This channel comprises two independent pores and closely apposed Ca2+ binding sites that are contained within each subunit of a homodimeric protein. Previously we characterized the influence of positively charged pore-lining residues on anion conduction (2+ concentration due to the alleviation of energy barriers for anion conduction. This phenomenon originates from Coulombic interactions between the bound Ca2+ and permeating anions and thus demonstrates that an electrostatic gate imposed by the vacant binding site present in the sterically open pore, is released by Ca2+ binding to enable an otherwise sub-conductive pore to conduct with full capacity.TMEM16A is a ligand-gated anion channel that is activated by intracellular Canduction . Here, w Whereas single mutations in the wide vestibules do not exert a recognizable effect on conduction, mutations at the border of the neck region result in a pronounced rectification of currents whose shape depends on the location of the altered residues  that saturates at an intermediate value, suggesting that the binding site is likely occupied by a single Mg2+ ion  to add a further positive charge to the binding site. Similar to PQ, this mutant likely binds only one calcium ion as the Hill coefficient is close to unity and the I-V relation retains its strong rectification even at saturating Catrations . Howeverd for PQ . Next, wd for PQ , reducesr PQ  and PR (PR5Q) backgrounds. At maximum Cackground , which eckground . The samee state , further2+ and permeating anions, we investigated the effect of the binding site mutations and the altered Ca2+ occupancy on the electrostatic profile of the pore using Poisson-Boltzmann calculations on the Ca2+-bound mouse TMEM16A structure  as the template. Double and triple mutants were generated sequentially. All constructs were verified by sequencing.HEK293T cells (ATCC CRL-1573) were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10 U/ml penicillin, 0.1 mg/ml streptomycin (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), and 10% FBS (Sigma-Aldrich) in a humidified atmosphere containing 5% COe method  using thRecordings were performed on inside-out patches excised from cells expressing the construct of interest. Recording pipettes were pulled from borosilicate glass capillaries  and were fire-polished with a microforge (Narishige) before use. Pipette resistance was typically 3–8 MΩ when standard recording solutions were used. Seal resistance was typically 4 GΩ or higher. Voltage-clamp recordings were performed using Axopatch 200B and Digidata 1550 (Molecular devices). Analogue signals were filtered through the in-built 4-pole Bessel filter at 5 kHz and were digitized at 10–20 kHz. Data acquisition was performed using Clampex 10.6 (Molecular devices). Solution exchange was achieved using a double-barreled theta glass pipette mounted on an ultra-high speed piezo-driven stepper (Siskiyou). Liquid junction potential was found to be consistently negligible given the ionic composition of the solutions and was therefore not corrected. All experiments were performed at 20 °C.2+-EGTA contained 150 mM NaCl, 5.99 mM Ca(OH)2, 5 mM EGTA and 10 mM HEPES at pH 7.40. Stock solution with EGTA contained 150 mM NaCl, 5 mM EGTA and 10 mM HEPES at pH 7.40. Free Ca2+ concentrations were adjusted by mixing the stock solutions at the required ratios, which were calculated using the WEBMAXC program (http://web.stanford.edu/~cpatton/webmaxcS.htm). Recording pipettes were filled with stock solution with Ca2+-EGTA, which has a free Ca2+ concentration of 1 mM.Recordings were performed under symmetrical ionic conditions. Stock solution with Ca2+ experiments, stock solution with Mg2+-EGTA contained 75 mM MgCl2, 23 mM (NMDG)2SO4, 5 mM EGTA and 10 mM HEPES at pH 7.40. Free Mg2+ concentrations were adjusted by mixing the stock solutions with Mg2+-EGTA and EGTA at the required ratios. For Gd3+ experiments, the final Gd3+ concentrations were reconstituted from a 1 M GdCl3 stock in a solution containing 150 mM NaCl, 10 mM HEPES at pH 7.40 with no added Ca2+ and metal chelators. In all cases, solutions were prepared in ultrapure molecular biology grade water having a resistivity of 15–18 MΩ cm at 25 °C (ELGA or Millipore).For Mg+120). When used judiciously, this procedure allows one to recover the true I-V relation and to correct for current fluctuation. Before and after each recording at the Ca2+ concentration of interest, we also recorded in zero Ca2+ to ensure that the quality of the patch did not deteriorate over time. Background subtraction was performed for constructs that do not display visible basal activity but was not possible for the mutants that do. In all cases, leaky patches were discarded.Concentration-response relations were constructed from steady-state current responses recorded using a rundown-correction protocol as described previously . For ins2+ and Gd3+. In contrast to the divalent cations Ca2+ and Mg2+, Gd3+ binding appears to be biphasic consisting of a reversible phase and an irreversible component that both affect the I-V relation of the instantaneous current . In order to titrate the second reversible site, we first incubated the patch with a submaximal concentration of Gd3+ (0.4 mM) to saturate the irreversible binding site. We then recorded at the test concentration of Gd3+ to obtain the corresponding instantaneous I-V relations I=zFA-100/+120), which allows a straightforward comparison with the I-V data.In this model, current rectification is governed by the two parameters mismatch . We haveequals 3 , which w2+ dependence of 2+ concentrations  was fitted to the Coulombic potential2+-bound state.The relationship between 2+ binding as a function of valence of the intracellular pore entrance,2+ binding when only affinity is affected, 2+. 2+-bound state. An analogous expression was used to account for the changes in the energy of Ca2+ occupancy to the ensemble current, the I-V data for WT were fitted to a weighted sum of I-V curves corresponding to zero, one and two calcium ions bound at the binding site,2+, G644P/E654Q at saturating Ca2+ and G644P at saturating Ca2+ respectively. Since WT does not display noticeable basal activity, the contribution of current corresponding to zero occupancy was considered negligible and the To estimate the contribution of open states with various Cahttp://www.numpy.org/). Data are presented as mean ± s.e.m. Fitted parameters are plotted and are reported as best-fit value ± 95% confidence interval.Data analysis was performed using Clampfit 10.6 (Molecular devices), Excel (Microsoft), and Prism 5 and 6 (GraphPad). Numerical calculations were performed using the NumPy package  and Alessio Accardi (Reviewer #2).Thank you for submitting your article "Ligand-dependent electrostatic control of anion access to the pore of the calcium-activated chloride channel TMEM16A" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:2+-binding sites act as an electrostatic barrier for the flow of anions once the pore has adopted the open conformation, thus acting as an additional activation gate in the absence of Ca2+. By introducing mutations in the Ca2+-sites in constitutively active mutants of TMEM16A and measuring their I-V relations at various Ca2+concentrations, the authors show that the binding of calcium reduces the two innermost energy barriers of an open channel through an electrostatic mechanism, allowing ions to flow. In the absence of Ca2+, the constitutively active mutants display pronounced outward rectification due to this electrostatic effect. Overall, these studies highlight a novel mechanism of regulation of ion permeation by a ligand. Nevertheless, the reviewers raise several concerns that should be addressed in the revised version.Lam and Dutzler discover that the negatively charged residues that form the CaEssential revisions:2+ affect only inward and not outward current? The energy profile in Figure 1D could not lead to rectification, because the height of each energy barrier is the same whether the ion is coming from the inside or the outside. Outward rectification requires an energy well between the barriers h and β so that the height of β is greater from the out>in than the in>out direction. It would seem that the depth of this well would be significant considering the degree of rectification observed.1) I am not sure that I understand the permeation model. Why do the electrostatics of Cahσ and βσ are voltage-independent, but Figure 1G seems to show that βσ is voltage-dependent. The statement in the next paragraph is rather inscrutable, as is the subsequent explanation about why the "decay" was omitted from the fits. What does "inaccurate estimation of βσ" mean? It would be useful to know how the conclusions are affected by changing n in Equation 2. And why is hσ not shown beyond Figure 1?2) Furthermore, the second paragraph of the subsection “Data analysis” states that 2+-binding on the rectification of the mutant channels could arise from these conformational changes rather than the simple electrostatic influence of the site and the bound Ca2+ ions. To test for this possibility, the authors could use Mg2+ instead of Ca2+, as it is reported to compete with Ca2+ without activating the channel. The authors should test whether the presence of internal magnesium in the context of either of the constitutively active mutants has the same effect as Ca2+, as expected from their proposed mechanism. Although it is not necessary, I think it would be also interesting to test whether a trivalent cation could also bind and have a larger effect than Ca2+.3) Even though the G644P and Q649A mutations increase the baseline open probability of the channel, it is unclear whether the protein with any of these mutations still undergoes some gating-associated conformational changes. If this were the case, some of the effects of Ca4) Large portions of the data shown depend on the use of a permeation model for analysis, together with the use of several equations for permeation and electrostatics. I think these play such a central role in the manuscript that it would make the reading easier if some of these models and equations were described with more detail in the main text, rather than the Materials and methods section. Essential revisions:2+ affect only inward and not outward current? The energy profile in Figure 1D could not lead to rectification, because the height of each energy barrier is the same whether the ion is coming from the inside or the outside. Outward rectification requires an energy well between the barriers h and β so that the height of β is greater from the out>in than the in>out direction. It would seem that the depth of this well would be significant considering the degree of rectification observed.1) I am not sure that I understand the permeation model. Why do the electrostatics of CaeLife, to which this work refers to . In this model, ions surmount energy barriers in a pore that does not contain deep energy wells and that cannot be saturated. We think that such model is appropriate for our analysis since we have not found evidence for tight ion binding sites in our structures and we have measured a KM of chloride conduction between 300-400 mM, thus underlining that there is no saturation in the applied conditions . The energy profile displayed in Figure 1D or Figure 1—figure supplement 1 represents the potential of mean force for permeation in the absence of an applied electric field, where, in symmetric ion concentrations, the system is at equilibrium and there is no net flux in either direction. Upon application of a potential difference, the model predicts ionic currents. In such case, rectification is a consequence of the asymmetric free energy profile. The location of the barrier maximum governs the shape of the I-V relation, which becomes increasingly outwardly rectifying when the barrier maximum moves closer to the inner end. We note that the I-V relation is a function of both occupancy and rate of barrier crossing. Unlike the pseudo-ohmic cases, in case of low symmetric barriers, where the total occupancy remains approximately constant, ion depletion occurs in cases of an increased intracellular barrier at negative voltages where the channel is less conductive while ion accumulation occurs at positive voltages . We have explained this model in detail in our previous manuscript published in voltages . Both bevoltages .h and σβ are voltage-independent, but Figure 1G seems to show that σβ is voltage-dependent.2) Furthermore, the second paragraph of the subsection “Data analysis” states that σ and  are both not intrinsically voltage-dependent as they denote the ratio of the rate constants in the case of a linear voltage drop . In Figure 1G they are only voltage-dependent because they change as a function of calcium binding and calcium binding itself is voltage-dependent. We have added the following sentences to our manuscript:2+ binding whose EC50 exhibits voltage dependence.”“β" mean? It would be useful to know how the conclusions are affected by changing n in Equation 2.The statement in the next paragraph is rather inscrutable, as is the subsequent explanation about why the "decay" was omitted from the fits. What does "inaccurate estimation of σ2+ not intrinsic to its high affinity binding, as we have observed in a previous study . As we are interested in the effect of calcium binding to the high affinity transmembrane site, the low affinity phase becomes irrelevant and was therefore omitted from analysis. Since the same treatment was applied consistently, this does not affect the outcome of the analysis. The number of barrier We note that the decay phase in general does not follow the same trend as the high affinity phase and occurs an order of magnitude above the concentration range where activation takes place . The decWe have added the following sentences:“We have previously shown that the model describes the behavior of WT best when the number of barriers 2+ concentrations . Since this decay might be related to processes not intrinsic to the transmembrane binding site , only the high affinity phase was analyzed.”“We observed for mutants G644P/E654Q, G644P/E654R, G644P/E654Q/E705Q and G644P/E654R/E705Q a low affinity decay of h not shown beyond Figure 1?And why is σ for most of our graphs since it was determined with higher precision than . As shown in Figure 1E, the 95% confidence interval of  is much wider than that of , indicating that the best-fit value of  is less well defined. In addition, the value of  increases monotonically  with the rectification index (I-100/+120) (We have selected 00/+120) , which a2+-binding on the rectification of the mutant channels could arise from these conformational changes rather than the simple electrostatic influence of the site and the bound Ca2+ ions. To test for this possibility, the authors could use Mg2+ instead of Ca2+, as it is reported to compete with Ca2+ without activating the channel. The authors should test whether the presence of internal magnesium in the context of either of the constitutively active mutants has the same effect as Ca2+, as expected from their proposed mechanism. Although it is not necessary, I think it would be also interesting to test whether a trivalent cation could also bind and have a larger effect than Ca2+.3) Even though the G644P and Q649A mutations increase the baseline open probability of the channel, it is unclear whether the protein with any of these mutations still undergoes some gating-associated conformational changes. If this were the case, some of the effects of CaWe agree that gating-associated conformational changes may still exist. However, since for our analysis we characterize instantaneous currents following a voltage jump prior to any conformational changes, the I-V relations correspond to the open populations of the channel.2+ binding on the conductance. For that purpose, we have recorded data at varying Mg2+ concentration in the absence of Ca2+. Consistent with previous observations by Chen and colleagues , we found that Mg2+ binds with an apparent affinity of around 1-2 mM. Although Mg2+ addition does not lead to activation of WT, its binding to the mutant G644P lowers the energy barrier for anion conduction at the intracellular part of the pore, consistent with the presence of a single Mg2+ ion in the site (as judged by the remaining rectification at high Mg2+ concentration). This result thus further confirms our proposed mechanism on the influence of divalent cations in the transmembrane binding site on the energetics of anion conduction in the open channel. In another set of experiments, we have also investigated the effect of the trivalent metal ion Gd3+ and found that the application of this ion at high µM concentrations exerts an even stronger effect than Ca2+, leading to slight inward rectification of currents. This behavior is consistent with a larger increase in the positive charge density after the binding of two Gd3+ ions. However, in this case the effect was not completely reversible upon washout indicating an unmeasurably slow off-rate for one of the bound ions. It should also be noted that in the case of Gd3+, we could not buffer residual Ca2+ in the solutions by addition of EGTA and we can thus not exclude the presence of traces of Ca2+ in the solutions. In light of our new data, we have added another subsection to our manuscript “The effect of the competitive antagonist Mg2+, and the trivalent cation Gd3+ on conduction”) and an additional accompanying figure .In our revised version, we have now investigated the effect of Mg4) Large portions of the data shown depend on the use of a permeation model for analysis, together with the use of several equations for permeation and electrostatics. I think these play such a central role in the manuscript that it would make the reading easier if some of these models and equations were described with more detail in the main text, rather than the Materials and methods section.We have added two sentences to the Results where we discuss general features of the model and extended its description in the methods. However, we have already explained the same model in great detail in our preceding study, which the current manuscript is referring to, and think that the link to this previous study would help interested readers to gain in-depth insight. We also think that a more extended description of the model in the main part of the manuscript might distract readers from the main findings of the work.The following sentences were added to the Results section:M for chloride conduction.”“The diffusion path does not contain deep energy wells and the model does not account for saturation of the pore, which is generally consistent with the high K“The rectification is a consequence of both pore occupancy and the rate of barrier crossing at the applied potential.”


Over 70 million women of reproductive age are undernourished in India. Most poverty alleviation programs have not been systematically evaluated to assess impact on women’s empowerment and nutrition outcomes. National Rural Livelihoods Mission’s poverty alleviation and livelihoods generation initiative is an opportune platform to layer women’s nutrition interventions being tapped by project Swabhimaan in three eastern Indian states—Bihar, Chhattisgarh and Odisha. A cross-sectional baseline survey covering 8755 mothers of children under-two years of age, one of the three primary target groups of program are presented. Standardized questionnaire was administered and anthropometric measurements were undertaken from October 2016 to January 2017. 21 indicators on women’s empowerment, Body Mass Index and Mid-upper Arm Circumference for nutrition status, food insecurity indicators as per the Food Insecurity Experience Scale and selected indicators for assessing women’s access to basic health services were included. National Rural Livelihoods Mission operates in contexts with stark social and gender inequalities. Self-help group members exhibited better control on financial resources and participation in community activities than non-members. Using Body Mass Index, at least 45% mothers were undernourished irrespective of their enrolment in self-help groups. Higher proportion of self-help group members (77%-87%) belonged to food insecure households than non-members (66%-83%). Proportion of mothers reporting receipt of various components of antenatal care service package varied from over 90% for tetanus toxoid vaccination to less than 10% for height measurement. Current use of family planning methods was excruciatingly low (8.2%-32.4%) in all states but positively skewed towards self-help group members. Participation in monthly fixed day health camps was a concern in Bihar. Layering women’s nutrition interventions as stipulated under Swabhimaan may yield better results for women’s empowerment and nutrition status under National Rural Livelihoods Mission. While this opportunity exists in all three states, Bihar with a higher proportion of matured self-help groups offers more readiness for Swabhimaan implementation. India had an estimated 71 million undernourished women of reproductive age and an estimated 30 million pregnancies . MajoritSwabhimaan is completely embedded in the government funded NRLM, using its administrative structures and thrift and credit processes. Launched in 2011, NRLM aims to enroll at least one woman from each household in the poorest administrative regions of the country under SHGs; it has reached over 50 million households nation-wide since inception , 9. Swab2, a 15% reduction in the proportion of mothers of children under two with a BMI <18.5 kg/m2, and a 0.4 cm improvement in mean Mid-Upper Arm Circumference (MUAC) among pregnant women and 2) between 5% and 20% increase in coverage of 18 key nutrition specific and sensitive interventions  for mothers of children under-two consisting of 128 questions and probes was developed, field tested and standardized for all states. Household survey questionnaire and interview schedule used with mothers of children under two are shared as supporting information . Data collection was completed between October 2016 and January 2017 across three states. Complete data set is available as supporting information  and a metre rod (100 cm). The mean standard errors of measurement for height, weight, and MUAC across all the data collection teams were insignificant and ranged between 0.001‒0.025  and analyzed using STATA 15.1. Univariate analysis was used to profile mothers of child under-two by different socio-economic and demographic characteristics. Bivariate analysis was used to show the distribution of SHG and non-SHG member mothers by different indicators of women empowerment, nutrition status, access to maternal health services and household food insecurity. The level of association of women’s empowerment, nutrition status, access to maternal health services and household food insecurity variables with participation of women in SHG was established through Chi square test of independence.The baseline survey protocol, methodology and tools were approved by the Institutional Ethics Committee of All India Institute of Medical Sciences (AIIMS) in the respective states of Bihar, Chhattisgarh and Odisha. The study has been registered with the Registry for International Development Impact Evaluations (RIDIE-STUDY-ID-58261b2f46876) and Indian Council of Medical Research (ICMR) National Clinical Trials Registry of India (CTRI/2016/11/007482). A consent form detailing the purpose of the study, agency and investigator involved and reasons for anthropometric measurements was developed as part of the study protocol approved by the Ethics Committee. Verbal consent was taken from all interviewees before conducting the interview and taking anthropometric measurements as most respondents were unable to write or sign. Investigators proceeded with the interview only when consent was given by the interviewee.In all states, majority of mothers of children under-two were in the 20–29 years age group. Odisha had the highest propotion of adolescent mothers (8.9%) among the three states. The proportion of mothers married before legal age was high in both Chhattisgarh and Odisha at 26.4% and 35.7%, respectively. Bihar had the highest proportion of mothers with parity of three or more (52.6%) compared with 38.9% in Chhattisgarh and 33.1% in Odisha. Of every 10 mothers, six in Bihar, five in Odisha and four in Chhattisgarh had never attended school. Mothers mostly belonged to Hindu households in Chhattisgarh and Odisha, and Muslim in Bihar. Over half of the sampled mothers belonged to Scheduled Tribes (ST) in Chhattisgarh and Odisha and Other Backward Castes (OBC) in Bihar. The penetration of PDS was lowest in Bihar with nearly half the mothers not having a ration card. Chhattisgarh had the highest proportion of Antyodaya card holders (17%) or those certified by the goverenment as poorest of the poor households. Nearly one in three mothers were members of SHGs in all states; VO membership was strongest in Bihar with nearly half the SHG members participating in VOs .Over 70% of mothers irrespective of SHG membership status were involved in household decision making in Odisha. Participation was near or over 70% in Chhattisgarh too, but lower in Bihar, particularly for decision making on social visits to family members and relatives (50% among SHG members and 44% among non-SHG members). When decision making was compared between SHG members and non-SHG members, in Bihar SHG members had greater involvement in decision making on major purchases, their own health care as well as planning social visits. In Chhattisgarh and Odisha, a higher proportion of SHG members could decide on use of money earned by husband than non-SHG members; however, there was no difference in decision making related to major purchases for the house between members and non-SHG members. In Chhattisgarh, like in Bihar, higher proportion of SHG members decided on visits to family members and relatives than non-members .Mothers did well on autonomy with nearly all being able to visit a health facility or going outside the house either alone or with someone in both Chhattisgarh and Odisha. In Odisha, a higher proportion of SHG members were able to undertake these tasks than non-SHG members. In both Chhattisgarh and Odisha, SHG members reported being able to go to market alone or with someone more frequently than non-SHG members.Endorsement for domestic violence was higher among mothers in Odisha than Chhattisgarh. Among the seven situations presented, in Odisha, mothers endorsed domestic violence most commonly for disrespecting in-laws (58% and 60%), followed by neglecting house or children (51% and 52%), arguing with husband (48% and 51%) and leaving house without informing husband (46% and 50%). They were least likely to endorse it for refusal of sex which was still relatively high at 29%. The situations endorsed by mothers in Chhattisgarh were similar but proportion mothers endorsing domestic violence were much lower and less than 20% across situations. Further, the endorsement for domestic violence lowered among mothers who were SHG members for four of the seven situations in Chhattisgarh, unlike one of seven situations in Odisha .In Chhattisgarh and Odisha, half the mothers had own income which they could independently spend and higher proportion of SHG members did so opposed to non-SHG members. Higher proportion of SHG members had a savings bank account than non-SHG members, 62% among SHG members opposed to 48% among non-SHG members in Chhattisgarh and 86% among SHG members opposed to 76% among non-SHG members in Odisha .More women in Chhattisgarh than Odisha were engaged in economically productive work in the 12 months preceding the survey. The proportion engaged in such work was similar among SHG members and non-SHG members in Chhattisgarh . However, higher proportion of SHG members were working in Odisha than non-SHG members (31% opposed to 25%) .A higher proportion of mothers organized or participated in community mobilization activities in Chhattisgarh than in Odisha. In both states, higher proportion of SHG members were involved in such activities than non-SHG members; 56% in Chhattisgarh and 28% in Odisha .Ownership of resources that is mobile phone, house and land was overall higher in Odisha than Chhattisgarh but comparable between SHG members and non-SHG members. The exception was Chhattisgarh where higher proportion of SHG members owned land than non-SHG members (38% opposed to 43%). Among investigated resources, ownership of mobile phone was lowest with about 12% and just over 20% owning mobile phones in Chhattisgarh and Odisha, respectively .2 (55% SHG members and 54% non-SHG members). Prevalence of thinness was also high in Bihar and Chhattisgarh with 45% and 55% mothers having BMI < 18.5 kg/m2. Using MUAC, prevalence of thinness was highest in Bihar (57% among SHG members and 61% among non-SHG members). Severe thinness too was considerably higher in Bihar compared with other two states (9% among SHG members and 8% among non-SHG members)  .Odisha had the least proportion of food secure households (13% SHG members households and 17% non-SHG members households) followed by Bihar and Chhattisgarh. Members of SHG more frequently belonged to food insecure households across all states. Among food insecure households, over 70% were moderate to severely insecure in Odisha, over 60% in Bihar and about 40% in Chhattisgarh. While state variations were observed, there was no difference between levels of food insecurity with respect to SHG membership status .Almost all pregnancies were registered in Chhattisgarh and Odisha, but over a quarter of mothers were not brought into the health system during their pregnancy in Bihar. Higher proportion of SHG members were registered than non-SHG members in all states. Less than a quarter of the mothers were registered in the first trimester in Odisha, about a third in Chhattisgarh and Bihar. SHG membership had no influence on the chance of receiving ANC in the first trimester. Weight measurement was done for over 85% of the mothers in Chhattisgarh and Odisha but less frequently in Bihar, with 75% SHG members and 70% non-SHG members reporting weight measurement. Less than one in five mothers underwent height measurement in Chhattisgarh and Odisha; it was rarely measured in Bihar (<3%). Both Blood Pressure (BP) measurement and hemoglobin estimation was reported by at least 80% of the mothers in both Chhattisgarh and Odisha. In Bihar, coverage of these services was lower with a little over 40% mothers reporting BP measurement and hemoglobin estimation. Not much difference was observed in reach of these services with respect to SHG membership status. Tetanus Toxoid (TT) vaccination was almost universal with SHG members covered better than non-SHG members in Bihar and Chhattisgarh. Receipt of IFA varied much across states being lowest in Bihar (53% non-SHG members and 61% SHG members), followed by Chhattisgarh (79% non-SHG members and 86% SHG members) and highest in Odisha (91% non-SHG members and 92% SHG members).Use of health facilities for delivery was lowest in Chhattisgarh (65% non-SHG members and 62% SHG members). It was relatively higher in Bihar and higher proportion of SHG members used health facilities (85%) than non-SHG members (75%). Use of modern family planning methods was excruciatingly low (10%) in Chhattisgarh and Bihar; in Bihar higher proportion of SHG members were using modern family methods (13%) compared with non-SHG members (8%).Village Health Sanitation and Nutrition Days (VHSNDs) were attended by 60% or more mothers in Chhattisgarh and Bihar with higher proportion of SHG members participating in both states. Participation was extremely poor in Bihar with less than 10% of mothers attending VHSNDs and no difference with respect to SHG membership .We found that at least one in four mothers were members of SHGs in Bihar, Chhattisgarh and Odisha; there may be more who came from households that had an SHG member as NRLM aims to have at least one woman member from each family linked to the SHGs. Our survey did not capture the latter. However, the context in which Swabhimaan operates is marked by social and gender inequalities. In all Swabhimaan intervention states, that is Bihar, Chhattisgarh and Odisha, sampled population belonged to government enlisted vulnerable communities of SC, ST or OBC. Over a third of mothers in Odisha were married before legal age and nearly one in ten were adolescent mothers. Schooling rates were low for mothers in all three states, one of the known drivers for early marriages and childbearing .In our survey, 11 of the 21 indicators for women’s empowerment in Chhattisgarh and 9 of the 21 in Odisha were skewed towards mothers who were SHG members while no difference was reported in the others. Estimates were available for only three of the 21 indicators in Bihar, all related to decision making and positively skewed towards SHG members. Indicators which consistently demonstrated better women’s empowerment status among SHG members in both Chhattisgarh and Odisha were: 1) Having a bank account, 2) Having own money and control of this money and 3) Organizing or participating in community level programs as a proxy for community leadership. Among women’s agency indicators a relatively high proportion of mothers reported autonomy as well decision making authority, however, acceptance of domestic violence was quite high especially in Odisha and it did not vary much with SHG membership. In our earlier work we had noted that guarding women from domestic violence is critical for successful implementation of women’s centered livelihood programs . The JEEWe observed high prevalence of undernutrition among mothers, scathing at 45% or higher across states. Using MUAC the situation worsened for Bihar with six in 10 women thin and one in ten severely thin. Unsurprisingly, not much variation was seen in nutrition status of SHG members and non-SHG members as there was no nutrition centered intervention in these areas except the universally applicable health and welfare schemes. Among the antenatal services, across all states, TT coverage was the highest and almost universal while height was most infrequently measured. Height <145 cm is an independent risk factor for low birth weight of babies and proposed to be used by the national government for screening nutritionally “at risk” women , 25, 26.NRLM’s poverty reduction program has some gains in improving women’s empowerment, particularly on increasing women’s control over financial resources and community leadership. Women’s tolerance to domestic violence in Odisha was a concern and needs to be addressed through NRLM or other social welfare platforms. Along with increasing SHG membership, need for improving reach of food security schemes or nutri-sensitive livelihoods is needed across all states. The gains in women’s financial control are yet to translate in improving their nutritional status across all states. SHG membership has varying influence in use of antenatal, partum and family planning services across Bihar, Chhattisgarh and Odisha. Services that require increased attention across all states include pregnancy registration in first trimester, height measurement of pregnant women, increased information and access to family planning methods and participation in VHSNDs. In Bihar, reach of all ANC services excluding TT vaccination need to be reviewed. The need for layering essential women’s nutrition interventions that is access to health services and care to nutritionally “at risk”, access to family planning services, improving dietary diversity, preventing micronutrient deficiencies and anemia and access to water, sanitation and hygiene services is immense. The context supports an inititaivet like Swabhimaan to layer these nutrition interventions on the NRLM platforms. This may be more effective in a state like Bihar where nearly half the SHGs have federated to VOs.S1 AppendixHousehold questionnaire.(PDF)Click here for additional data file.S2 AppendixMothers of under two years questionnaire.(PDF)Click here for additional data file.S3 Appendix(RAR)Click here for additional data file.


Chloroflexus aggregans, is widely distributed in neutral to slightly alkaline hot springs. Sulfide has been suggested as an electron donor for autotrophic growth in microbial mats dominated with C. aggregans, but remarkable photoautotrophic growth of isolated C. aggregans has not been observed with sulfide as the sole electron source. From the idea that sulfide is oxidized to elemental sulfur by C. aggregans and the accumulation of elemental sulfur may have an inhibitory effect for the growth, the effects of an elemental sulfur-disproportionating bacterium that consumes elemental sulfur was examined on the autotrophic growth of C. aggregans, strain NA9-6, isolated from Nakabusa hot spring. A sulfur-disproportionating bacterium, Caldimicrobium thiodismutans strain TF1, also isolated from Nakabusa hot spring was co-cultured with C. aggregans. C. aggregans and C. thiodismutans were successfully co-cultured in a medium containing thiosulfate as the sole electron source and bicarbonate as the sole carbon source. Quantitative conversion of thiosulfate to sulfate and a small transient accumulation of sulfide was observed in the co-culture. Then the electron source of the established co-culture was changed from thiosulfate to sulfide, and the growth of C. aggregans and C. thiodismutans was successfully observed with sulfide as the sole electron donor for the autotrophic growth of the co-culture. During the cultivation in the light, simultaneous consumption and accumulation of sulfide and sulfate, respectively, were observed, accompanied with the increase of cellular DNAs of both species. C. thiodismutans likely works as an elemental sulfur scavenger for C. aggregans, and C. aggregans seems to work as a sulfide scavenger for C. thiodismutans. These results suggest that C. aggregans grows autotrophically with sulfide as the electron donor in the co-culture with C. thiodismutans, and the consumption of elemental sulfur by C. thiodismutans enabled the continuous growth of the C. aggregans in the symbiotic system. This study shows a novel symbiotic relationship between a sulfide-oxidizing photoautotroph and an elemental sulfur-disproportionating chemolithoautotroph via cooperative dissimilatory sulfide oxidation to sulfate.A thermophilic filamentous anoxygenic photosynthetic bacterium,  Chloroflexus aggregans, in the phylum Chloroflexi, is widely distributed in neutral to slightly alkaline hot springs . However, no strains of C. aggregans showed marked photoautotrophic growth with sulfide.The thermophilic filamentous anoxygenic photosynthetic bacterium,  springs . This ba springs . Genome phically . HoweverOK-70-fl  and Chloain MS-G . RecentlChloroflexus spp. are generally thought to heterotrophically grow on organic compounds produced by cyanobacteria  at Nakabusa hot springs . This strain shows good photoautotrophic growth with H2 as the electron donor and 98.7% identity of 16S rRNA gene sequence with the type strain of C. aggregans (MD-66 = DSM 9485T). C. aggregans NA9-6 was cultivated in a medium lacking an organic carbon source. The autotrophic medium was composed of (L−1) 0.38 g KH2PO4, 0.39 g K2HPO4, 0.5 g NH4Cl, 4.2 g NaHCO3, and 0.5 g Na2S2O3⋅5H2O, buffered to pH 7.5. 5 ml of basal salt solution and 1 ml of vitamin mixture (−2⋅s−1) under an H2:N2:CO2  atmosphere. C. thiodismutans strain TF1 (−1 ferrihydrite under N2:CO2 (80:20) atmosphere. Ferrihydrite was prepared as previously described . The initial optical density (OD) at 610 nm was adjusted to be 0.03. 0.05 g of sublimed sulfur  sterilized at 110°C for 1 h three times or 1 mmol L−1 of sulfide was aseptically added into the medium to evaluate the effect of each compound on the growth. The inoculated tubes were cultivated at 60°C in the light . OD at 610 nm of the culture tube was periodically measured during cultivation. Measurements were made after allowing tubes to stand at 60°C for 5 min in the dark to precipitate sulfur globules.C. aggregans NA9-6 and C. thiodismutans TF1 were separately cultivated under autotrophic conditions as described above. The two cultures were inoculated together into 5 ml of the autotrophic medium containing thiosulfate as a sole electron source under an N2:CO2 (80:20) atmosphere in 30 ml glass test tubes, capped with butyl rubber stoppers and screw caps. The tubes were cultivated at 60°C in the light  for 7–20 days. 1 ml of the culture was repetitively subcultured in 5 ml of fresh medium under the same conditions to remove residual ferrihydrite from the original culture of C. thiodismutans. OD of the cultures was measured with a photometer . 0.3 ml of the culture solution was sampled during cultivation for measurements of the amount of sulfur compounds in the culture.C. aggregans NA9-6 and C. thiodismutans TF1 in the medium containing thiosulfate, 1 ml of the co-culture solution was inoculated into 5 ml of the autotrophic medium containing 1 mmol L−1 of Na2S instead of thiosulfate in 30 ml glass test tubes, capped with butyl rubber stoppers and screw caps. The gas phase of the tube was N2:CO2 (80:20). The initial OD at 610 nm was adjusted to 0.03. The tubes were cultivated at 60°C in the light . 0.3 ml of the culture solution was sampled during cultivation for measurements of the amount of sulfur compounds in the culture and extraction of DNAs from the cells.After repetitive subcultures of the co-culture of −1 carbonate-bicarbonate buffer (pH 10.0) immediately after collecting from the culture tube to prevent the loss of hydrogen sulfide. The mixture was centrifuged at 19,600 × g for 2 min at 4°C to remove bacterial cells. 200 μl of the supernatant was transferred to 400 μl of solution A  and stored at 4°C until measurements were made. The solution was alkalized by adding 400 μl of 40 mmol L−1 NaOH. After centrifugation of the mixture at 19,600 × g for 2 min, the precipitate was solubilized in 552 μl of solution A. Finally, 48 μl of Cline reagent  was added and mixed well. After 20 min incubation at room temperature, the absorbance at 665 nm was measured with a spectrophotometer .Sulfide concentration in culture solution was measured using a methylene blue formation method as described previously  with somg for 2 min. The supernatant was filtrated with a 0.45-μm-pore-size membrane filter. The solution was analyzed with a suppressed anion-exchange chromatography system  equipped with a LC-20AD liquid chromatography, a DGU-20A3 degasser, a SIL-10AF auto sampler, a CT-20AC SP column oven, a CDD-10A SP conductivity detector, and a SCL-10A VP system controller. A Shim-pack IC-SA2  was employed for separation of anions. The conditions of analyses were as follows; column temperature, 30°C; injection volume, 50 μl; eluent, 12 mmol L−1 NaHCO3 + 0.6 mmol L−1 Na2CO3; flow rate, 1.0 ml min−1. Data were collected and analyzed using Smart Chrome .Thiosulfate and sulfate concentrations were determined by ion chromatography. 100 μl of culture was diluted with 900 μl of MilliQ water and centrifuged at 19,600 × −1 Tris-HCl, 50 mmol L−1 EDTA, pH 8.0), SDS, and benzyl chloride. Culture cell suspensions were prepared in 2 ml tubes and incubated at 50°C for 2 h with mixing at 5 min intervals. Next, approximately 0.1 g of 0.1 mm diameter zirconia-silica beads  were added and the beating treatment was performed twice for 1 min at 2,500 rpm using a Mini-beadbeater (Biospec Products). DNA was recovered by phenol and chloroform extraction followed by alcohol precipitation with Ethachinmate . Finally, DNA was solubilized in TE buffer  and stored at −20°C until use.DNA was extracted using the benzyl chloride method  combinedC. aggregans and C. thiodismutans: agg-F (5′-CAAACGTGGTCTCAGTGCAGATCGG-3′) and agg-R (5′-TTAGCACACGGACTTCAAGCATTAG-3′) for C. aggregans; C-CmicF (5′-TACAATGGGGGGTACAGAGG-3′) and C-CmicR (5′-TGAGATAGCGACTTCGGGTG-3′) for C. thiodismutans. Specific amplification using each primer set was confirmed by DNA sequence analyses of the PCR fragments obtained using a mixture of both genomic DNAs. StepOne Real-Time PCR system  was used with FastStart Universal SYBR Green Master . The reaction mixture was composed of 10 μl of FastStart SYBR Green Master, 1 μl of 10 μM primers, 7 μl of water and 1 μl of DNA solution. Real-time PCR was performed under the following conditions; initial denaturation at 95°C for 10 min followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Fluorescence was detected at the end of the extension reaction. The purified DNA fragments amplified using the primer sets, agg-F and agg-R or C-CmicF and C-CmicR for the respective strain were spectrophotometrically quantified using Biospec-nano (Shimazu) and used as a template DNA to obtain the standard curve. The copy numbers of genome in the culture were calculated based on the copy number of 16S rRNA gene per genome, i.e., three in C. aggregans (NC_011831) and one in C. thiodismutans  under N2 gas, and the vials were sealed with butyl rubber stoppers and aluminum seals. After pre-heating the vials at 65°C in the dark for 1 h, NaHCO3 and Na2S solutions were added to the vials  and the vials were incubated at 65°C in the light  or dark. Sodium molybdenum oxide was added to the vial  when indicated. Periodically 0.2 ml of the solution was collected from the vials for determining sulfide and sulfate concentrations in the cell-free supernatant, as described above.Microbial cell aggregates  that develop in hot spring water at 65°C at Nakabusa hot spring, Nagano, Japan were collected. The major member of these mats was eviously . ApproxiC. aggregans NA9-6 with H2 as the sole electron source (closed circles). However, no marked growth was observed with sulfide (crosses), and the addition of sulfide on the growth with H2 showed some inhibitory effect (closed triangles). No significant increase in OD was detected in the absence of sulfide and H2 (data not shown). From these observations, we supposed that the absence of the growth on sulfide as the sole electron source is possibly due to the early formation of elemental sulfur from the oxidation of sulfide by C. aggregans. This was partly supported by the observation that the H2-dependent growth was largely suppressed in the presence of sublimed sulfur  atmosphere. After more than 10 subcultures in medium without ferrihydrite, microscopic observation of the culture found no particles of ferrihydrite and iron sulfide. Only cells of both bacterial species, i.e., 200–300 μm length multicellular filamentous cells (C. aggregans) and 1.0–2.0 μm × 0.5–0.6 μm rods (C. thiodismutans) were observed (data not shown). Bacterial growth of the co-culture was evaluated by measurement of OD at 610 nm (C. aggregans did not show such growth under the same conditions in the presence of thiosulfate (open triangles). No marked growth of the co-culture was observed in the dark (closed circles) and in the medium lacking thiosulfate or CO2 in the light (data not shown).The culture solution of −1 of thiosulfate was consumed and 3.1 mmol L−1 of sulfate was produced during 8 days of cultivation. 0.3 mmol L−1 of sulfide was detected at 6 days of cultivation, but the sulfide concentration was below 0.08 mmol L−1 after 8 days.C. aggregans grew using sulfide produced by C. thiodismutans as the electron source. C. aggregans worked as a biotic sulfide scavenger for C. thiodismutans through the photo-oxidation of sulfide, as previously described in co-culture of a sulfur-disproportionating bacterium in the genus Desulfocapsa with a purple sulfur bacterium in the genus Lamprocystis that oxidized sulfide to sulfate :(C. thiodismutans cells) was roughly calculated to be 30:1 ∼ 40:1 based on the qPCR results. This was consistent with microscopic observation  targeting the 16S rRNA gene of each strain after repetitive subcultures with sulfide. ervation . These rC. aggregans under co-culture conditions indicated that C. aggregans grew autotrophically using sulfide as the electron source. The doubling time was 41.4 ± 0.9 h−1. This growth rate was approximately 2.5-fold higher than that of photoautotrophic growth using H2 in the axenic culture of C. aggregans (strain NA9-6) as shown in C. thiodismutans and accumulation of sulfate in the co-culture suggested that C. thiodismutans utilized elemental sulfur produced by C. aggregans as the electron source. Chloroflexus aurantiacus OK-70-fl which is phylogenetically close to C. aggregans grows photosynthetically with sulfide as the electron source, but lacks the ability to oxidize elemental sulfur  with other bacteria which adhere to solid surface (fixation . Recentlfixation . Howeverf a lake , 2005. I surface  in sulfiC. aggregans-dominated microbial mats. Chloroflexus-dominated microbial mats were collected from under hot spring water at Nakabusa and a piece of the microbial mat was incubated anaerobically in artificial sulfidic hot spring water at 65°C. In the light, sulfide concentration gradually decreased with the increase in sulfate concentration within 12 h of incubation (We also examined the photo-oxidation of sulfide in cubation . The inccubation  which iscubation . The addC. aggregans and C. thiodismutans in vitro with isolated strains as well as in microbial mats in situ. Sulfide is externally provided as the sole electron donor and C. aggregans converts it to elemental sulfur using light energy autotrophically. The elemental sulfur is used by C. thiodismutans with anaerobic chemolithoautotrophic growth producing sulfide and sulfate simultaneously through the process called disproportionation. The produced sulfide can then be used by C. aggregans again, and with all of these processes, externally provided sulfide is converted to sulfate by the symbiotic autotrophic system with two different autotrophic organisms performing photosynthesis and chemosynthesis. The electrons externally supplied with sulfide should mostly be used by C. aggregans to fix CO2 finally and only a few percent of electrons will be used by C. thiodismutans based on the observation that the amount of genome copies (C. aggregans was about 30 times larger than that of C. thiodismutans and the cell mass should be roughly parallel to the electron input to CO2. This explanation is consistent with the idea that most ATP is supplied by light-induced cyclic phosphorylation in C. aggregans and, on the other hand, ATP is produced by energetically unfavorable and electron-consuming process of the disproportionation of elemental sulfur to sulfide and sulfate in C. thiodismutans.e copies  and celle copies  of C. agSK, NK, KM, and SH designed the study and analyzed and interpreted the data. SK performed the experiments in co-cultures. NK performed the experiments in natural microbial mats. SH supervised the experiments. SK, KM, and SH wrote and revised the manuscript. All the authors approved the final version.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.


Several axes of the UPRmt have been described, creating some confusion as to the nature of the different responses. While distinct molecularly, these different axes are likely mutually beneficial and activated in parallel. This review aims at describing and distinguishing the different mammalian MSR/UPRmt axes to define key processes and members and to examine the involvement of protein misfolding.Mitochondria are sensitive to numerous environmental stresses, which can lead to activation of mitochondrial stress responses (MSRs). Of particular recent interest has been the mitochondrial unfolded protein response (UPR Mitochondria are highly regulated cellular organelles that fulfill numerous metabolic functions, including the production of ATP by respiration. Function and quality of mitochondria need to be tightly controlled to ensure the supply of metabolic building blocks and to prevent the production of harmful agents such as reactive oxygen species (ROS), produced at increased rates upon malfunctioning respiration , 2. MitoMitochondria are cellular organelles separated from the extra-mitochondrial environment by two membranes—the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM). The compartment enclosed by the IMM is called the matrix, and the space between the OMM and IMM defines the intermembrane space (IMS). Due to the archetypic origin of mitochondria and the resulting physical separation from the cytosol, the mitochondrial matrix forms a largely independent protein compartment providing its own translation and protein quality control machinery including chaperones and proteases , 10–12. ER) have been extensively studied and reviewed [ER that elicits a multi-axis response mediated by several receptors and leading to different effects such as induction of pro-folding factors and inhibition of translation [mt also appears to contain several axes with distinct molecular outcomes  [In 1996, the Hoogenraad laboratory discovered a stress response that is specific to mitochondrial protein misfolding and that was later named the UPRd HSP10) , to be cd HSP10) , 29. Thimt in Caenorhabditis elegans have uncovered molecular mechanisms involved in signaling the mitochondrial stress to the nucleus, causing induction of a transcriptional response [mt, leading to its accumulation in the nucleus and activation of the transcriptional UPRmt [mt pathways in C. elegans have been comprehensively reviewed [Extensive studies into the UPRresponse . This real UPRmt , 31. Thereviewed , 32, butmt axis and forms the canonical UPRmt. Its outcome is the induction of genes increasing the folding capacity in mitochondria. Employing misfolding-prone deletion mutants of the mitochondrial protein ornithine transcarbamylase (OTCΔ) aided in defining the UPRmt in mammalian cells and determined its role in response to protein misfolding [mt [CHOP [The transcriptional response to mitochondrial protein misfolding described above remains the best understood mammalian UPRsfolding . Exogenosfolding , 34. Chasfolding , 33. Anamt [CHOP .mt signaling is highly specific, as documented by the fact that the OTCΔ-triggered activation of CHOP does not increase BiP transcript levels, an ER chaperone induced by UPRER in a process also mediated by the ISR [mt to the nucleus in a function similar to ATFS-1 in C. elegans [mt signaling [mt signaling in cells have not been determined. Luciferase reporter assay testing of potentially UPRmt-regulated promoters pointed towards further genes possibly being activated by the UPRmt [mt revealed that induction of the canonical UPRmt leads to specific and extensive transcriptional rearrangements affecting a wide range of biological pathways, mainly involved in protein folding and cellular homeostasis [mt response as an extensive transcriptional response in the nucleus, triggered by mitochondrial unfolded proteins inducing chaperonin transcription via a mechanism involving a CHOP element in the promoter region.Despite the dependence on ISR factors, i.e., CHOP, UPR the ISR , 33. Whi the ISR , increas the ISR . Recentl the ISR , 38, was elegans . Additioignaling . Which the UPRmt , some ofhe UPRmt . Recent eostasis . Combinemt signaling is surprising as CHOP is also a key member of the ISR, which is induced by various other stresses and also forms part of the UPRER [mt [CHOP upon acute UPRmt induction mediated by mitochondrial HSP90 inhibition [mt-mediated CHOP induction. The distinct regulation of CHOP hints towards other factors playing important roles in shaping the transcriptional outcome of the UPRmt, possibly by binding to MURE1 and MURE2 sites. Due to the lack of understanding of co-regulating factors and the inherent non-specificity of CHOP, the induction of CHOP must not be used as a readout for the induction of the UPRmt. Indeed, numerous mitochondrial stresses that are not related to protein misfolding, but instead inhibit central functions like respiration, mitochondrial membrane potential, import, and translation, can quickly induce CHOP (via ATF4) without leading to chaperonin activation [mt, likely regulating hitherto unknown aspects of the modulation of cellular processes by the UPRmt, they alone are not sufficient to elicit the canonical UPRmt transcriptional response, which likely depends on additional signaling factors.The central role of CHOP in UPRhe UPRER . OverexpPRER [mt . Howeverhibition . This imtivation , 43. Thumt defined by chaperonin induction: (1) overexpression of misfolding-prone deletion mutants of OTCΔ; (2) inhibition of mitochondrial HSP90 [mt in mammalian cells, unless the UPRmt is induced chronically, or highly quantitative methods such as mass spectrometry are used [mt axis, as documented by numerous publications [mt to increase the mitochondrial folding capacity in response to protein misfolding; however, this is not the only mitochondrial response to protein misfolding.The following conditions causing mitochondrial protein misfolding have been shown to subsequently elicit the canonical UPRal HSP90 , 44, 45;al HSP90 , 46, whial HSP90 ; and (4)al HSP90 , 48. Dueal HSP90 , the anaare used . Howeverare used , and areications , 48, 50.mt transcriptional response—cells employ a second mechanism of decreasing the unfolded protein load, e.g., achieved by a reduced uptake of proteins into the organelle (for protein-importing compartments) or by reduced translation (for compartments containing a translation machinery). While work in C. elegans has shown a decrease in mitochondrial protein import and translation [mt that reduces the folding load: taking advantage of two inhibitors targeting the mitochondrial HSP90 and LONP1 [mt, we discovered a translational UPRmt axis that controls the folding load within mitochondria upon UPRmt activation [mt, we observed a rapid decrease of MRPP3 transcript and protein levels, causing a markedly lower level of mitochondrial pre-RNA processing and ultimately a reversible reduction in mitochondrial translation [In addition to increasing folding capacity through induction of chaperones—the canonical UPRnslation , 51, thend LONP1 , 46 to ativation . Whole stivation , 52, 53.nslation .mt axis, which limits the protein folding load by regulating mitochondrial translation, constitutes an interesting new aspect to the UPRmt: due to its post-translational regulation within a single mitochondrion, there is no requirement to pass a cellular signaling threshold for activating the transcriptional UPRmt. Instead, it acts locally in single, damaged mitochondria and could thus form a first line of defense against mitochondrial damage that is most likely independent of extra-mitochondrial stimuli function ; and (3)function , 65. Str+ levels, thereby causing an elevated mitochondrial antioxidant activity in both C. elegans and mammalian cells [mt [mt transcriptional response [mt sirtuin axis is likely highly complementary to the canonical UPRmt transcriptional response in securing mitochondrial health.SIRT1/SIRT 3 had been known to exert an antioxidant effect by controlling the activity and localization of the transcription factor FOXO3A , 67. Deaan cells , 73. Recan cells . Additioells [mt . Importaresponse . With prIMS. Its underlying features have been largely described by the use of endonuclease G (EndoG), an IMS endonuclease released from mitochondria to fragment DNA, causing caspase-independent apoptosis upon conditions such as heat and oxidative stress [IMS) that appears to be independent of the matrix UPRmt and does not cause induction of CHOP or HSP60 [mt transcriptional response. Instead, its signaling is dependent on estrogen receptor alpha (ERα) and mediated by ROS-dependent phosphorylation of ERα by AKT [CHOP and HSP60 similarly to the effects observed upon inducing matrix protein misfolding [mt transcriptional response becomes activated directly by unknown mechanisms, or that the severe accumulation of misfolded IMS proteins causes matrix protein misfolding that activates the canonical UPRmt transcriptional response as an indirect response to perturbed IMS proteostasis. The UPRIMS–ERα axis defines a distinct response from the UPRmt axes, attempting to specifically modulate IMS proteostasis to improve folding. Depending on the environment causing protein misfolding, it may act in parallel to the UPRmt axes. UPRmt induction upon UPRIMS failure shows the important role IMS proteostasis exerts on folding in the matrix and suggests possible links between these responses.The IMS is separated from the matrix by a membrane forming a distinct compartment in which protein misfolding can occur, leading to a distinct mitochondrial UPR—the UPRe stress –77. Expre stress , 79. Thiα by AKT , the mitα by AKT , (2) eleα by AKT , and (3)α by AKT , 79. Strsfolding , suggestmt by relying on overlapping pathways  are not apparently induced by mitochondrial protein misfolding, but still show a certain degree of similarity to the UPRays Fig. . Of partactivity . This re pathway . Howeversfolding . DARS2-d the ISR . Strikinnock-out . (3) Ovenock-out , inducesnock-out . Increasnock-out . Thus, aand LONP , 90. Surand LONP . Thus, tongevity , 91, poiperonins .Fig. 4Mimt transcriptional response  MSRs and UPRmt, depending on the significant/primary involvement of mitochondrial protein misfolding as causative agent, and (2) the UPRmt axes studied by clearly determining and describing the UPRmt axes monitored and activated.Together, these described MSRs are able to induce adaptation mechanisms, largely mediated by retrograde signaling (via the ISR) and transcriptional modulation, and some potentially utilize signaling through the canonical UPRnse Fig. . There amt signaling through the ISR is overlapping with the activation of the ISR by EIF2A kinases. Some significant differences between classic activation of the ISR by EIF2A kinases and activation by mitochondrial stress have already been described, leading to the proposal of a specific mitochondrial ISR (ISRmt), distinct from the canonical ISR [mt in the UPRmt and to determine the temporal control and cross-activation of the ISRmt and the different UPRmt axes in response to mitochondrial misfolding stress.Many of the MSRs signal, at least in part, via the ISR. One of the future challenges is to determine to what extent MSR and UPRical ISR . Furthermt. Strikingly, inhibition of mitochondrial HSP90 to induce protein misfolding and the UPRmt has been recently explored as a potent therapeutic strategy to target cancer [mt axes demonstrate that the UPRmt is a multi-pronged response modulating several aspects of mitochondrial proteostasis in an attempt to alleviate folding stress (Fig. mt axes are distinct with specific sets of defining factors, allowing and encouraging researchers to clearly define, describe, and validate activation of the proposed axis studied. The different UPRmt axes are pro-survival and attempt to maintain mitochondria. However, there must also be destructive pathways activated upon failure to restore proteostasis. Strikingly, there have now been numerous reports of mitochondrial protein misfolding triggering LC3B lipidation, induction of autophagy genes, and mitophagy [mt. However, substantiated evidence proving a causative relation between mitochondrial protein misfolding and autophagy is still lacking, and the same applies to insight into the mechanisms involved. Thus, the relationship between non-selective autophagy, mitophagy, and the UPRmt will require further investigation. It is tempting to speculate that these studies will reveal autophagy pathways forming an additional UPRmt axis that might initiate upon more severe or prolonged mitochondrial protein misfolding, when the described UPRmt axes fail to restore proteostasis, or are overburdened.Various conditions, where mitochondrial protein misfolding is the primary cause of mitochondrial stress, have been shown to activate axes of the UPRt cancer , 94. Theess Fig. . The UPRitophagy , 94, 95,


Calcium ions are highly versatile intracellular signals that regulate many cellular processes. The key to achieving this pleiotropic role is the spatiotemporal control of calcium concentration evoked by an extensive molecular repertoire of signalling components. Among these, reactive oxygen species (ROS) signalling, together with calcium signalling, plays a crucial role in controlling several physiopathological events. Although initially considered detrimental by-products of aerobic metabolism, it is now widely accepted that ROS, in subtoxic levels, act as signalling molecules. However, dysfunctions in the mechanisms controlling the physiological ROS concentration affect cellular homeostasis, leading to the pathogenesis of various disorders. The first is the cooperation of two different sources of Ca2+ in the generation of [Ca2+]cyt fluctuations: the extracellular medium, a virtually unlimited reservoir with a [Ca2+] of ∼1 mM [2+] > 100 μM, which allow rapid release of Ca2+ through store-resident channels [2+]cyt variations, such as pumps, channels, Ca2+-binding signalling molecules, enzymes, and buffering proteins . Importantly, while complex molecules can be chemically altered, the only mechanism that exerts control over Ca2+ are chelation, subcellular compartmentalization and cell extrusion. The consequence is a very steep [Ca2+] gradient across the plasma membrane and the intracellular stores [2+]cyt are maintained within very low values of ∼100 nM, while the extracellular space generally presents a [Ca2+] of over 1 mM  can reach ∼0.8 mM, depending on the cell type. Rapid release of Ca2+ from these compartments ensures [Ca2+]cyt rises required for specific cellular functions  up to a specific [Ca2+] threshold, above which further increases in [Ca2+] play an inhibitory function  to return to its resting condition [2+ into the ER/SR stores via the activity of ER/SR Ca2+ ATPase pumps (SERCAs) or extrude Ca2+ in the extracellular milieu via plasma membrane Ca2+ ATPases (PMCA pumps) by exploiting ATP-derived energy. A second mechanism utilizes the electrochemical gradient of Na+ across the plasma membrane to provide the energy to transport Ca2+ to the extracellular space through the Na+/Ca2+ (NCX) and Na+/Ca2+-K+ exchangers (NCKX) [Once Caondition . ATPase s (NCKX) .2+]cyt also for other membrane-bound compartments such as the Golgi apparatus, endolysosomes, and mitochondria  [2+] is about 10-100 nM in resting conditions and reaches values of 2-3 μM during cell stimulation, the role of mitochondria in Ca2+ homeostasis was considered marginal. Therefore, the plasma membrane and the ER became the major players in the Ca2+ signalling scene [2+] in intact living cells were developed, allowing to uncover the role of mitochondria in Ca2+ handling [2+]mit in basal resting condition is very low, comparable to the cytosolic one (10-100 nM), upon cell stimulation, mitochondria are able to rapidly and efficiently accumulate Ca2+ at levels that exceed that of the bulk cytosol that, in some cell lines, can reach also [Ca2+] of 100 μM [2+ uptake and the prompt response of mitochondria to [Ca2+] increases was later solved by the demonstration that mitochondria are located in close proximity to the Ca2+ channels that elicit the rise in [Ca2+]cyt, the InsP3Rs, and the RYRs on the ER and SR [2+] compatible with the low affinity of the MCU and that dissipates rapidly, thus preventing mitochondrial Ca2+ overload or vicious Ca2+ cycling across the mitochondrial membrane  . Since cng scene . The sithandling , 17. Indf 100 μM . The disR and SR , 19. Indmembrane , 19.2+ to cross the outer mitochondrial membrane (OMM) plays a crucial role. The OMM permeability to solutes is prevalently due to the high expression of the voltage-dependent ion channels (VDACs), permeable to solutes smaller than 5 kDa and, thus, also Ca2+ [2+]mit, whereas VDAC downregulation has the opposite effect  at both sides of IMM cyt . The idepothesis . The gatpothesis . They al2+ entry and the sensitivity to ruthenium red/Ru360 (RuRed/Ru360), a compound that inhibits the activity of the uniporter  , ensurinogenases .2+-binding EF-hand domains with MICU1 and MICU2 cyt cyt .Saccharomyces cerevisiae, an organism that lacks mitochondrial Ca2+ uptake. Its role in MCU complex regulation is highly debated, since it has been shown, both in yeast and mammalian cells, that it is involved in complex IV assembly . Therefore, when [Ca2+] overcomes this threshold, mROS production becomes detrimental and compromises mitochondrial bioenergetics and cell functions cyt increases beyond a certain value and induces mitochondrial Ca2+ overload, triggering the mitochondrial “permeability transition.” In this condition, the mitochondrial membrane becomes permeable to any molecule less than 1.5 kDa in size. Consequent dissipation of ΔΨm leads to a permanent membrane depolarization, decreased ATP production, and eventually cell apoptosis. Moreover, mitochondrial membrane depolarization leads to crista unfolding, uncoupling of oxidative phosphorylation, and the reverse electron transport (RET). RET is evoked when electrons from ubiquinol are transferred back to respiratory complex I, reducing NAD+ to NADH. This process generates a significant amount of ROS mit, suggesting that oxidative stress, and thus ROS accumulation, plays a positive feedback role in modulating MCU activity [2+]mit at resting [Ca2+]cyt. In detail, they identified a conserved cysteine in metazoan at position 97 (Cys-97) in the NTD of the MCU protein sequence to be the only reactive thiol in human MCU that undergoes redox modification (S-glutathionylation). The Cys-97 residue is surface-exposed and primed for an oxidative posttranslational modification that induces a conformational change of MCU that promotes the clustering of MCU channels and their persistent activation mit by preventing Ca2+ overload mit by affecting the activation of the Krebs cycle during increased workload cyt), which accelerates mitochondrial Ca2+ efflux via the mitochondrial Na+/Ca2+ exchanger (mNCX) cyt could be explained by lower Na+/K+ pump activity, consistent with a decreased Na+/K+ pump expression in some models of HF [+]cyt is due to elevated diastolic Na+ influx rather than altered Na+/K+ pump activity [+]cyt modifies the direction of the sarcoplasmic NCX transport by inducing the increase in cytosolic Ca2+ that partially compensates the decreased SR Ca2+ load and release in failing myocytes  in knockout mitochondria is only partially reduced cyt increases after the positive inotropic and chronotropic responses to β-AR stimulation cyt cyt  and enha+ influx , thus gee stress .2+ uptake  leading to apoptosis and necrosis mit associated with an increase in ROS production has been proposed as a possible mechanism for skeletal muscle fiber death in Duchenne muscular dystrophy (DMD) [2+]cyt that, in turn, induces mitochondrial Ca2+ overload , mitochondrial permeability, and dysfunction culminating in muscle loss , and even small defects in Ca2+ homeostasis, hallmark of aging and neurodegenerative diseases, are able to impair neuronal activity [2+]cyt increases in neurons principally occur through Ca2+ entry from the plasma membrane through ligand-gated glutamate receptors, such as the N-methyl-d-aspartate receptor (NMDAR) or various voltage-dependent Ca2+ channels (VDCCs), as well as from the release of Ca2+ from intracellular stores  and [Na+], for example, after having sustained NMDA receptor activation mit mit . This in2+]mit . In addill death . These f2+ homeostasis are considered hallmarks of cancer cells mit, metastatic cell motility, and matrix invasiveness. Most importantly, in MDA-MB-231 xenografts, deletion of MCU greatly reduces tumor growth and metastasis formation and this is associated with a decrease in mitochondrial ROS production, suggesting that mitochondrial ROS might play a crucial role in cell malignancy regulation by mitochondrial Ca2+ uptake [α) expression, thus negatively affecting the expression of HIF1-α target genes involved in cancer progression [2+ uptake homeostasis [2+ level and ROS production [2+ uptake not only represents a fundamental mechanism to regulate cell survival and metabolism but also plays a pivotal role in the regulation of autophagy that plays both a negative and a positive role in cancer [Tumor formation and progresssion are directly related to mitochondrial dysfunction . Furtherer cells . Multipltastasis . Althougsurvival . Howeversurvival . This stsurvival . Coheren+ uptake . In addigression . Elevategression . Akt or eostasis . Akt-medoduction . In addin cancer , 153. Inn cancer . Increasn cancer . Even thn cancer –160.It is important to underline that dysregulation of mitophagy represents a scenario that characterizes not only cancer but also different spectra of diseases including neurodegenerative diseases, motor neuron disorders, autosomal dominant optic atrophy, I/R injury, diabetes, aging, and cancer . The dis2+ levels and tumor development and strongly suggest that alteration in the activity of the MCU complex components represents a critical checkpoint of metastatic behaviour and thus a potential pharmacological target to combat aggressive cancers.Altogether, these data reveal the importance of the association between aberrant mitochondrial CaMitochondria are key intracellular organelles that play a fundamental role in energy production and control many cellular processes from signalling to cell death. The function of the mitochondrial electron transport chain, the major source of ATP in the cell, is coupled with the production of ROS that are maintained at physiological levels by highly efficient mitochondrial antioxidant systems. Moreover, these antioxidant defences rely on mitochondrial metabolism that supplies the reducing equivalents needed for their activity.In the last years, several studies demonstrated that quick changes in ROS levels, coupled with essential cellular functions, are fundamental participants of physiological signalling. Importantly, mitochondrial calcium, by impinging on aerobic metabolism, plays a crucial role in this process, since it joins the cellular activation stimuli and ROS production. This phenomenon plays a crucial role in the maintenance of cellular homeostasis in several tissues, as discussed above.2+ overload. For these reasons, the physiopathological role of mitochondrial Ca2+ uptake and mROS production has been extensively studied in the past years.When the balance between ROS production and clearance is altered by either overproduction of mROS or impairment of the antioxidant defence, mitochondrial dysfunction occurs, leading to the induction of the cell death cascade. Indeed, the overproduction of mROS and the change in mitochondrial redox homeostasis have been shown to be involved in several pathological conditions, which often are associated with mitochondrial Ca2+ in organ physiology. In particular, in this review, we highlighted the contribution of the MCU complex activity, and thus the regulation of mitochondrial Ca2+ uptake, in cardiovascular, skeletal muscle, and neurodegenerative diseases and cancer. Although after the discovery of the molecular identity of the MCU complex many studies have confirmed the crucial role of mitochondrial Ca2+ signals in the regulation of cell survival, metabolism, and autophagy, many findings are controversial, and many questions are still open. In this regard, the characterization of the mechanisms responsible for the survival of the total MCU KO mouse only in the outbred strain and the analysis of the mechanisms underlying the different phenotypes of the MICU1 KO mice will be fundamental important to finely dissect the physiological role of mitochondrial Ca2+. Furthermore, the structural and functional complexity of the MCU complex needs to be clarified. Thus, the study of the physiological role of the different MCU complex components might be useful to better characterize the regulation of mitochondrial Ca2+ uptake in different physiopathological conditions thus resulting in the identification of novel therapeutic strategies to cure pathologies characterized by dysregulation of mitochondrial Ca2+ homeostasis. In line with this, the discovery of drugs that modulate the activity of the MCU complex shall be extremely relevant for the future development of MCU-targeting therapies.The molecular and functional characterization of the MCU complex components highlights the importance of the dynamic regulation of mitochondrial Ca


The majority of pet rabbits have flatter, shorter faces than wild rabbits. However, rabbits with flat faces are at higher risk of developing considerable health problems, including painful dental problems. The aim of this research was to identify which type of rabbit face people actually prefer, in order to better understand why rabbits with flat faces might be bred and purchased. Images of 25 rabbit faces were assigned to a face-type group by 134 veterinary professionals. Through an online questionnaire, people then rated each of the 25 images according to preference for the rabbits’ faces, and a total of 20,858 questionnaires were completed globally. Through statistical modelling, we show for the first time that mildly flat-faced rabbits  are more preferred globally than any other kind, and that the longest faced rabbits are the least preferred. Aside from the shape, other features of rabbit faces that were preferred include a soft, medium-light fur appearance and being generally short-furred. These results support the theory that the human preference for the baby-like features of flat-faced rabbits has driven their popularity. We would encourage breeders to avoid breeding extremely flat-faced rabbits due to the associated health problems and to focus instead on breeding more preferred mildly flat-faced, erect-eared rabbits such as the Havana breed.Domesticated rabbits typically exhibit shorter, flatter skulls than their wild counterparts . However, brachycephaly is associated with considerable health problems, including problems with dentition. The aim of this study was to establish which type of rabbit face people prefer, with a particular emphasis on skull morphology and brachycephaly. We grouped 25 images of rabbit faces by cephalic degree based on ratings assigned by 134 veterinary professionals. An online questionnaire was then launched, in which people could rate each of the 25 images according to preference for the rabbits’ faces, and a total of 20,858 questionnaires were completed globally. Repeated-measure, multi-level general linear modelling revealed mildly-brachycephalic rabbits to be the most preferred type of rabbit, and moderately-dolichocephalic (longer skulled) rabbits to be the least preferred. The preference for brachycephalic rabbits was stable across continents, and as such it is highly plausible that human preference has been a driver for the shortening of the skull typically seen in domestic rabbits, perhaps as a result of the ‘baby-schema’. Additional features of rabbit faces that were preferred include a soft, medium-light fur appearance and being generally short-furred. These novel insights may prove useful in the improvement of the public understanding of rabbit health and welfare. The relationship between preference and skull shape is particularly pertinent to future work concerning rabbit health, given the cross-species evidence that having a flat face is associated with chronic health conditions. Recent research estimates that over a quarter of a million pet rabbits are bred annually in the United Kingdom (UK) for online sales alone . Within Where breed standards are used to classify and select dogs for specific morphological traits, they are known to contribute directly  or indirPerhaps one of the most extreme morphological changes within companion animal breeds is brachycephaly, which results in wholesale changes to the jaw and skull, in turn affecting the orbits and cranial cavity. The majority of domestic rabbits have shorter skulls than their wild counterparts, some more extreme than others, which has a noted impact on mastication performance, predisposing them to dental health problems . BrachycOne of the possible drivers for the popularity of, and selection for, BC animals is the baby-schema (Kindchenschema) . First pAlthough the factors driving rabbit breed selection are relatively unexplored, dog breed popularity is partly driven by social influences and fashion ,31. BC dFor cats, although images of BC individuals have been shown to be less preferred when compared to their mesocephalic  and dolichocephalic  counterparts, substantial preferences for BC cats persisted in those individuals that currently owned a BC breed . Again, Given the relative paucity of data concerning domestic rabbits, this study sought to explore if and how preferences for rabbit facial structures may mirror those already established in cats and dogs. If the baby-schema is the main driver for preferences for rabbit faces, we would expect to see higher preference ratings for BC rabbits compared to mesocephalic (MC) or dolichocephalic (DC) rabbits. Alternatively, if rabbits are considered the same as cats when it comes to facial feature preference, we would expect to see higher scores for mesocephalic rabbits, and significantly lower scores for rabbits with extreme facial conformations. Considering the popularity of BC rabbit breeds in the pet population and the considerable health risks of extreme brachycephaly, particular emphasis will be placed on investigating the factors that influence preference for extremely-BC rabbits.Photographic images of rabbit faces were collected by the authors, including the authors’ own rabbits and images that were freely available on the internet under creative commons licences  based upon methods presented in Farnworth et al. [Two online surveys were designed using the platform SurveyGizmo . Both surveys were anonymous, collecting no personal data. Participants gave their consent by ticking a box associated with a statement regarding the anonymous nature of the survey, which meant that we would not be able to delete anyone’s data upon request. At the end of each survey, participants were, however, given the option to confidentially share an email address with us for the purpose of informing them of the results upon publication. Consent was gained here (again by ticking an unchecked box) specifically for the purpose of storage at the University of Nottingham (accessible only to N.D.H) and the use of this email only to disseminate the results, after which the emails would be deleted.Brachycephalic: having a head with a markedly flattened, short face;Mesocephalic: having a head of medium proportions, not markedly brachycephalic or dolichocephalic;Dolichocephalic: having a relatively long skull, with a length longer than it is wide.Due to the varying nature of the photographic images  and inclusion of long-haired rabbit breeds , physical measurements were unable to be consistently taken from the images. Therefore, in order to create a cephalic index, an anonymous veterinary survey was designed for veterinary professionals to rate the 25 rabbit facial images . It was also disseminated through social media (Facebook and Twitter) and remained live for a period of four weeks from the 21st of May to the 18th of June, 2018.An anonymous questionnaire was developed based on a previous study by Farnworth et al.  that wasThe questionnaire was written in English and was also translated into Spanish (translation by G.M.P). The survey was distributed from the 6th of July to the 28th of August 2018, through social media (Facebook and Twitter) and a University of Nottingham press release.beanplot [p < 0.001, a stricter threshold than is standard.All data distributions between groups were visually checked to ensure that the distributions were comparable  as per best practice recommendations  using thbeanplot . OverallThe median cephalic rating provided to each rabbit image by the veterinary consensus was used in the analysis to group the images according to their cephalic type. Images were also assigned to phenotypic categories dependent on whether the rabbits in them were lop-eared or not, had all short fur or areas of long fur, and whether they appeared to be coloured solid white, solid dark, solid grey, solid medium-light, or patterned.Univariable associations between phenotypic features  and preference scores were analysed using Kruskal-Wallis (for variables with three or more categories) or Mann-Whitney tests (for variables with two categories). To investigate how respondent-related variables  may be associated with preferences for extremely-BC rabbits, Mann-Whitney or Kruskal-Wallis tests were used as appropriate for testing for associations with scores given to the rabbits classified as extremely-BC only .Phenotypic features and respondent variables that were significant to < 0.2 were taken forwards  into mulOver a four-week period, the veterinary survey received 134 completed responses from veterinary professionals, of which 16% (21) considered themselves to be rabbit specialists and another 16% were not sure if they classified as such . Sixty-nSix of the images were assigned a median cephalic rating of 1—extreme brachycephalism, three were assigned a median of 2—moderate brachycephalism, six achieved a median rating of 3—mild brachycephalism, five were assigned a median of 4—mesocephalic, three images were assigned a median of 5—mild dolichocephalism, two were assigned a median of 6—moderate dolichocephalism, and no images achieved a median of 7—extreme dolichocephalism . InteresA total of 20,858 complete responses to the preference questionnaire were received internationally . Countryp < 0.001), with the highest scores given to rabbits in the Mildly-BC group and the lowest given to those in the Moderate-DC group , the group of rabbits least likely to receive a ‘top’ preference rating were those rated Moderately-DC. The most likely to be rated 9 or 10 were images of Mildly-BC rabbits, which were 3.31 times more likely to receive a top preference rating compared to the images in the Moderately-DC group . MeanwhiOnce all the phenotypic variables were controlled within a repeated-measures multi-level analysis, all phenotypic variables  contributed to the preference ratings  without A univariate analysis revealed significant associations for every demographic variable , face shape (42.5%), and fluffiness (36.7%) were the main reasons given for assigning a high preference score . Being tThe face shape was 7.5 times less likely to be cited as a factor in why people disliked rather than liked Extremely-BC rabbits . HoweverThe ear form (lop or erect) did not appear to have a clear association with the skull length. Preference for ear type was found to increase in some skull forms,  but had converse outcomes for other images .Colour type was far more likely to be selected as a reason for liking than disliking a rabbit across all cephalic groups, but particularly for the Mildly-BC group. Eyes were 4 times more likely to be selected as a reason for liking a Mildly-BC rabbit than disliking one of these rabbits and looking soft was the most impactful reason overall for liking any group of rabbit, with face shape coming in second. Rabbits were not likely to be disliked for not being fluffy, but could be disliked for being too fluffy, and a past experience with similar looking rabbits had very little (conscious) impact on whether people liked or disliked an image.Domesticated rabbits typically exhibit shorter, flatter skulls than their wild counterparts, a difference suggested to be due, in part, to differences in diet . Here, wThis study received an exceptional level of engagement with the public, with 20,858 people spread over every continent completing the preference questionnaire and rating all 25 rabbit images. Through this exceedingly rich dataset, we were able to show that mildly-BC rabbits are the most commonly preferred rabbits, closely followed by moderately-BC rabbits, whilst the least preferred types of rabbits according to skull morphology are moderately-DC rabbits, followed by extremely-BC rabbits. Although extremely-BC rabbits were the second lowest scoring group, their face shape was the least often ‘disliked’ factor of their appearance.When investigating the demographic factors that influence preference for rabbits with extremely-BC faces, we showed that the preference scores are relatively stable across continents, with only participants from Europe and Oceania differing from the rest of the world by scoring these images significantly lower overall in the multivariable models . In compWith the face morphology seen to be an important factor in the scoring of all rabbit types, it is likely that this, along with other prevalent factors that include fluffiness, ear position and softness, will influence decisions when acquiring pet rabbits. Rabbits, commonly, are bought more impulsively than other domestic pets ,43,44; akawaii originated in Japan and is typically characterised by the use of images and cartoons of small fluffy creatures and infants [Traditionally, rabbits are bought as pets for children ; therefo infants . However infants ,28. It i infants . With apOne finding from this study was that people who work in an animal health-related profession or had an advanced degree were likely to provide lower median preference scores for extremely-BC rabbit images. This finding mirrors the research into preferences for BC cats by Farnworth et al. . These aWhen focusing on how people scored extremely-BC rabbits , the face shape was commonly cited as a reason for having a strong preference for them (57%) but was not commonly cited as a reason for disliking them (7%). This finding differed from other types of rabbits, with face shape more likely to be disliked as skulls became elongated. Such a finding suggests that a preference for rabbits is in large part driven by their facial conformation, and that extreme brachycephalism is not widely considered to be a disliked trait, even by those who did not like those rabbits’ appearance at all. In fact, the main reason given for disliking the extremely-BC rabbits was being lop-eared (36%), a trait that is independent of the skull length, which suggests that the popularity of extremely-BC rabbits could increase if un-coupled from them being lop-eared. This finding may explain the popularity of the Netherland dwarf (an erect-eared extremely-BC rabbit breed) as the second most common breed in the UK .As a web-survey largely distributed through social media, the data described here were collected through self-selection, which introduces its own form of bias . In addiFuture research in this area would do well to develop a rabbit cephalic index. Cephalic indices have proven to be useful measures for investigating conformation-related problems in dogs , and thWhen considering ‘what makes a rabbit cute?’, the baby-schema effect would suggest that rabbits with more extreme-BC features would be rated as being more preferred. Here, we provide new evidence that mildly-BC rabbits are the most preferred type of rabbit considering skull morphology. Additionally, despite the popularity of lop-eared rabbits, we show that erect-eared rabbits tend to be more preferred. Given that BC and lop-eared features are also significantly associated with greater health risks that compromise animal welfare, we would strongly advocate breeding away from extreme-BC conformations in rabbits, and breeding fewer lop-eared breeds. Whilst the overall trend showed a preference of mild brachycephalism in rabbit faces, a large proportion of people still assigned strong preference scores to images of rabbits with extremely-BC facial conformations, with current rabbit owners more likely to prefer extremely-BC rabbits. The preference scores for BC rabbits was stable across continents, and as such it is highly plausible that this preference has been a strong driver for the shortening of the skull typically seen in domestic rabbit species compared to their wild counterparts. Additional features of rabbit faces that were preferred include a soft, medium-light fur appearance, as well as being generally short-furred. Knowing that people have a general preference for BC rabbits, targeted education campaigns could be developed to raise awareness of conformation-associated health issues and to suggest more moderate or mesocephalic alternatives for prospective rabbit owners.