 and 40% gives a nucleation rate of the order of 1023 cm−3 s−1, which is consistent with the peak temperature-dependent rate.Freezing-point suppression is an 0–102 s following cooling to 200–230 K. When ice eventually becomes detectable, its diffraction intensity saturates in as little as 0.2–0.4 s. Assuming that ice detection arises from a single nucleation event shortly before that detection, nucleation rates between 200 and 230 K are in the region of 106 cm−3 s−1, which is ∼1010–1017 times smaller than in water nanodrops comparable in size to the solvent cavities in apoferritin. The most conservative assumptions, that ice is first detectable when the ice fraction reaches 2%, that nucleation occurs steadily until that threshold is reached, that each nucleus involves 100 water molecules and that nuclei do not grow, give a nucleation rate of ∼1019 cm−3 s−1, which is roughly four orders of magnitude smaller than in water nanodrops below 215 K. The grain sizes of internal ice in apoferritin crystals deduced from diffraction peak widths are in the range ∼200–800 Å, spanning many unit cells, so the actual nucleation rates are likely to lie between these limits. The dramatically reduced nucleation rates under nanoconfinement that we infer are qualitatively consistent with simulations  decrease to a minimum near ∼220 K, and neither cooling to 100 K nor the use of glycerol provide clear improvements . For both proteins, the crystal mosaicities generally increase with decreasing temperature  and are smaller by factors of ∼2–6 at 220 K than at 100 K for all glycerol concentrations except 40%(v/v). Between 200 and 260 K, glycerol-free crystals of both proteins tend to have the lowest mosaicities.For ice-free apoferritin and thaumatin crystals, the Wilson 3.3.a) and 5b)]. For both proteins, the contraction of the protein volume on cooling to 180 K is small (∼0.5–1%) and is nearly independent of glycerol concentration [Figs. 5c) and 5d)]. The solvent-cavity volume contraction is much larger and is only weakly dependent on the glycerol concentration [Figs. 5e) and 5f)].For ice-free apoferritin and thaumatin crystals at all glycerol concentrations, the unit-cell volumes measured ∼3–5 s after cooling contract monotonically between 300 and 180 K . For all 29 of these crystals, ice formed within ∼2 s of the start of cooling, suggesting that it nucleated during cooling, and many of the raw diffraction patterns had a component that was azimuthally ‘lumpy’ [Fig. 6a)], indicating a small number of large ice grains. In some cases, residual frozen solvent was clearly visible on the crystal surface. We thus attribute the appearance of hexagonal ice as arising from nucleation in residual external solvent, not internal solvent.In the present experiments, we attempted to remove all external solvent. For glycerol concentrations of 0 and 10% and 6f)]. The Ih peaks near 3.4 and 2.1 Å were absent or strongly suppressed, and a smooth shoulder was instead observed near the position of the 3.4 Å peak. Ice diffraction in this case was always uniform and isotropic [Figs. 6b) and 6c)], indicating a large number of small, randomly oriented grains. For the glycerol-free apoferritin crystals that developed these ice diffraction patterns, the mean time to ice formation was 6.4 s and the standard deviation was 8.3 s, suggesting delayed and stochastic nucleation. These systematics, together with estimated freezing temperatures for bulk-like internal solvent, indicate that the ice arises from nucleation within deeply supercooled internal solvent. Poor fits to the diffraction patterns obtained in refinement indicate that it is neither cubic nor hexagonal, nor a simple mixture of the two.By far the most common pattern of ice diffraction, which was observed in 41 of 98 apoferritin crystals and in 36 of 51 thaumatin crystals between 180 and 230 K, consisted of a strong but broadened peak near et al., 2012et al., 2012et al., 2016c direction [Fig. 7a)].Similar diffraction patterns have been observed in experiments on ice formed on cooling in deeply supercooled water microdrops and nanodrops ]. The solid lines in Figs. 6e) and 6f) are refined ice diffraction fits assuming this model (Supporting Information Section S7) calculated using the program DIFFaX  and 7c)] for the substantial number of crystals analysed (Supplementary Table S1) indicates that this stacking fraction is robust. A near-random stacking of cubic and hexagonal layers may result because the free energies of nucleating cubic and hexagonal ice planes on an ice-crystal surface are similar, so that competitive nucleation under conditions of deep supercooling leads to stacking that is dominated by kinetics rather than thermodynamics . The ratio of these quantities from ice and protein crystal diffraction then gives the fraction of the X-ray-illuminated volume occupied by ice. Using the refined crystallographic models for the protein structure, the fraction of the unit cell occupied by solvent can be determined. This allows the fraction of internal solvent that forms ice to be determined with high accuracy.One metric of the perturbation of water is its ability to participate in a crystalline network , the thickness of the layer of noncrystallizable solvent adjacent to the protein surface can be determined. This thickness is of the order of 6 Å for all three proteins Table 2, which i4.We have connected the behaviour of water and ice in protein crystals to results from previous studies of water in nanoporous inorganic matrices and in microdrops and nanodrops. Protein crystals have significant advantages over other nanoconfining systems. The ∼100 000 known protein crystal structures offer tremendous variety in pore size, pore geometry  and chemical properties. A majority have excellent long-range order, so the full atomic structure of the confining matrix is known from crystallography and available for simulations. Ordered inorganic nanoporous matrices are typically synthesized as micrometre-size powders with large ratios of external surface area to volume, significant (5–10%) pore-size variations and substantial defect densities. Measurements on nanoconfined solvent require the use of packed powders, often filled from the vapour phase to minimize internal bubbles and overfilling, introducing substantial uncertainties and restricting study to pure water and other volatile liquids. Ice formation in solvent on the surface of individual grains is likely to corrupt the measurement of ice-nucleation rates.In contrast, protein crystals are typically tens to hundreds of micrometres in size. Single crystals are sufficient for many measurements, and surface solvent can be optically detected and removed. The X-ray diffraction methods demonstrated here should allow ice nucleation and growth, grain sizes and crystallized solvent fractions to be tracked during and following cooling in single crystals with thermal response times of <100 ms. The composition of the internal solvent of a crystal can be changed by serial soaking in aqueous solutions containing salts, sugars, alcohols and polyols, including at concentrations (including nearly pure water) which cause crystal dissolution and/or protein unfolding, and often still retain excellent order, and the effects of, for example, the preferential hydration of protein surfaces and solute rejection by growing ice crystals on crystallizable solvent fractions can be determined. These features make protein crystals attractive model systems for studying the effects of confinement on ice formation, especially under biophysically relevant conditions.in situ cooling with intense synchrotron X-ray beams and fast X-ray detectors, complete structural data sets for high-value targets including membrane proteins and large complexes may be collected at ∼220 K that have much lower mosaicities and comparable B factors, and that may allow more confident identification of ligand binding than in current cryocrystallographic practice. This same crystal-based strategy of abrupt cooling and fast data collection before ice formation may enable a variety of temperature-dependent biophysical studies of protein structure, conformational ensembles and function, including at temperatures near Th that are inaccessible when studying proteins in solution or in vivo.Even in protein crystals with ∼70 Å solvent cavities, which are larger than those in ∼98% of current PDB depositions, nanoconfinement dramatically suppresses freezing temperatures (to ≤240 K) and ice-nucleation rates, with the latter allowing internal solvent to remain as a (supercooled) liquid for at least several seconds at temperatures between 200 and 240 K. By combining abrupt 5.et al. , Nakasako (2004et al. (2000et al. (2018et al. (2003et al. (2013et al. (2002et al. (2011et al. (1995et al. (2001et al. (2016et al. (2007et al. (2015et al. (1998et al. (2015et al. (2001et al. (1992et al. (1996et al. (2016et al. (2006aet al. (2005et al. (2017The following references are cited in the supporting information for this article: Adams  al. 2010, Alcorn  al. 2010, Amaya e al. 2017, Ashioti al. 2015, Atakisi al. 2018, Bagchi  al. 2018, Barbosa al. 2018, Bartell al. 2018, Charron al. 2002, Chen et al. 2010, Chukin  al. 2010, Crichto al. 2010, Datta e al. 2001, Doster  al. 2001, Douzou  al. 1975, Ebbingh al. 2007, Emsley  al. 2010, Erko et al. 2012, Espinos al. 2014, 2016 ▸,Click here for additional data file.10.1107/S2052252519001878/tj5020sup1.aviSupplementary Movie S1. Animation of structure of cubic apoferritin. DOI: Click here for additional data file.10.1107/S2052252519001878/tj5020sup2.mpgSupplementary Movie S2. Animation of solvent cavities in cubic apoferritin crystals. DOI: Click here for additional data file.10.1107/S2052252519001878/tj5020sup3.aviSupplementary Movie S3. Animation of tetragonal thaumatin structure. DOI: Click here for additional data file.10.1107/S2052252519001878/tj5020sup4.mp4Supplementary Movie S4. Animation showing the solvent cavities in tetragonal thaumatin crystals. DOI: Click here for additional data file.10.1107/S2052252519001878/tj5020sup5.aviSupplementary Movie S5. Animation of tetragonal lysozyme structure. DOI: Click here for additional data file.10.1107/S2052252519001878/tj5020sup6.mpgSupplementary Movie S6. Animation of solvent cavities in tetragonal lysozyme. DOI: Click here for additional data file.10.1107/S2052252519001878/tj5020sup7.xlsxSpreadsheet S1. Refinement statistics for apoferritin data sets. DOI: Click here for additional data file.10.1107/S2052252519001878/tj5020sup8.xlsxSpreadsheet S2. Refinement statistics for thaumatin crystals. DOI: 10.1107/S2052252519001878/tj5020sup9.pdfSupporting information, Supplementary Tables and Figures, and detailed captions to Supplementary Movies. DOI:


Purified rCari p 1 can be used a marker allergen for component-resolved molecular diagnosis. Further immunological studies on Cari p 1 are warranted to design immunotherapeutic vaccine for the clinical management of papaya allergy.Papaya has been reported to elicit IgE-mediated hypersensitivity via pollen inhalation and fruit consumption. Certain papaya sensitive patients with food allergy were found to experience recurrent respiratory distresses even after quitting the consumption of fruits. This observation prompted us to investigate the allergens commonly present in fruits and pollen grains of papaya. A discovery approach consisting of immunoproteomic detection followed by molecular characterization led to the identification of a novel papaya allergen designated as Cari p 1. This allergen was detected as a 56 kDa IgE-reactive protein from pollen as well as fruit proteome through serological analysis. The protein was identified as an endopolygalacturonase by tandem mass spectrometry. Full length Cari p 1 cDNA was isolated from papaya pollen, cloned in expression vector, and purified as recombinant allergen. The recombinant protein was monomeric and displayed pectinolytic activity. Recombinant Cari p 1 reacted with IgE-antibodies of all the papaya sensitized patient sera. In addition to IgE-reactivity, rCari p 1 displayed allergenic activity by stimulating histamine release from IgE-sensitized granulocytes. CD-spectroscopy of rCari p 1 revealed the presence of predominantly β-sheet characters. The melting curve of the allergen showed partial refolding from a fully denatured state indicating the possible presence of conformational IgE-epitopes characteristic of inhalant allergens in addition to the linear IgE-epitopes of food allergens. The expression of this allergen in papaya fruits was detected by immunoblot with anti-Cari p 1 rabbit IgG and reconfirmed by PCR. In an  IgE-antibody mediated type-I hypersensitivity has assumed a pandemic dimension with a prevalence rate of 10–30% of the global population . A substIn all the cases, the efficacy of accurate diagnosis and specific immunotherapy of allergy largely depends on the availability of the molecular and structural details of the allergen molecule. In the past few decades, considerable advancement in recombinant DNA technology , bioinfoCarica papaya L.) of Caricaceae family is globally cultivated for its edible fruits with high nutritious value , known as ‘Pusa nanha,’ was grown in the experimental garden of Bose Institute, Kolkata, India. The tricolporate pollen grains were isolated (with less than 1% non-pollen contaminants) from the mature inflorescence of the experimental male plants. Fruits were collected from female plants at two different stages of edible form such as pre-ripening (PR) stage  and late ripening (LR) stage .The dioecious and edible Indian variety of papaya (n = 7) suffering from respiratory allergy (atopic asthma and rhinitis) were collected from the chest clinic of B. R. Singh Hospital and Centre for Medical Education and Research, Kolkata, India. Five of them were reported having mild to moderate allergic reactions to papaya fruits. These patients were diagnosed with an elevated level of specific IgE-antibody against the fruit and pollen extracts of papaya as determined by direct ELISA . For papaya fruit proteome, the lyophilized pulp, and peel were homogenized in liquid nitrogen and mixed with extraction buffer  for overnight. The supernatant was then mixed with equal volume of tris-phenol  and homogenized on a shaker for 30 min at 4°C. After centrifugation, the upper phenol phase was collected, and the protein was precipitated with 5 volumes of 0.1 M ammonium acetate in methanol. The protein was dissolved in urea buffer. The protein content was estimated by Bradford assay (Biorad), and the quality of extracted protein was checked in 12% SDS-PAGE.Proteins on PVDF membrane were blocked with 3% BSA and exposed to individual patient sera at 1:10 v/v dilution. The bound IgE-antibodies were detected with monoclonal anti-human IgE secondary antibody with AP conjugate produced in mouse  at 1:1000 v/v and NBT-BCIP (Abcam) substrate .TM (version 3.4) program as inputs into the MASCOT search engine version 2.4  for protein identification. Spectral baseline subtraction, smoothing, and centroiding were performed. The parameters for MASCOT search include database, NCBInr; taxonomy, Viridiplantae; enzyme, trypsin; universal modification, cysteine carbamidomethylation; variable modification, methionine oxidation; peptide charge state, ‘1+’; missed cleavage, 1; mass tolerance of precursor ions, 100 ppm, and mass tolerance of fragment ions, 0.7 Da. The confidence level of protein identification was based on significant probability score, and minimum 2 unique matched peptides. Protein identification was validated if the peptide ion scores were above the threshold of the significance level of p < 0.05, which indicated a 95% confidence level.The 56 kDa band corresponding to the IgE-reactive Cari p 1 band on immunoblot was excised from the SDS-PAGE of pollen protein and was trypsin digested as described in Escherichia coli DH5α strain using LB plates containing 100 μg ml-1 ampicillin. Positive transformants containing inserts were confirmed by double digestion. The accuracy of Cari p 1 reading frame was confirmed by Sanger sequencing .The total RNA was extracted from freshly harvested papaya pollen following the Trizol method (Invitrogen), and genomic DNA contamination was removed by DNAse-I digestion. The first strand cDNA was synthesized using iScript cDNA synthesis kit (Biorad). The full-length cDNA of Cari p 1 was PCR amplified using gene specific primers (shown in Supplementary Table E. coli BL21(DE3) , and the expression of recombinant Cari p 1 (rCari p 1) was induced with 0.5 mM IPTG for 16 h at 20°C. Cells were lysed by sonication in 25 mM Tris-Cl (pH 8.0), 7 M Urea, 300 mM NaCl, 10% glycerol and 10 mM imidazole. The hexahistidine-tagged rCari p 1 was purified by Ni-NTA resin (Qiagen) packed column under the denaturing condition as mentioned in manufacturers protocol. The denatured rCari p 1 was refolded into its soluble form by rapid dilution method (n = 7).The Cari p 1 cDNA clone was transformed into n method . BrieflyThe enzymatic activity of the refolded rCari p 1 was studied by a standard ruthenium red based endopolygalacturonase assay as described by 280 of the eluted fractions.About 1 mg of refolded rCari p 1 was injected into Superdex 200 increase 10/300 GL column connected to AKTA pure FPLC system (GE Lifesciences). The column was calibrated with standard protein mix. The chromatogram was recorded by measuring the A2PO4, pH 7.0. The CD spectra were recorded in a Jasco Corp. J-815 CD spectropolarimeter at 25°C within a wavelength range of 200 – 260 nm. The CD spectra were analyzed using CAPITO server  were transferred on PVDF membranes and then exposed to either polyclonal rabbit anti-Cari p 1 antisera or preimmune sera (as a negative control) at 1:100 v/v dilutions. Bound IgG antibodies were detected with anti-Rabbit IgG (whole molecule)–alkaline phosphatase conjugate produced in goat  at 1:30000 v/v dilutions and NBT-BCIP (Abcam) substrate. For reconfirmation at transcript level, the total RNA was extracted from the freshly harvested fruit tissues by SDS-phenol method  followedin vivo allergen challenge protocol described in The mouse model of papaya allergy was established following an p < 0.05) of the inflammatory changes in the histology of Cari p 1 challenged versus PBS treated mice was assessed by paired Students’ t-test using GraphPad Prism version 6.Statistical significance  commonly present in both pollen and fruit proteome. Hence, these two seroreactive components of papaya pollen and fruits were thought to elicit IgE-mediated hypersensitivity in the respiratory and gut mucosa of these patients respectively. In the present study, we concentrated on the 56 kDa IgE-reactive protein for further identification and characterization at the molecular level.In the present study, we selected a group of patients who have elevated level of serum IgE-antibody against papaya pollen as well as fruit. These patients reported to experience typical food allergic reactions to papaya fruits. However, the clinical investigations revealed the occurrence of recurrent respiratory symptoms in these patients even after giving up the consumption of papaya fruits. This observation led us to conclude the presence of common allergenic protein(s) in both pollen and fruits of papaya. We, therefore, performed a serological analysis in which the proteomes of papaya pollen and ripe fruit were separately confronted with two of these patient sera to detect the common IgE-reactive component(s). The immunoblot in Carica papaya . The yield of rCari p 1 was about 4 mg L-1 of culture. The denatured rCari p 1 was refolded into a soluble form followed by hexahistidine tag removal for downstream immunobiochemical studies. The immunoreactivity of rCari p 1 was checked by western blot with seven individual sera from papaya allergic subjects as shown in Figure 3B. The rCari p 1 displayed strong binding with serum IgE-antibodies of all the patients suggesting this protein as an important allergen of papaya. No reactive bands were observed in either control serum or buffer control blot.The complete amino acid sequence of Cari p 1 allergen was searched in NCBI database using the tBLASTn program, and the full-length cDNA of Cari p 1 (NCBI Acc. No. GQ479794) was retrieved as shown in Figure 4A. Under physiological condition (pH 7.2), rCari p 1 eluted from the column as a single fraction with an apparent molecular weight of 56 kDa as determined from the calibration curve of standard mix shown in Supplementary Figure Figure 3A). The ruthenium red based enzyme assay was performed with 0.16 μM of rCari p 1 to confirm whether the putative allergen retained the functional activity. Purified rCari p 1 showed substrate specificity toward PGA. Typical enzyme kinetics for increased concentration of PGA was observed and plotted according to Lineweaver–Burk shown in Figure 4B. The Vmax and Km of the substrate-saturated enzyme reaction were determined to be 561.7 μg ml-1 min-1 and 0.055% respectively.The aggregation status of rCari p 1 was checked by applying gel filtration chromatography as shown in Figure 4C) showed a properly folded protein with predominantly β-sheet characters as the minima were obtained at 215 nm. The denaturation pattern and refolding behavior of rCari p 1 were studied by generating a melting curve  of the protein with increasing temperature. It was observed that the denaturation took place at ∼62°C and the protein became unfolded entirely at 75°C. After cooling back the system to 25°C, a certain increase in the CD signal at λ215 was observed indicating a partial refolding of rCari p 1 from a fully denatured state .The CD spectra of rCari p 1 . The highest percentage of degranulation was observed at a concentration of 1000 ng/ml in which the histamine release took place within a range from 30 to 72%  among the four patients tested. Further increasing the allergen concentration  led to a sharp decrease in histamine release. No release was observed with control sera as shown in Supplementary Figure In addition to IgE-reactivity, the biological activity of rCari p 1, in terms of its allergenicity, was tested by degranulation assay. The rCari p 1 displayed an efficient basophil activation property leading to histamine release by degranulation. Due to unavailability of fresh uncoagulated blood, a passive sensitization protocol was followed. Firstly, the prebound IgE-antibodies were stripped off the FC𝜀R receptors on basophils of a healthy volunteer, and this was followed by addition of IgE-antibody present in the papaya sensitive patient sera. These IgE-sensitized effector cells upon stimulation with rCari p 1 resulted in a dose-dependent release of histamine  by immunoblot. As shown in Figure 6B, Cari p 1 was detected in the peel and pulp proteins of papaya fruits at two different harvesting stages, i.e., PR and LR stage. No reactive bands were detected in immunoblot with corresponding preimmune sera. In addition to protein level, the presence of Cari p 1 transcripts were also confirmed in the papaya fruits by gene-specific PCR as shown in Figure 6C.The polyclonal rCari p 1-specific rabbit antisera was used to screen for the presence of this allergen in fruit tissues at two different maturation stages  and mucus secretions  were observed in the lung and duodenum tissues of the mice after nasal and oral challenge respectively. The degree of lung inflammation was quantified in terms of eosinophil infiltration in the peribronchial spaces and mucus-secreting goblet cell proliferation inside the bronchial basement membrane. The degree of gut inflammation was quantified in terms of eosinophil infiltration in the lamina propria and mucus-secreting goblet cell proliferation inside the epithelial tissue of duodenum . The histopathological evidences revealed that rCari p 1 induced inflammatory changes in respiratory and gut mucosa were almost comparable  to the corresponding tissue sections of ovalbumin challenged mice. The results clearly demonstrate the dual role of Cari p 1 as a potent respiratory as well as food allergen.The IgE-mediated sensitization potential of rCari p 1 was studied The role of papaya plant in causing IgE-mediated hypersensitivity was studied by several workers from different parts of the world as described elsewhere . Howeverin vivo model, rCari p 1 displayed its effective role by eliciting inflammatory changes in respiratory as well as in gastrointestinal mucosa.Till now, the worldwide diagnosis of papaya allergy still relies on the use of outdated, less-characterized, and less-standardized crude extracts. In our study, rCari p 1 was found to retain its immunoreactivity as well as allergenic activity. Therefore, the recombinantly purified functional allergen can be considered as a suitable candidate for component-resolved diagnosis. The presence of conserved endopolygalacturonase like catalytic domain in Cari p 1 sequence was validated by enzyme assay for pectinolytic activity similar to an earlier reported plane tree allergen Pla a 2 . The bioTaken together, our present study reports Cari p 1 as the first major allergen of papaya. The dual role of Cari p 1 as ingestant and inhalant allergen makes it an ideal candidate for molecular diagnosis of papaya mediated allergic disorders. Further antigenic analyses of Cari p 1 for mapping its immunodominant epitopes may lead to the design of immunotherapeutic vaccines for effective disease management.This study was carried out in accordance with the recommendations of ‘ICMR guidelines for research involving Human Participants, Bose Institute Human Ethics Committee’ with written informed consent from all the subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the ‘Bose Institute Human Ethics Committee.’ This study was carried out in accordance with the recommendations of ‘The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines, Bose Institute Animal Ethics Committee’. The protocol was approved by the ‘Bose Institute Animal Ethics Committee’.GS and MS conceived the study and designed the experiments under the supervision of SB. MS, GS, NG, AD, KJ, and AD performed the experiments. MS and GS analyzed the data. GS, MS, and SB wrote the paper. All the authors have seen and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Microseira wollei (basionym Lyngbya wollei) was found at two of 15 sites in Butterfield Lake and analyzed for microcystins, anatoxins, cylindrospermopsins, and paralytic shellfish poisoning toxins (PSTs). Only PSTs and trace levels of anatoxin-a were detected in these samples. This is the first published report of PSTs within a New York State lake. To evaluate the environmental and temporal drivers leading to the observed toxicity, PST content at the two sites was examined in detail. There were distinct differences in the total PST content, filament nutrient, filament chlorophyll, and relationship to environmental drivers between the sites, as well as distinct differences in the total PST content measured using different analytical techniques. A multivariate model containing site, temperature, and filament chlorophyll explained 85% of the variation in PSTs observed over the growing season. This work emphasizes the importance of proper site selection and choice of analytical technique in the development of monitoring programs to protect lake users from the occurrence of benthic cyanobacteria toxins.Butterfield Lake is a mesotrophic lake in New York State where residents and pets have experienced unexplained health issues.  Paralytic shellfish poisoning toxins (PSTs) are a class of algal toxins which inhibit the sodium channel of higher organisms, including humans and marine mammals ,2,3. ProDolichospermum (basionym Anabaena) , Raphidiopsis (basionym Cylindrospermopsis) (Brazil), Microseira (basionym Lyngbya) (USA and Canada), Scytonema , and Cuspidothrix (basionym Aphanizomenon) (USA). PST production is highly variable within each clade as even closely related species do not always produce toxins [PSTs were originally thought to predominately occur in marine environments. This assumption has been challenged as reports of PSTs produced by freshwater cyanobacteria grow in number. Freshwater PST-producing cyanobacteria occur in Australia , Brazil e toxins .Microseira wollei (basionym Lyngbya wollei) [M. wollei collected from the Guntersville Reservoir, Alabama, USA [Benthic cyanobacteria produce a number of cyanotoxins including microcystins, anatoxins, cylindrospermopsins, and PSTs . Benthic wollei)  have beeama, USA . These nama, USA ,21, but ama, USA . Freshwaama, USA . BenthicButterfield Lake (44°19′10.4″ N 75°46′29.0″ W) is a mesotrophic lake in the foothills of the New York State Adirondack Mountains . The regSpirogyra leaving the source of this toxin uncertain. In 2016–2018, large plumes of Mougeotia were observed in the water column, likely associated with infusions of phosphorus from the hypolimnetic waters into surface waters during spring and fall turnover. Despite few reports of pelagic cyanobacteria toxins, there is a history of health issues associated with Butterfield Lake. In 2015, a CSLAP volunteer reported rashes on their arms after working on the lake [In 2015–2018, samples from Butterfield Lake were tested for microcystins, cylindrospermopsins, and anatoxin-a through the CSLAP program . One pelthe lake . In 2018the lake . In 2017the lake . The cauM. wollei in Butterfield Lake; however, the spatial extent of its distribution and its production of toxins was unknown. As pelagic cyanobacterial toxins did not appear to be associated with the reported health concerns, we investigated whether benthic cyanobacteria could be a contributing factor. Here, we describe the spatial, temporal, and between-site variation of M. wollei and its cyanotoxin production in Butterfield Lake, as well as a preliminary investigation into the environmental drivers associated with PSTs occurrence in M. wollei.We had previously observed M. wollei mats (M. wollei was found at the other 13 sites. One site (Channel) was located at a man-made channel inside of a wetland that extended approximately 300 m long and was 5 m wide. This channel was used to connect the lake to several homes and a private boat launch and marina. A second site (Dock) was located at the southern end of the lake by the public boat launch and fishing dock. M. wollei at the Dock site was patchier and growing amongst more aquatic vegetation when compared to the Channel site. M. wollei at the Channel site was found growing on or in the muddy substrate, whereas M. wollei at the Dock site was found on the muddy substrate, on aquatic vegetation, and in detached clumps floating on or near the shoreline.Benthic rake collections made at 15 locations in shallow waters along the western and southern portions of the lake identified two sites with large lei mats . No M. wSix samples, three from each site, were analyzed for PSTs by LC-MS/MS, ELISA, and HPLC with fluorescence detection (HPLC-FL). Higher concentrations of PSTs were found in Dock site samples relative to the Channel site samples using all three methods; however, the absolute concentrations as measured by the three analytical methods were markedly different . The relM. wollei from the Dock and Channel sites. Samples from both sites yielded partial 16S rRNA gene sequences  that were identical over the 678 bp alignment. These sequences showed greater than 99% identity to 16S rRNA gene of M. wollei originating from the Guntersville Reservoir, a known PST producer [M. wollei, the Butterfield Lake sequences formed a highly supported cluster with the strain from Guntersville Reservoir  if the sites without detectable PSTs were excluded from the regression. The relationship between total PSTs per area or filament chlorophyll per area to biomass/cm2 was less clear when the 54 m and 72 m samples were included in the regression (R2: 0.14–0.16). None of the samples tested positive for microcystins, anatoxins or cylindrospermopsins.Transects along the length of the Channel site demonstrated that hick mat . M. woll Channel A with an dry wt. C. These ocations C,D. FiveA comparison of the basic water quality parameters for the Dock and Channel sites are shown in M. wollei measurements included average filament chlorophyll, filament nitrogen, filament phosphorus, N:P ratio, total PSTs per g dry wt., and PSTs per chlorophyll. All were higher at the Dock site than at the Channel site for most of 2017  [M. wollei filaments, indicating variation in the degree of sediment contamination of the samples collected from the two sites was unlikely. of 2017 . Trace lectively . The N:Pctively) ,37, nor M. wollei at the Dock site had approximately 30% more filament chlorophyll per g dry wt. at the same concentration of filament phosphorus. There was a negative relationship between PSTs and filament phosphorus, and for the same concentrations of filament phosphorus, there were approximately 2.5 times more PSTs in M. wollei collected at the Dock site than at the Channel site. Temperature  regression models with either filament phosphorus, filament nitrogen or temperature as independent variables had complex relationships with site, filament chlorophyll and total PSTs . Filamenperature E had a sperature F, with t2, 0.02).The minimally adequate model needed to predict total PST concentrations consisted of three parameters listed in their order of importance: site, temperature, and filament chlorophyll . The modThe choice of analytical method for PST analysis is non-trivial. Four analytical methods for PST have replaced the original mouse bioassay (AOAC 959.08): HPLC with fluorescence detection after pre-column chemical oxidation (AOAC 2005.06)  or post-M. wollei samples from Butterfield Lake were measured by post-column HPLC-FL, LC-MS/MS, and ELISA. As expected, HPLC-FL, with its ability to respond to a broader range of PSTs, reported a higher concentration of total PSTs than the ELISA assay. Concentrations of PSTs measured by LC-MS/MS analysis were also lower than total PSTs as measured by HPLC-FL. This was expected, as additional toxins such as LTX-2 through LTX-6 were present in the HPLC-FL analysis that were not included in the LC-MS/MS analysis. Despite these quantitative differences, the methods were internally consistent. Higher levels of PSTs were observed at the Dock site relative to the Channel site with all three techniques.Six Because of the limited availability of freshwater PST standards and the abundance of the LTX analogs in our samples, we chose HPLC-FL with post-column oxidation as our primary analytical tool. One complication is that different analogs have different conversion efficiencies in forming fluorescent product(s). We adopted a single response factor based on STX, fully recognizing that some variants (GTX-2 and GTX-3) had a higher response factor, whereas other variants  had a lower response factor relative to STX. In this approach, the HPLC-FL method was operated as an assay , mimicking the ELISA which quantified total PSTs in µg STX eq. using a single congener (STX) for the standard curve. This allowed us to estimate PST concentrations where standards were not available.M. wollei in Northeastern USA and the first documented case of benthic production of PSTs within New York [M. wollei dominated in mats in Butterfield Lake and PSTs were present in most samples. M. wollei was the most likely source of the PSTs identified in Butterfield Lake based on the similarity of the 16S rRNA gene sequence of M. wollei from Butterfield Lake to the PST-producing M. wollei strain from Alabama, the similarity of the PST congener profiles between the Alabama M. wollei and the profiles observed in the two Butterfield Lake sites, and the dominance of M. wollei in the benthic mat from both sites. Genetic work to confirm the presence of the stx biosynthetic operon in the M. wollei from Butterfield Lake is currently in progress.Butterfield Lake represents the second documented case of PST production by New York . The conNew York . M. wollAnabaena and Phormidium species were associated with anatoxin-a production in wadeable streams in California and New Zealand [We tested for several other types of cyanobacteria toxins in Butterfield Lake. Only trace levels of anatoxin-a were detected in addition to the PSTs. There were no detectable levels microcystins, homo-anatoxin, cylindrospermopsins or deoxycylinderospermopsin in these samples. The source of anatoxin-a was uncertain. While  Zealand ,47,48, tM. wollei was more likely to occur at the Dock site than at the Channel site due to its proximity to people. However, even at the Dock site, most M. wollei was located far enough from the shoreline to limit potential human contact. Unlike pelagic cyanobacteria blooms, swimmers were unlikely to encounter the toxic benthic mats unless they detached from the bottom substrate. Additionally, the highest toxin concentrations occurred later in the season, coinciding with decreasing water temperatures. Lower temperatures would correspond with less recreation at the lake, further decreasing the risk of direct exposure to M. wollei containing high levels of toxin.We considered both intra- and extracellular toxin concentrations when evaluating the risk to humans from exposure to these toxins. Routes of exposure to PSTs in Butterfield Lake are comparable to their pelagic counterparts ,49, wherA second route of exposure is through consumption of drinking water contaminated by PSTs. Many lake residents pump water directly from the lake into their homes. The residents use different approaches to treat their water, with filtration being the most common but some using UV-disinfection or chlorination ,51. PSTsThe presence of benthic cyanobacteria mats in Butterfield Lake was very consistent at the two sites but offers a different challenge for monitoring when compared to a traditional pelagic bloom. The New York CSLAP program is well suited to monitoring pelagic algal blooms and their corresponding toxins; using a combination of temporal sampling over the growing season and spot sampling of visible blooms to track the temporal and spatial variation of the bloom. Due to its original purpose as a monitoring program for tracking long-term changes in water quality, CSLAP samples are usually collected from a limited number of open water sites in the center of the lake. A similar approach was used in the EPA National Lakes Assessment to evaluate cyanobacterial toxins . These gM. wollei filaments were generally nitrogen-limited, with N:P ratios below 7.2 for the majority of samples. Only three samples, all at the Dock, had N:P ratios above the Redfield ratio. Changes in filament nitrogen content were closely linked to changes in total PSTs and filament chlorophyll, while filament phosphorus content had no relationship with filament chlorophyll at either site. Benthic cyanobacteria grow in close proximity to the sediments and phosphorus content in Butterfield Lake sediments can be quite high . Thus Mwere low . Unlike were low , M. wollsediment ) and filtoxicity .M. wollei. Boyer et al. attributed this increase to a decreased growth rate that in turn led to an increase in toxin content per cell. A similar relationship may occur with M. wollei in Butterfield Lake.Despite this lack of phosphorus limitation, there was still a weak negative relationship between total PSTs and filament phosphorus. Boyer et al.  observedM. wollei with submerged aquatic vegetation at the Dock site, where aquatic vegetation was largely absent in the Channel.The relationships between PSTs, environmental parameters, and site were diverse. Still the choice of site remained the most important variable in explaining the PST concentrations in Butterfield Lake. The similarity in 16S rRNA segments between the Dock site and Channel site indicate that changes in toxin production were not due to changes in species, but that environmental and/or biological differences between the sites may be important. Although there were important relationships between nutrients and total PSTs, models to predict total PST content did not include the filament nutrient concentrations as predictors, only environmental variables in addition to filament chlorophyll. Filament chlorophyll, which may reflect filament nitrogen content, was a better predictor of total PSTs than the filament nitrogen content itself. Toxin concentration was primarily driven by site, temperature, and filament chlorophyll, with significant loss in explanatory power when each of these terms was removed. Calcium content, an important predictor of biomass and toxicity in culture , was notM. wollei PSTs were interesting in the context of freshwater pelagic blooms, where pelagic bloom size is closely related to phosphorus, while microcystins and anatoxin-a content is more closely related to nitrogen availability [Lyngbya  samples from two large fluvial lakes in the St. Lawrence River and examined the correlation of several environmental parameters to LTX-1. They identified depth, dissolved organic carbon, and the 1% light level as important contributors for toxin occurrence, while water flow was important in mat proliferation. Water flow did not change in Butterfield Lake, while the 1% light level was at or below the sediment water interface at both sites. Further studies would be needed to better define the role of light in this lacustrine system. Hudon et al. reported no significant differences in LTX-1 concentrations between their two fluvial lakes. This was very different from what we observed in Butterfield Lake where LTX-1 was absent and there was a large variation in the other lyngbyatoxins. This emphasizes the importance of a representative and balanced sample design that employs a broad-spectrum analytical technique and multiple sites when trying to assess the overall risk from benthic PSTs.The environmental drivers of lability ,61,62,63lability . The drilability ,65. Thislability  collecteM. wollei. The collections sites were 50–300 m apart on the western shore, and 300–1000 m apart on the eastern shore. At each of these 15 locations, 1–5 rakes were tossed in different directions from the boat, dragged across the bottom and handsorted back in the boat to find M. wollei. For temporal analysis, the rake was tossed three times into different portions of the M. wollei mat and a ~10 g wet weight portion of cyanobacteria from each toss was combined to form one sample. Samples were collected every two weeks between July and October. Plant material and detritus were handpicked out from the samples of M. wollei, and the sample was washed in the field to remove sediments. For spatial coverage, a petite ponar  was dropped vertically into the M. wollei mat and the resulting mud mixture/biomass mixture was placed into a bucket. Variation due to sampling technique was evaluated by collecting three replicates in close proximity. Cyanobacteria mat material was picked out by hand, washed using lake water and returned to the lab. In the lab, the samples were cleaned again to further remove sediment. Both rake and ponar samples were placed on ice until their return to the lab. Once in the lab, the samples were weighed, immediately frozen at −80 °C, and freeze-dried. The freeze-dried samples were homogenized in a mortar and pestle using liquid nitrogen, and the powder stored at −20 °C until further analysis. Measurements of water column chlorophyll, phycocyanin, temperature, pH, and conductivity were collected with a Hydrolab DS 5X Sonde,  at each collection site.Ten locations along the southwestern shoreline and five locations on the eastern shoreline of the lake were investigated for the presence of M. wollei powder. Material was fragmented by sonification (3 × 20 s at 32 watts), and a ~20 mL aliquot sonicated further (12 × 20 s at 32 watts) to lyse the filament prior to analysis. Lake water was collected and maintained in the field at 4 °C for analysis of total phosphorus and total nitrogen, or sterile filtered (0.22 µm) in the field and maintained at −20 °C for later analysis of total dissolved nitrogen. Total phosphorus (EPA method 365.1) and total nitrogen (EPA method 353.2) were measured using these samples using a SEAL autoanalyzer model AA3 .Filament total nitrogen and phosphorus were measured in duplicate on ~30 mg of portions of lyophilized M. wollei powder was heated to 75 °C overnight in 10 mL concentrated nitric acid. Samples were then centrifuged at 10,000× g for 5 min, diluted to a final concentration of 7.5% nitric acid, and infused into the ICP-OES (Perkin Elmer Optima 3300DV) for measurement of the common soil elements: iron, aluminum, calcium, magnesium, and sulfur.Chlorophyll-a was determined by extraction of duplicate lyophilized powders using a modification of EPA method 445.0 and analyzed using a Turner Design TD-700 fluorimeter . Total carbon was determined on ~2 mg of sample with a Thermo Scientific FlashEA 1112 elemental analyzer  interfaced with a thermal conductivity detector. Filament-associated metal content was determined by ICP-OES . Approximately 50 mg of w/v) sucrose for 2 h on ice. Lysozyme (25 µL of 100 mg mL−1) was added and incubated at 37 °C for 20 min. Proteinase K (50 µL of 1 mg mL−1) and 50 µL of 10% SDS (w/v) were added and incubated at 50 °C for 2 h. DNA was collected with three extractions using phenol/chloroform/isoamyl alcohol  followed by two extractions of chloroform/isoamyl alcohol . DNA was precipitated with sodium acetate (0.3 M) and 100% ethanol, and then washed with 70% ethanol using standard methods [Genomic DNA was extracted from freeze-dried environmental samples using a method modified from Kurmayer et al. . Ten mil methods . DNA was methods . PCR rea methods . Sequencv/v). Ten milliliters was added to ~700 mg of cyanobacterial powder prior to probe sonication (3 × 20 s at 32 watts) on ice. The slurry was centrifuged at 15,000× g for 10 min, passed through a 0.45 µm nylon syringe filter, and was the same sample used for HPLC-FL, ELISA, LC-MS, and LC-MS/MS analyses. SPATT bags were constructed as described in Lane et al. [M. wollei samples and whole water samples, respectively. Method LODs for anatoxin-a and homo-anatoxin were 0.005 µg/g and 0.02 µg/L for M. wollei and whole water samples, respectively. Cylindrospermopsin and deoxycylinderospermopsin LODs were 0.05 µg/g and 0.1 µg/L, respectively. PST LODs were 0.15 µg/g and 0.10 µg/L, respectively. Primary PST standards were purchased from the NRC Canada  and United States Food and Drug Administration (FDA) . FDA STX was diluted 1:50 to a concentration of 4 µM prior to use. STX standard response converted into an equivalent M. wollei concentration was linear between 1.3 and 184.5 µg/g using five points. NRC standards of STX (66.3 µM), dcSTX (2.6 µM), GTX-1 (2.3 µM), GTX-2 (4.1 µM), GTX-3 (1.7 µM), GTX-4 (0.7 µM), GTX-5 (2.3 µM), dcGTX-2 (4.0 µM), dcGTX-3 (1.2 µM), LTX-1 (17 µM), and C1+C2 (5.9 µM) were used to calculate relative response factors. Uncalibrated neosaxitoxin was used as a retention time standard. Response factors for other variants of PSTs relative to STX ranged from 0.1 to 4.6 with an average of 1.44. All PSTs, both known and unknown, were quantified using an STX standard curve with single injections of STX standards to verify the stability of the response factor. For comparison of analytical methods, three samples were chosen from the Dock site and three samples were chosen from the Channel site, with three samples from the beginning, middle, and end of the sampling period each. PSTs were analyzed by LC-MS/MS per the method described in Armstrong et al. [Microcystins, cylindrospermopsins, anatoxins, and PSTs were extracted 50% methanol containing 1% acetic acid . Durbin–Watson tests assessed autocorrelation from temporal pseudoreplication in parameters used in the ANCOVA and multivariate models, with significant autocorrelation found only in total PSTs collected from the Channel (p < 0.05). Small deviations from normality were observed in the residuals for some tests, but were not deemed sufficient enough to warrant transformation. Transformation did not change the significance or interpretation of the tests or results, with the loss of information in the magnitude of differences deemed excessively detrimental. ANCOVA maximal models started with predictor, site, and predictor site interaction, and were reduced to a minimum explanatory model using F tests (p < 0.05). The explanation of the variation in PSTs from both sites was performed by a multivariate linear regression containing biological and environmental variables collected from the M. wollei and the surrounding water. The full model started with the following predictors: filament chlorophyll, filament nitrogen and phosphorus, temperature, site, temperature/site interaction, and CSLAP water column total phosphorus and total nitrogen, and was simplified to be minimally adequate using manual and automated forwards and backwards stepwise addition or removal of parameters. Model simplification was done by a combination of F tests and AIC . Coefficients for the final model were intercept (25.71), temperature (−1.53), site (149.92), chlorophyll (0.015), and interaction (−5.66). Final model statistics . Filament chlorophyll and filament nitrogen were covariate so only one term was kept in the reduced model. Non-nested models containing either filament chlorophyll or filament nitrogen were compared using Akaike information criterion (AIC) and predicted residual error sum of squares (PRESS) [Statistical analyses were performed in R version 3.5.1  with base package tools. Multiple comparisons for paired  (PRESS) . Relativ