Supplemental Digital Content is available in the text. “Obesity paradox” refers to an association between obesity and reduced mortality . A common explanation is collider stratification bias: unmeasured confounding induced by selection bias. Here, we test this supposition through a realistic generative model.We quantify the collider stratification bias in a selected population using counterfactual causal analysis. We illustrate the bias for a range of scenarios, describing associations between exposure (obesity), outcome , mediator  and an unmeasured confounder.Collider stratification leads to biased estimation of the causal effect of exposure on outcome. However, the bias is small relative to the causal relationships between the variables.http://links.lww.com/EDE/B51.Collider bias can be a partial explanation of the obesity paradox, but unlikely to be the main explanation for a reverse direction of an association to a true causal relationship. Alternative explanations of the obesity paradox should be explored. See Video Abstract at  This has been observed in patients with coronary artery disease,5: body fat helping patients survive periods of low nutrition; the nonobese population including patients who have lost weight as a result of more severe illness; body mass index (BMI) poorly representing body fat6; BMI cut-offs not being appropriate7; and obese people being diagnosed earlier.Proposed explanations for the paradox include8 explanation: a correlation induced between an exposure and confounder when stratifying on a third variable (collider) that is associated with, and downstream from, both.9 If the confounder also affects the outcome, conditioning on the collider can induce a false, strengthened, or reversed association between exposure and outcome.This article focuses on the collider stratification bias8 However, it is not known whether the conditions under which effect reversal occurs are realistic. The aim of this article is to investigate the plausibility of collider stratification bias as an explanation for the obesity paradox under a realistic generative model.Existing literature has demonstrated that collider stratification bias can occur in principle.11 Referring to Figure A  and the outcome Y , complicated by a mediator M  and a confounder U, which may be unmeasured. U is assumed  independent of A. Suppose that U and A have distributions FU and FA, respectively. While our derivations allow the variables U and A to take any form, the mathematics is clearer if we consider the binary case, with , , and the other two variables generated by regression equations:We give a general description of collider stratification bias, using the obesity paradox to illustrate, beginning with some definitions from counterfactual causal analysis. logit, with inverse: .with A on Y , conditioned on M being at level m (diabetes status), i.e., a comparison in which A is set  to level a or a* :We are interested in the causal effect of δ represents a difference between the two expectations. However, we calculate the association:where observed at exposure levels a and a*. Effect sizes of interest may be a (log) risk ratio or (log) odds ratio. The obesity paradox explained by collider stratification bias argument follows from the noninequality of the above measures. A scenario of particular interest is when the association is the reverse of the causal effect.in which we compare individuals ΔCE and ΔAS is possible becauseThe nonequality of while because conditioning on M induces a relationship between U and A.In general, 12 the above can be computed:Following similar lines to the study of Vanderweele,ΔCE we haveWith a logit link, for andwhere if .In particular ΔAS:For similarly, and , but the weights differ depending on the status of A and U, so a spurious association may be observed.Both the causal effect and association are a weighted average of M = 1 . We visualized the discrepancy between the association and causal effect for a range of parameter values. The collider bias variables αA, αU, αAU, and βU were varied over a grid from −3 to 3; this range captures the salient features, and covers the range of parameters that may reasonably be observed in practice. We considered two scenarios for βA no causal effect (βA = 0), and some causal effect (βA = 1). Throughout we set pU = pA = 0.5, , and ), so that the prevalences of all variables remain close to 50% . We set βM =βAM = 0, without loss of generality.The model is described by Figure 13; code is in eAppendix 1 (http://links.lww.com/EDE/B48).We presented all effect sizes as odds ratios. The illustrations were produced using R 3.1.0A and Y when there is no causal effect. Figure αA, αU, αAU, and βU, one at a time; in each case the remaining variables are set to 1, except αAU = 0. The left panel of Figure αA is positive , the observed association between A and Y is negative. This represents a bias because the causal effect is zero. In the diabetes example, this means that in a diabetes population where A (obesity) and U both increase the risk of M (diabetes), and U also increases the risk of Y (death), but A has no effect on Y except through M, we observe a negative association between A and Y. Similar results are seen for the other parameters. In Figure αA, while each column corresponds to a value of βU. Within each graph, αU is varied from −3 to 3, and we consider no interaction  and antagonistic interaction . The bottom right panels of Figure αA,αU, and βU parameters are positive, the association between A and Y becomes negative.Figures A and Y when there is a causal effect, βA = 1. Obesity paradox occurs when the association has the opposite sign to the causal effect. The direction of bias between the association and causal effect are the same as when the causal effect was zero. Notably, obesity paradox is happening only for configurations such as αA = 3, αU = 3, βU = 3 .Figures Contrary to much recent literature, our results suggest that collider bias alone cannot fully explain the obesity paradox, with only small discrepancies between the association and the causal effect observed. For large discrepancies to occur , the parameters on the collider bias pathway must be large compared with the true causal effect. This could only happen if the true causal effect is small, and therefore unlikely to be important; or the effect of the unmeasured confounder on both the mediator and the outcome is very large, therefore unlikely to be missed from the analysis.14 also demonstrated that collider bias must be very strong to lead to an association that reverses the causal effect, and Greenland15 gave a formula for calculating the maximum observable bias. Banack and Kaufman16 studied the strength of collider bias required to reverse a particular causal effect. While they concluded that such a reversal was plausible, strong relationships along the collider stratification bias pathway are nevertheless required. Collider stratification bias does not apply when the population is unselected, so our finding is supported by a similar protective effect of obesity in the general population.17Glymour and Vittinghoffx axis in right panel, Figure 19For certain nonzero configurations of the model parameters, there is no bias in the estimation of the causal effect , and continuous exposure (BMI).U and A) were assumed independent; however, dependence between these variables may affect the degree of the collider bias.16 There may be multiple confounding variables; we have considered only one. There may be latent subtypes of the mediating disease.20 Finally, we have not considered the time-varying nature of obesity.We have given a simple exposition here, based on a minimal set of four variables. Two of these variables (21When examining the relationship between an exposure and outcome in a subpopulation, the real interest is in whether this relationship differs from the population as a whole, i.e., the relationship is moderated by the mediator. This can only be assessed by modeling the whole population, with interaction terms between exposure and moderator. However, statistical interaction does not necessarily imply a true biological interaction.Our results show that the paradoxical observation of a protective effect of obesity on mortality is unlikely to be fully explained by collider stratification bias.


Individual differences in working memory capacity (WMC) are associated with speech recognition in adverse conditions, reflecting the need to maintain and process speech fragments until lexical access can be achieved. When working memory resources are engaged in unlocking the lexicon, there is less Cognitive Spare Capacity (CSC) available for higher level processing of speech. CSC is essential for interpreting the linguistic content of speech input and preparing an appropriate response, that is, engaging in conversation. Previously, we showed, using a Cognitive Spare Capacity Test (CSCT) that in young adults with normal hearing, CSC was not generally related to WMC and that when CSC decreased in noise it could be restored by visual cues. In the present study, we investigated CSC in 24 older adults with age-related hearing loss, by administering the CSCT and a battery of cognitive tests. We found generally reduced CSC in older adults with hearing loss compared to the younger group in our previous study, probably because they had poorer cognitive skills and deployed them differently. Importantly, CSC was not reduced in the older group when listening conditions were optimal. Visual cues improved CSC more for this group than for the younger group in our previous study. CSC of older adults with hearing loss was not generally related to WMC but it was consistently related to episodic long term memory, suggesting that the efficiency of this processing bottleneck is important for executive processing of speech in this group. Communication is vital for social participation but may be hampered by adverse listening conditions. Such conditions arise when target speech is accompanied by background noise, possibly including other talkers, and when the listener has a hearing impairment  and without (A-only) a video of the talker's face. Presentation may take place in quiet or in background noise adjusted to a level at which good stimulus intelligibility is maintained. At the end of each list, participants recall two (low memory load) or three numbers (high memory load) depending on instructions eliciting one of two different executive functions (updating and inhibition). In young adults with normal hearing thresholds, CSCT performance is generally better for inhibition than updating, and low than high memory load , 14 males and 10 females, completed the testing. All participants had sensorineural hearing loss (Air-Bone gap < 10 dB HL) and the average pure-tone threshold (PTA4) across 0.5, 1, 2, and 4 kHz was 34.5 dB HL (SD = 3.6), see Figure 4: 26–40 dB HL; WHO, 4: 41–60 dB HL; WHO, Twenty-seven adults with mild-to-moderate hearing loss and no reported tinnitus consented to participate in the study. They were all recruited from the hearing clinic at Linköping University Hospital, Sweden. Two participants opted to drop out of the testing and one participant was excluded due to poor vision. Thus, 24 participants  noise, having the same long term average spectrum as the stimuli (numbers). The modulated noise was the International Speech Testing Signal (ISTS), which contains concatenated short segments of speech in six different languages  for all speech in noise tests with auditory presentation was amplified using the Cambridge prescriptive formula (Cameq) for linear hearing aids  is not included in the scoring. Thus, all scoring in the CSCT is based on correct report, in any order, of two numbers.All participants performed the CSCT with stimulus presentation in quiet (no noise), SSSW noise and ISTS noise. Thus, there were a total of 24 conditions of presentation in the CSCT with two executive tasks , two memory loads , two modalities  of presentation and three noise conditions  in a 2 × 2 × 2 × 3 design.The CSCT was administered using DMDX software . The task prompt remained on screen until the participant pressed a button to continue to the test. At the end of each list, an instruction “Respond now” appeared on the screen and the participant was required to say the target numbers. Corrections to reported numbers were allowed and responses were audio recorded. The participant then pressed another button when they were ready to continue. All the participants practiced each task with two lists before doing the test. The participants were specifically instructed to keep looking at the screen during stimulus presentation. This applied even during presentation in the A-only modality where a fixation cross was provided at center screen. If they looked away from the screen, the test was stopped after presentation of the list and the participants were reinstructed to keep looking at the screen.The participants read series of sentences which appeared on the computer screen one at a time  on the CSCT scores was conducted. The inter-correlation among the cognitive tests and the association between cognitive functions and CSCT was assessed using Pearson's correlations.SD = 1.39). The mean intelligibility levels were 94.5% (SD = 3.0) and 88.3% (SD = 3.0) for the SSSW and ISTS noise, respectively. The difference between these levels was statistically significant, t(46) = 7.05, p < 0.01.The mean SNR for CSCT presentation in noise was −0.17 dB , confirming internal and construct validity.Mean raw scores are shown in Figure F = 30.00, MSE = 0.23, p < 0.001, showing higher CSCT scores in inhibition than updating conditions; memory load, F = 71.71, MSE = 0.35, p < 0.001, showing higher CSCT scores in low than high memory load conditions; modality, F = 26.23, MSE = 0.14, p < 0.001, showing CSCT scores were higher in AV than A-only conditions and noise, F = 23.78, MSE = 0.18, p < 0.001. Pair-wise comparisons with Bonferroni adjustment for multiple comparisons were conducted in order to identify statistically significant differences in performance between the three noise types. They showed that CSCT performance in quiet was better than in both ISTS and SSSW noise (p < 0.05), but there was no difference between performance in ISTS and SSSW noise (p = 0.13). All main effects were in line with our prediction. It should be borne in mind here that intelligibility was significantly higher in SSSW noise than in ISTS noise. To test whether this difference in intelligibility influenced memory performance, we examined whether there was a difference in recall of the first list item in the high memory load conditions when items were presented in SSSW noise compared to ISTS noise. There was no statistically significant difference, t(46) = 0.01, p > 0.05. This suggests that the lack of difference in CSCT performance in SSSW and ISTS noise is not an artifact of intelligibility differences. There were no statistically significant Two-Way or Three-Way interactions.The overall repeated measures ANOVA revealed main effects of all four factors: executive function, SD = 3.20) out of 54 possible responses, demonstrating adherence to instructions. We excluded the delayed recall of reading span score of one participant that was more than two standard deviations above the mean score.Table 4 threshold and age. PTA4 threshold was associated with age, and reading span was associated with letter memory and delayed recall of reading span, see Table Table p = 0.63). However, TRT was associated with performance in inhibition conditions. A higher score in TRT indicates poorer performance. Therefore, the negative correlation shows that better TRT performance is associated with better CSCT performance in the inhibition condition. Working memory as measured by the reading span test correlated significantly with the performance of CSCT in ISTS noise conditions. Reaction times on the congruent trials of the Simon task, which was our measure of processing speed, did not correlate significantly with CSCT performance. We did not include a measure of motor processing speed in the present study and thus we cannot exclude the possibility that cognitive processing speed was confounded by differences in motor skills.Table In the present study we investigated CSC for speech heard in quiet and in noise in adults with hearing loss in AV and A-only modality of presentation. We did this by administering the CSCT  between Simon and CSCT performance under inhibition conditions in a study in which noise was not presented in any condition  and the mean PTA4 3.2 dB HL (SD = 3.2). There were statistically significant differences in age  and PTA4  with the participants in the present study being older and having higher hearing thresholds.The reanalysis included the participants in the present study and the 20 young adult participants in a previous study  test. In order to test simple main effects in accordance with our a-priori hypothesis, planned comparisons were carried out. Independent sample t-tests were used to compare performance across groups on the cognitive test battery.The data were analyzed using a mixed repeated measures ANOVA on CSCT scores with the two groups of participants as a between subjects variable. Where significant interactions were obtained in ANOVAs, the simple main effects observed were investigated using SD = 1.39) and for the young adults, the mean SNR of presentation was −2.17 dB  and 88.3% (SD = 3.0) for the SSSW and ISTS noise respectively. For the young adults, the mean intelligibility level was 93.8% (SD = 3.0) and 92.3% (SD = 2.9) for the SSSW and ISTS noise, respectively,  = 43.86, MSE = 0.01, p < 0.001, indicating that the intelligibility levels were higher in SSSW compared to ISTS noise. The Two-Way interaction between group and noise was also statistically significant, F = 12.49, MSE = 0.01, p = 0.001. Post-hoc Tukey HSD tests assessing this interaction revealed that in the SSSW noise there was no statistically significant difference in intelligibility levels between groups, but in ISTS noise, the intelligibility level for the participants in the present study was significantly lower than that for the young adults with normal hearing, statistically.The mean SNR for CSCT presentation in noise for the older adults with hearing loss was −0.17 dB  = 7.78, MSE = 0.02, p < 0.01, showing that the participants in the present study had lower CSCT scores compared to young adults with normal hearing thresholds. In line with results for the two groups separately, main effects of executive function, F = 35.80, MSE = 0.25, p < 0.001; memory load, F = 93.44, MSE = 0.29, p < 0.001; modality, F = 26.31, MSE = 0.12, p < 0.001, and noise, F = 37.84, MSE = 0.18, p < 0.001, were observed. Pair-wise comparisons with Bonferroni adjustment for multiple comparisons revealed that the CSCT scores in quiet were significantly higher than the scores in ISTS noise (p = 0.001) which in turn were significantly higher than the scores in SSSW noise (p < 0.001). The Two-Way interactions between group and memory load , group and modality , and group and noise  were statistically significant, see Figure Post-hoc Tukey HSD tests assessing these Two-Way interactions revealed, in line with our predictions, that although the participants in the present study had significantly lower CSCT scores compared to the younger adults in high memory load conditions, there was no statistically significant difference in performance between groups in low load conditions. Further, although participants in the present study had significantly lower CSCT scores in the A-only modality than the younger adults, there was no statistically significant difference in performance between groups in AV conditions. Participants in the present study had lower CSCT scores than younger adults in ISTS noise but there was no statistically significant difference in performance between groups in quiet or in SSSW noise.The repeated measures ANOVA revealed a main effect of group, F = 9.25, MSE = 0.11, p < 0.01] and the Three-Way interaction between memory load, modality and noise , see Figure The Two-Way interaction between modality and noise [F2, 8 = 9.25, Post-hoc Tukey's HSD tests investigating the Two-Way interaction between modality and noise revealed that visual cues significantly enhanced performance in SSSW noise (p < 0.01) and ISTS noise (p < 0.01) but not in quiet. Investigation of the Three-Way interaction revealed that the findings of the Two-Way interaction were modulated by memory load. In particular, although visual cues enhanced CSCT scores in ISTS noise (p < 0.05) when memory load was high, this was not the case when memory load was low. Visual cues enhanced CSCT scores in SSSW noise (p < 0.01) in low memory load conditions, but in high memory load conditions the difference in scores did not reach significance with post-hoc testing.t-tests showed that, in line with our prediction, the performance of the participants in the present study was significantly poorer in all the cognitive tests than that of the young adults with normal hearing thresholds (Mishra et al., Independent sample On combining the data of CSCT performance by the older adults with hearing loss in the present study and the young adults without hearing loss in the previous study (Mishra et al., The findings of the present study further our understanding of CSC. Using the CSCT, we found lower CSC in older adults with hearing loss compared to the younger adults with normal hearing whom we had tested with the same experimental paradigm in a previous study (Mishra et al., As we had predicted, factors previously found to decrease CSC in young adults with normal hearing (Mishra et al., The finding of a stronger effect of increasing memory load on CSC for older adults with hearing loss compared to younger adults with normal hearing is in tune with work showing differences in the deployment of cognitive resources for younger and older adults in response to changes in memory load. In particular, imaging studies have shown that older adults display a greater increase in brain activity in response to cognitive load than younger adults, and that brain activity reaches a plateau at a lower level in older adults than in younger adults Grady, .Importantly, visual cues enhanced CSC for older adults with hearing loss. Further, this effect was stronger for this group than for young adults with normal hearing. This finding demonstrates that for older adults with hearing loss, visual cues can support the kind of executive processing of speech that may be used in everyday conversation (c.f. Frtusova et al., It is worth noticing that although the older adults with hearing loss generally had reduced CSC compared to young adults; this did not apply in all conditions. When CSCT tasks were performed in quiet, with low memory load and in presence of visual cues, there was no significant difference across groups. This suggests that when listening conditions are optimized, there is no difference in CSC between older adults with hearing loss and younger adults with normal hearing.In the present study, we found that better CSCT performance in older adults with hearing loss was associated with better updating skills, but only in those conditions in which the participants performed worse than the younger adults with normal hearing who took part in our previous study using the same experimental paradigm (Mishra et al., Older adults with hearing loss have lower CSC than young adults without hearing loss, probably because they have poorer cognitive skills and deploy them differently. However, visual cues and efficient episodic LTM enhance CSC more for the older group.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.


Hearing loss affects 360 million people worldwide and it is estimated that this number will exceed 900 million by 2025 . Hearing loss has different etiologies; it severely affects quality of life by reducing individual communication, a fact that has different implications depending on age.Age-related hearing loss (ARHL), also called presbycusis, is an increasing health, social, and economic problem as the affected population represents a continuously increasing percentage of the world population. Associated with ARHL is an acceleration of cognitive decline, and its links with developing neurodegenerative diseases, including Alzheimer's and frailty, are currently under study. Excessive exposure to noise and/or ototoxic drugs are additional factors in the worldwide increase of ARHL. Both noise-induced hearing loss (NIHL) and ARHL share common molecular mechanisms that involve redox imbalance and inflammation.Sensorineural hearing loss is associated with damage or death of cochlear cells, including neurons and sensory hair cells. Hearing insults decrease cell survival pathways and promote apoptotic programs. In the first article of this eBook, “Aging, neurogenesis and neuroinflammation in hearing loss and protection,” the mechanisms behind sensorineural cell damage are reviewed (Wong and Ryan, People suffering from profound hearing loss can benefit from using hearing aids, the importance of which is being recognized by numerous awards including the 2013 Lasker-DeBakey Clinical Medical Research and the 2015 Fritz J. and Dolores H. Russ Prize Awards for the development of the modern cochlear implant. There is no other specific therapy available, but there is a boom of research efforts aimed at developing cell therapy, gene therapy, and small-molecule based pharmacological approaches. These exciting developments are based on the knowledge generated from basic neurobiology and developmental studies. Laboratory animals are essential for generating accurate models of human hearing loss. One of the articles included in this eBook describes novel models for studying NIHL, based on the use of different sound stimuli, which provide solid ground on which to study potential therapeutic molecules (Sanz et al., Finally, authors herein illustrate and discuss small molecule-based novel therapies directed to confer otoprotection or reduce injury. The role of oxidative stress in hearing loss has prompted study of the potential of a combination of antioxidants and vasodilators on hearing loss remediation (Alvarado et al., This work was supported by Spanish grant from the Ministerio de Economía y Competitividad SAF2014-53979-R and European FP7-PEOPLE-IAPP-TARGEAR. The cost of this publication has been paid in part by FEDER funds.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.


GJB2) to the genetic load of deafness and its mutation spectra vary among different ethnic groups.The contribution of Gap junction beta-2 protein  among GJB2 mutations, followed by p.R143W (26.8%) and p.V37I (9.8%). Among those probands without the p.V37I allele in a trans configuration who showed some degree of residual hearing, the mean air conduction thresholds at 250 and 500 Hz were 57 dB HL and 77.8 dB HL, respectively. The c.235delC mutation showed a particularly wide spectrum of hearing loss, from mild to profound and significantly better hearing thresholds at 250 Hz and 2k Hz than in the non-p.V37I and non-235delC nonsyndromic hearing loss and deafness 1(DFNB1) subjects.Of the 130 subjects, 22 (16.9%) were found to carry at least one mutant allele of Despite its reputation as the cause of severe to profound deafness, c.235delC, the most frequent DFNB1 mutation in our cohort, caused a wide range of hearing loss with some residual hearing in low frequencies. This finding can be of paramount help for prediction of low frequency hearing thresholds in very young DFNB1 patients and highlights the importance of soft surgery for cochlear implantation in these patients. The incidence of profound pre-lingual hearing loss is about 1 per 1000 at birth and 1 per 300 children by 4 years of age .GJB2) , which encodes connexin 26, are responsible for nearly half of the cases of hearing loss in many populations ) and p.V37I (4/41 [9.8%])  . The mos [9.8%]) . These tr cohort .GJB2 mutation (+) group (n = 22) consisted of 14 males (63.6%) and 8 females (36.4%) with ages ranging from 9 months to 25 years; 95.5% (21/22) of the subjects were within the age range of 0–15 years. The mean age at recruitment to the study was 3.1 (±3.5) years. Among these 22 subjects, 19 subjects were confirmed to be DFNB1, carrying two mutant alleles of GJB2 either as a homozygous or a compound heterozygous state  showed clearly better hearing thresholds than the other DFNB1 subjects (trans configuration with p.V37I showed the best threshold (mean threshold: 24.2 dB), as compared to the other subjects with p.V37I  exhibited a mean hearing threshold of 93.5 dB , again csubjects . Breakinquencies . Althougquencies . When wequencies .GJB2 are known to be responsible  for non-syndromic SNHL, which is commonly related with a severe to profound degree of autosomal recessive inheritance ..GJB2 mutGJB2 mutant alleles associated with the better residual hearing.Furthermore, two homozygotes for c.235delC showed a significantly different degree of SNHL . Such phGJB2-related hearing loss showed a better auditory outcome after cochlear implantation than age-matched children [GJB2 can potentially provide us the clue to the audiologic phenotype, especially at low frequencies which is sometimes not feasible to evaluate in infants.The presence of residual hearing can affect the planning and placement of cochlear implants. Recent studies demonstrated that children with children . This wachildren  or an ischildren , 40. OurGJB2 mutations. This can be of importance when planning the insertion depth of cochlear implants in infants, in whom the measurement of low-frequency hearing thresholds may not be feasible.This study showed wide range of hearing loss and low frequencies residual hearing in the DFNB1 subjects with the c.235delC mutation. Especially for patients with residual hearing, it is important to try to preserve this residual hearing during cochlear implantation. Clinicians should keep in mind that there may be substantial residual hearing, especially at low frequencies, in DFNB1 subjects related to c.235delC in Koreans. A further study with a larger cohort and more longitudinal data will improve our knowledge on the residual hearing associated with various S1 FigAll three patients showed profound hearing loss and, rarely, residual hearing.(TIF)Click here for additional data file.


Chlamydomonas reinhardtii, CrDGTT2, can produce a large amount of TAG when expressed in yeast or higher plants, suggesting a unique ability of CrDGTT2 to enhance oil production in a heterologous system. Here, we performed metabolic engineering in Saccharomyces cerevisiae by taking advantage of CrDGTT2. We suppressed membrane phospholipid biosynthesis at the log phase by mutating OPI3, enhanced TAG biosynthetic pathway at the stationary phase by overexpressing PAH1 and CrDGTT2, and suppressed TAG hydrolysis on growth resumption from the stationary phase by knocking out DGK1. The resulting engineered yeast cells accumulated about 70-fold of TAG compared with wild type cells. Moreover, TAG production was sustainable. Our results demonstrated the enhanced and sustainable TAG production in the yeast synthetic platform.The current fascination with algal biofuel production stems from a high lipid biosynthetic capacity and little conflict with land plant cultivation. However, the mechanisms which enable algae to accumulate massive oil remain elusive. An enzyme for triacylglycerol (TAG) biosynthesis in  Saccharomyces cerevisiae is an advanced model unicellular eukaryotic microorganism for metabolic engineering as well as basic molecular biological studies on metabolism.Biofuel production is highly demanded as an alternative to the limited fossil fuels. The current fascination with eukaryotic algae as a resource of biofuel production stems from a high lipid biosynthetic capacity and little competition for land required for plant cultivation. However, technical difficulty in gene manipulation of many eukaryotic algae species hampers potential usefulness of these organisms as a synthetic platform of metabolic engineering to produce biofuel. On the other hand, sn-1,2-diacylglycerol (DAG) by the catalysis of acyltransferase in many organisms such as S. cerevisiae, Chlamydomonas reinhardtii, and Arabidopsis thaliana  is a major source of biodiesel  and Type 2 with five isogenes (CrDGTT1-5)  unless otherwise stated. Cells harboring the URA3 and LEU2 marker plasmids were grown in synthetic complete media lacking uracil and leucine . The OD600 in Figure 600 of 0.1 and cultured for 60 h at 30°C. In Figures 600 of 0.05 and grown for 60 h to the late stationary phase, then time-course observation was started. The dilution was conducted twice at 60 h intervals. Data are mean±SD from three biological replicates.Cells were grown in YPD media  was performed using Phusion polymerase  under the following condition; an initial denaturing step of 5 min at 95°C followed by 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 3 min and then a final extension at 72°C for 5 min. The plasmids and primers are listed in Table The yeast expression vector pCH108 was created by replacing the ScPAH1 (YMR165C) was amplified with the primers CH367 and CH368, cloned into pENTR/D-TOPO , digested with AscI and NotI, and inserted into AscI/NotI sites of pCH108 to construct pCH109. This plasmid contains GPD promoter for constitutive overexpression , a 932-bp fragment of opi3Δ::HIS3 was amplified with the primers CH430 and CH431 from pRS313, and transformed into dgk1Δ::KANMX. The plasmid pCH109, which overexpresses ScPAH1 (OE-PAH1), was transformed into CHY034  to produce CHY138 (Δdgk1Δopi3 OE-PAH1). Then, the plasmid pCH091, which overexpresses CrDGTT2 (OE-CrDGTT2), was transformed into CHY138 to produce CHY101, CHY140, CHY141, and CHY142 (Δdgk1Δopi3 OE-PAH1 OE-CrDGTT2). The transformation was performed as described previously by mixing the plasmid with the PLATE reagent, salmon sperm DNA and yeast cells . Cell suspension was vortexed vigorously and left for 30 min at room temperature. Organic phase was recovered and residual lipids in cell pellet were extracted twice by vigorous vortex with chloroform. The combined organic extracts were vortexed with 1% KCl solution, and separated organic phase was washed once with dH2O. Lipids were dried up under nitrogen stream, dissolved in chloroform:methanol  and stored at −30°C till use. Each lipid class was separated by thin layer chromatography . Separation of TAG was as described previously . Cells were lyophilized using freeze drier  and dry cell weight was measured with fine weighing scale . Prior to the lipid extraction, cells were treated with pre-heated isopropanol containing 0.01% butylated hydroxytoluene at 75°C for 15 min to inactivate phospholipase activity. Total lipids were extracted essentially as described previously using chloroform-methanol solvent system  containing 8% glucose at 30°C for 48 h. Samples were frozen in a high-pressure freezer (Leica EM PACT2) at 2000–2050 bar. Freeze-substitution was performed in anhydrous ethanol  with an automatic freeze substitution system (Leica EM AFS2). The samples were first kept at −85°C for 3 days, then switched to −60°C, −20°C, 0°C, and room temperature at 1-day intervals. After 2 times of rinse with ethanol for 12 h, the samples were embedded by infiltrating LR White resin. Ultrathin sections (70–90 nm) were prepared with use of Reichert Ultracut S or Lecia EM UC7  and collected on 100-mesh copper grids. The sections were stained with 5% uranyl acetate in 50% methanol for 10 min and 0.4% lead citrate for 4–6 min. Sections were observed under a transmission electron microscope (Philips CM 100) at 80 KV and images were taken with use of a Gatan Orius CCD camera.S. cerevisiae by considering the following 3 points: (1) suppressing metabolic flux to membrane lipid biosynthesis at the log phase, (2) enhancing metabolic flux to TAG biosynthesis at the stationary phase, and (3) suppressing TAG hydrolysis for membrane lipid biosynthesis on growth resumption to maintain accumulated TAG  as compared with the wild type  biosynthesis by knocking out OPI3, which encodes an enzyme catalyzing the final step of PC biosynthesis  to stimulate PA to DAG conversion . The TAG level was increased 9.7-fold in this strain as compared with the wild type  by transforming the plasmid to overexpress CrDGTT2 into the Δdgk1Δopi3 OE-PAH1. When maximal TAG level is produced at late stationary phase (60 h), these cells produced 64.8- to 73.2-fold increase  as a substrate to produce phospholipids which are required for rapid growth of cellular membranes (Athenstaedt and Daum, dgk1Δopi3 OE-PAH1 OE-CrDGTT2 cells, quantity (Figure dgk1Δopi3 OE-PAH1 OE-CrDGTT2 than the wild type (Figure dgk1Δopi3 OE-PAH1 OE-CrDGTT2 cells reached stationary phase at 48 h after growth resumption with OD600 of 4, whereas wild type cells reached stationary phase at 36 h with OD600 of 10. At 60 h, these cultures were diluted again to resume the growth, which reproduced similar growth profile through the following two repeats of growth resumption. TAG level of the Δdgk1Δopi3 OE-PAH1 OE-CrDGTT2 cells in full growth at 60 h was 13.2 to 14.5% of dry cell weight (Figure dgk1Δopi3 OE-PAH1 OE-CrDGTT2 cells could grow with reduced degradation of accumulated TAG, which favored sustainable TAG production.To demonstrate whether sustainable TAG production is possible with the Δy Figure  and qualy Figure  of TAG wy Figure  through e Figure ; the ΔdgS. cerevisiae resulted in about 70-fold increase in TAG level, which is estimated to be 12.2% of dry cell weight (Figure S. cerevisiae. For example, overexpression of a glycerol kinase and DAG acyltransferases achieved 8.2% of TAG with 2% glycerol as carbon source (Yu et al., S. cerevisiae produces similar amounts of TAG and sterylesters as major lipid content (Beopoulos et al., S. cerevisiae strain diluted from full growth resumed growth with TAG accumulated (Figure Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, and Yarrowia lipolytica can produce up to 20% lipids per dry biomass (Runguphan and Keasling, dgk1Δopi3 OE-PAH1 OE-CrDGTT2 cells were not superior to these oleaginous microorganisms, several advantages of using S. cerevisiae over other yeasts or microalgae have been pointed, such as genetic tractability, commercially available whole-genome deletion strain collection or a proven track record in different industrial applications (Tang et al., S. cerevisiae. Since algal species are highly diverse, a robust enzyme similar to CrDGTT2 may be found in other oleaginous algal species. Since CrDGTT2 showed much higher expression level than any other DGTT isoforms in S. cerevisiae even though they were all expressed by the same promoter system, it is possible that the robustness of CrDGTT2 may be due to higher stability of mRNA in a heterologous system (Hung et al., Our metabolic engineering to overexpress an algal gene in genetically manipulated yeast t Figure . This yid Figure . This suCrDGTT2, to reconstitute enhanced and sustainable TAG production in a yeast S. cerevisiae by metabolic engineering.In conclusion, we employed an algal gene resource, YN conceived research; KK and YN designed experiments; CH performed experiments and analyzed data; KK provided technical assistance to CH, KK, and YN wrote the manuscript; all author commented on the manuscript and approved the contents.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.


Mycoplasma hyopneumoniae infections in pigs. However, it is not known whether the efficacy of vaccinating against M. hyopneumoniae can be influenced by the weaning process when vaccination is applied at the day of weaning. The present study assessed the efficacy of a single M. hyopneumoniae vaccination (Ingelvac MycoFLEX®) three days before weaning (V1) or at weaning (V2) against experimental challenge infection. Four weeks after vaccination, groups V1 and V2 (n = 20 pigs each) and a non-vaccinated, positive control group (PCG) (n = 20) were endotracheally inoculated with a virulent M. hyopneumoniae field strain. Five pigs were used as a negative control group. All pigs were euthanized 5 weeks after challenge. The main parameters investigated included macroscopic and histopathological lung lesions at necropsy, immunofluorescence (IF) staining and quantitative real-time PCR (qPCR) on broncho-alveolar lavage (BAL) fluid for quantifying M. hyopneumoniae.Commercial bacterins are widely used at weaning to control P > 0.05). The average lymphohistiocytic infiltration scores in groups V1, V2 and PCG were 2.95, 3.16 and 3.61, respectively (P < 0.05). The average IF scores were: V1 = 1.13, V2 = 1.19 and PCG = 1.25 (P > 0.05), the qPCR values were: V1 = 102.94, V2 = 102.76 and PCG = 103.23 (P > 0.05). All pigs of the negative control group remained negative throughout the study.The average macroscopic lung lesion scores in groups V1, V2 and PCG were 0.54, 0.88 and 1.04, respectively (M. hyopneumoniae organisms in the BAL fluid compared to the PCG. However, no firm conclusions could be made on whether weaning negatively influences the efficacy of M. hyopneumoniae vaccination, since significant differences between the treatment groups were only obtained for the histopathological lung lesions. This could be attributed to the fact that milder macroscopic lung lesions were produced in the inoculated pigs, when compared to previous trials conducted by the same group. Further research under field conditions is warranted to assess possible differences between the two vaccination strategies.Both vaccinated groups had lower numbers of macroscopic and histopathological lung lesions, and lower numbers of  Mycoplasma hyopneumoniae is the etiological agent of enzootic pneumonia (EP), a chronic respiratory disease affecting mainly grow-finishing pigs [M. hyopneumoniae infections have been detected in almost all countries with intensive production systems and are responsible for major economic losses in the pig industry [Pasteurella multocida, Actinobacillus pleuropneumoniae, Trueperella pyogenes and Streptococcus suis [M. hyopneumoniae is also an important pathogen involved in the porcine respiratory disease complex (PRDC) [ing pigs , 2. M. hindustry , 3. Thescus suis . M. hyopx (PRDC) , 6.M. hyopneumoniae infections can be achieved in a number of ways, such as optimizing management and housing conditions, antimicrobial medication and vaccination. In many countries, more than 70 % of pig herds are vaccinated against M. hyopneumoniae in an effort to control the disease [Control of  disease –12. DiffCurrently, both double and single vaccination strategies are widely practiced. Single vaccination is often administered either at 1 week of age or at weaning. Vaccination is often done at weaning because the piglets are handled then anyway, so vaccination can easily be included in routine farm management practices , 13. On M. hyopneumoniae can be influenced by the weaning process when vaccination is applied at the day of weaning. The objective of this trial was to investigate the efficacy of a one-shot vaccination applied either at weaning or three days before weaning in pigs experimentally infected with a virulent M. hyopneumoniae field strain.Consequently, one important question that remains to be answered, and whose answer could contribute to the optimization of existing vaccination schemes, is whether the efficacy of vaccinating against P > 0.05).In total, two pigs died during the trial, after receiving the endotracheal challenge infection (one in the V2 group and one in the PCG group). These pigs were not included in the analysis. In groups V1, V2 and PCG, coughing started approximately six days after challenge and continued to increase towards the end of the trial. In groups V1 and V2, coughing peaked between 66 and 70 days of age, whereas in the PCG, the peak was noticed at 80 days of age. The respiratory disease score (RDS) values for the period before challenge and the period after challenge, as well as for the overall period, are shown in Table P > 0.05) between the groups.The average daily weight gain (ADG) results of groups V1, V2 and PCG during the period before challenge and during the period after challenge, as well as during the overall period, are presented in Table P > 0.05) were found between groups V1, V2 and PCG  in histopathological lung lesions were present between groups V1, V2 and PCG , 1.19 (group V2) and 1.25 (PCG), were not significantly different (P > 0.05) between groups (Table The results from the IF testing that was used to semi-quantitatively assess the load of M. hyopneumoniae organisms quantified by qPCR in the BAL fluid was not significantly different (P > 0.05) between groups V1, V2 and PCG  compared to group PCG (103.23 copies/mL). No M. hyopneumoniae DNA was detected in pigs of the negative control group.The number of CG Table . Lower nBordetella bronchiseptica  and polybacterial cultures  were obtained. In the PCG, Haemophilus parasuis (1/20 pigs), S. suis (1/20 pigs) and polybacterial cultures (1/20 pigs) were obtained. The bacteriological culture remained negative for all pigs in the negative control group.Several bacteria were isolated from BAL fluid. In groups V1 and V2, M. hyopneumoniae at 21, 48 and 83 days of age are presented in Table M. hyopneumoniae. At 48 days of age, 40 % of the pigs in each of the vaccinated groups V1 and V2 had seroconverted. None of the pigs in the PCG had seroconverted to M. hyopneumoniae at that time. At 83 days of age, all the pigs in groups V1 and V2 were seropositive, while 95 % of the pigs in the PCG had seroconverted. All pigs in the negative control group were serologically negative at all time-points.The serological results for The present experimental trial investigated whether the efficacy of one-shot vaccination is influenced by the weaning process. Differences between V1 (vaccination three days before weaning), V2 (vaccination at weaning) and the PCG (no vacination) were small and mostly statistically not significant, except for the microscopic lung lesions, which were lower in V1.Apart from the transport related to the weaning process, the piglets in the present study were also transported to the experimental facilities, a trip that lasted anywhere from one-half to two hours. In fact, age- and site-segregated pork production is a complex process that involves movement of the piglets from the farrowing house to the nursery facilities, which are situated in different locations . It can Vaccination at the moment of weaning, when the piglets are being handled anyway, is a common practice, as it can be implemented easily in the daily management of a pig herd. The pigs of V1 were vaccinated three days before weaning. This vaccination strategy is also commonly practiced by some pig producers in order to avoid any possible negative effects of the weaning process, and/or to avoid too much handling or interventions on the same day . A diffeM. hyopneumoniae was similar to that used in previous trials [Mycoplasma-like macroscopic lung lesions at necropsy and were positive for IF staining. However, the macroscopic lung lesions observed in the present trial were milder compared to trials that had been previously conducted using the same inoculation dose of M. hyopneumoniae F7.2C field strain, experimental facilities and way of allocation [M. hyopneumoniae throughout the trial, thus confirming that the study piglets were free of M. hyopneumoniae.The experimental infection model used for s trials –24. Challocation –25. The The RDS values in the PCG were similar to those mentioned in other experimental trials , 24. ComM. hyopneumoniae vaccination is effective in reducing lymphohistiocytic infiltration and increasing the percentage of air in the lung. This reduction of the lymphohistiocytic infiltration scores in the vaccinated groups is in accordance with previous experimental trials [M. hyopneumoniae reduces the infiltration of macrophages in the lung tissue [Group V1 had the lowest numbers of macroscopic and histopathological lung lesions, and the lowest IF scores when compared to groups V2 and PCG. Nevertheless, significant differences between groups V1, V2 and PCG were only obtained for the histopathological lung lesions. This was confirmed by the results of the percentage of air parameter in the lung tissue measured by means of an automatic image analysis system. The statistically significant differences between groups V1, V2 and PCG proved that l trials , 24. Theg tissue .M. hyopneumoniae in the BAL fluid of groups V1 and V2 confirms that vaccination alone is not able to prevent colonization of the pig’s respiratory tract [Although lower qPCR values were recorded in the vaccinated groups when compared to the PCG, it was not possible to conclude whether vaccination was associated with the reduced bacterial load in the lungs, since the differences between the groups were only numerical. However, the detection of ry tract , 22, 24.M. hyopneumoniae on the day of transportation to the experimental facilities. At the time of inoculation, 40 % of the pigs in each of the vaccinated groups V1 and V2 had seroconverted. This is in agreement with other studies which have reported that vaccination with commercial bacterins induces seroconversion rates ranging from 30 to 100 % [The pigs of groups V1, V2 and PCG were seronegative for to 100 % . At necrM. hyopneumoniae vaccination. Field trials make it possible to include more animals and to study the entire course of M. hyopneumoniae infections up to the time of slaughter.Overall, no firm conclusions could be made as to whether group V1 performed better than groups V2 and PCG, since only the microscopic lung lesions were significantly lower in the pigs in group V1. Further research under field conditions is necessary to elucidate the possible effect of weaning on the efficacy of one-shot M. hyopneumoniae. A future field trial could possibly provide further insight on the same topic.In conclusion, the differences between the vaccinated groups V1 and V2 were small and mostly statistically not significant. Thus, no firm conclusions could be made as to whether the process of weaning had a negative impact on the efficacy of vaccinating against M. hyopneumoniae and PRRSV. Sows and fattening pigs of different age groups in the herd had been repeatedly monitored clinically and serologically, and then by means of macroscopic pneumonia lung lesions at slaughter, and no evidence for the presence of M. hyopneumoniae or PRRSV in the herd was found at 3 weeks of age (day seven), the piglets were weaned and transported for one-half to two hours to the animal facilities of the Faculty of Veterinary Medicine, Ghent University, Belgium. From this point onwards, all the pigs received ad libitum a commercial, antibiotic-free feed.Sixty-five cross-bred piglets (Topigs 20 sows and Pietrain boars) were purchased from a herd that was free of n = 20), V2  and a non-vaccinated positive control group . Five pigs were used as a negative control group, to verify that they remained free of M. hyopneumoniae. They were not vaccinated. The pigs in groups V1 and V2 received a single-shot intramuscular (i.m.) injection (1 mL) of a commercial M. hyopneumoniae bacterin vaccine . The pigs in PCG were left untreated and were not injected with a placebo to simulate field conditions. Upon arrival at the Faculty of Veterinary Medicine, each group was housed in a different experimental room equipped with a high efficiency particulate air filter to avoid possible transmission of the pathogen between groups.At 14 days of age, prior to purchase, piglets from 20 different litters were individually ear-tagged and randomly allocated to four different groups. The three main treatment groups were: V1 .Each treatment group was examined daily for a minimum of 15 min for the presence of clinical signs throughout the entire trial period. Coughing severity was evaluated in a blinded manner by means of an RDS on a scale from 0 to 6 . AccordiIndividual bodyweights were measured at 20, 48 and 83 days of age to determine the ADG of each treatment group during the three following periods: from day 20 until day 48, from day 48 until day 83 and from day 20 until day 83.Mycoplasma-like macroscopic lung lesions were quantified in a blinded manner using a lung lesion score diagram [After necropsy,  diagram . TheoretTwo lung tissue samples per lobe  were collected from the right lung of each pig for histopathology and IF testing. Samples were collected from the edges of the lung lesions, if present .M. hyopneumoniae lesions were scored on a scale from 1 to 5 [Mycoplasma infections. Scores 3, 4 and 5 were considered to be associated with Mycoplasma infections  pneumonia, surrounding the bronchioles but extending to the interstitium, with lymphofollicular infiltration and mixed inflammatory cell exudates). After three lobes per pig were investigated, the average score per treatment group was calculated.For histopathology, the tissue was fixed in 10 % neutral buffered formalin and routinely processed and embedded in paraffin. One slide was made per lobe sample. Using light microscopy, the severity of peribronchiolar and perivascular lymphohistiocytic infiltration and nodule formation consistent with m 1 to 5 . Scores The percentage of lung area occupied by air (percentage air) was examined by means of an automatic image analysis system . This percentage is inversely proportional to the severity of peribronchiolar and perivascular lymphohistiocytic infiltration in the lung tissue and the amount of intrabronchiolar – intrabronchial exudate. An average percentage per treatment group was calculated .M. hyopneumoniae in the lungs. The scores given could range from 0 to 3: 0 (no IF), 1 (limited IF), 2 (moderate IF), and 3 (intense IF) [A direct IF assay was performed to assess the presence of ense IF) .M. hyopneumoniae organisms by qPCR [M. hyopneumoniae DNA was used. Values below the last dilution were considered as negative.During necropsy, BAL fluid was collected from the left part of the lungs before the tissue samples for histopathology and IF were taken . The rec by qPCR . DNA wasActinobacillus and Haemophilus spp., and other pathogens which may affect the respiratory tract, such as S. suis, B. bronchiseptica, P. multocida and T. pyogenes [The second aliquot of BAL fluid was used for routine bacteriological culture to detect the presence of pyogenes .M. hyopneumoniae, using a blocking ELISA  [buffer-control were considered positive, while sera with OD values ≥ 50 % of the ODbuffer-control were considered negative.Blood samples were collected from all pigs at 21, 48 and 83 days of age to measure antibodies against oid, UK) . Sera win = 5) were not included, as they were only employed to ensure that the animals remained free of M. hyopneumoniae throughout the experiment. The RDS data were analyzed using repeated measures analysis of variance (ANOVA). Levene’s test was used to assess the homogeneity of the variances between the different groups. The ADG, macroscopic and histopathological lung lesions, percentage of air in the lungs, IF testing and qPCR were analyzed with a non-parametric Kruskal-Wallis ANOVA. These five continuous variables did not fulfill the criteria for homogeneity of variances. Statistical results were considered significant when the P-values were ≤ 0.05 (two-sided test). The statistical package SPSS 21.0 for Windows  was used to analyze the data.The statistical analysis was performed on groups V1, V2 and PCG. Pigs of the negative control group (


Cannabis is one of the most prevalent drugs used in industrialized countries. The main effects of Cannabis are mediated by two major exogenous cannabinoids: ∆9-tetrahydroxycannabinol and cannabidiol. They act on specific endocannabinoid receptors, especially types 1 and 2. Mammals are endowed with a functional cannabinoid system including cannabinoid receptors, ligands, and enzymes. This endocannabinoid signaling pathway is involved in both physiological and pathophysiological conditions with a main role in the biology of the central nervous system. As the retina is a part of the central nervous system due to its embryonic origin, we aim at providing the relevance of studying the endocannabinoid system in the retina. Here, we review the distribution of the cannabinoid receptors, ligands, and enzymes in the retina and focus on the role of the cannabinoid system in retinal neurobiology. This review describes the presence of the cannabinoid system in critical stages of retinal processing and its broad involvement in retinal neurotransmission, neuroplasticity, and neuroprotection. Accordingly, we support the use of synthetic cannabinoids as new neuroprotective drugs to prevent and treat retinal diseases. Finally, we argue for the relevance of functional retinal measures in cannabis users to evaluate the impact of cannabis use on human retinal processing. The cellular level of these ligands is regulated by several enzymes, especially fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MGL), and cyclooxygenase-2 (COX-2) [Cannabis is one of the most prevalent drugs used worldwide. The main constituents of cannabis are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) –3. They  (COX-2) . Togethe (COX-2) , 15. As  (COX-2) –18. For  (COX-2) . Consequ (COX-2) . Afterwa (COX-2) , 14. Exo (COX-2) , 14. In  (COX-2) .The retina is anatomically and developmentally an extension of the CNS, and the retina and the brain are connected by the optic nerve, the axons of the ganglion cells, through the lateral geniculate nucleus . The retIn the CNS, the manipulation of endocannabinoids has been reported to attenuate brain damages induced by a variety of insults. Moreover, the cannabinoid system signaling pathways is considered as a critical process involved in neuron survival. For instance, an upregulation of the endocannabinoid system is known as an adaptive response that attenuates CNS damages in the context of trauma and neurodegenerative diseases , 37 and The cannabinoid system has been detected in ocular tissues and other critical stages of visual information processing. In ocular tissues, CB1 receptors have been localized in the ciliary body of rat, bovine, and human and in the trabecular meshwork in bovine and human –42. CB1 This review focuses on the neurobiological role of the cannabinoid system in the retina. We first review the distribution of the cannabinoid receptors, ligands, and enzymes in the retina. Then, we review studies that have examined the role of the cannabinoid system in retinal neurotransmission, neuroplasticity, and neuroprotection. Based on these results, we argue for the development of synthetic cannabinoids as therapeutic agents for the treatment and the prevention of retinal diseases. Finally, we support potential effects of cannabis use on human retinal processing and we present several functional measurements allowing the rigorous assessment of the retinal function in cannabis users.In order to thoroughly explore the role of the cannabinoid system in the retina, a search for relevant articles was conducted in the PubMed, ScienceDirect, and Google Scholar databases using the following keywords: (“cannabinoid system” or “endocannabinoids” or “cannabinoids” or “cannabinoid ligands” or “cannabinoid receptors” or “cannabinoid enzymes”) and (“neurotransmission” or “neuroplasticity” or “neuroprotection”) and . All results up to June 1, 2015, were examined for the selection process. Relevant publications were chosen through an individual independent selection of titles by the following authors: Thomas Schwitzer, Raymund Schwan, Anne Giersch, and Vincent Laprevote. The articles selected had to be written in English and be related to the topic of the review. Additionally, a manual search was performed on the bibliography of each selected article.Numerous studies have shown the presence of the cannabinoid receptors CB1 and CB2 in the retina of several animal species such as tiger salamander, goldfish, rat, mouse, chick, and monkey , 48–58. Although the presynaptic location of CB1 receptors was readily described, especially in gabaergic and glutamatergic neurons, there are several evidences also indicating a postsynaptic localization. For example, Chaves et al. showed that retinal ablation induced an increase in level of CB1 proteins in the optic tectum and other retinorecipient visual areas in the adult chick brain with no change in CB1 mRNA levels . This inCB2 receptors are detected in the retina of several species. In the rat retina, CB2 receptors are expressed in photoreceptors, horizontal cells, amacrine cells, inner nuclear layer, inner plexiform layer, retinal pigment epithelium, and retinal ganglion cell layer, especially in the somas of retinal ganglion cells , 55. In In addition, CB1 and CB2 receptors are expressed in the human retina. CB1 receptors are detected in outer segments of photoreceptor cells, inner plexiform layer, outer plexiform layer, two synaptic layers of the retina, inner nuclear layer, ganglion cell layer, and retinal pigment epithelium cells , 43, 60.The two main endogenous cannabinoid ligands acting on cannabinoid receptors, namely, anandamide and 2-AG, are found in the retina of tiger salamander, goldfish, rat, mouse, chick, bovine, porcine, and monkey , 61, 62.The cellular level of retinal endocannabinoids is regulated by several main enzymes: FAAH, MGL, and COX-2. These enzymes are detected in the retina and enable the degradation of cannabinoid ligands . FAAH isPreliminary findings are consistent with the detection of MGL in inner plexiform layer, rod bipolar cells, amacrine cells, and ganglion cells in the mouse retina , 64. Simα has been detected in the two synaptic layers, the outer plexiform layer, and the inner plexiform layer of the mouse retina [α is localized in postsynaptic terminals of type 1 OFF cone bipolar cells whereas the expression of DAGLβ appears to be restricted to retinal blood vessels in the mouse retina [α is detected early in postnatal development in the rat retina in photoreceptors—cones and rods—cone bipolar cells, horizontal, amacrine, and ganglion cells [Enzymes allowing the synthesis of cannabinoid ligands are also expressed in the retina , 67. Fore retina . Importae retina . DAGLα iThe endocannabinoid system is detected in critical stages of retinal information processing such as photoreceptors, bipolar cells, and ganglion cells. These findings support a role of endocannabinoids in the modulation of retinal neurobiology as well as in the regulation of vertical transmission of the retinal information.Neurotransmission is characterized by the transmission of a nerve impulse across a synapse. The modulation of ionic channels or enzymatic activity, to name a few, can affect neurotransmission. Previous studies have outlined the involvement of the cannabinoid system in these mechanisms, thus allowing the regulation of the retinal neurotransmission , 15.Several inward and outward ionic channels are known to play a major role in retinal physiology . For exaA direct action of cannabinoids on retinal enzymatic activity or retinal transmitter release has also been described , 75–79. Finally, studies on the goldfish retina argue for a role of the cannabinoid system in the regulation of the phototransduction cascade, the dark and light retinal sensitivity, the dark and light retinal adaptation, and the retinal contrast sensitivity , 70, 80.The role of the cannabinoid system in the regulation of short-term and long-term plasticity in the CNS has been readily described –84. HoweNeuroprotection is a protecting mechanism that consists in preventing the death of damaged neurons and their degeneration, due to a hostile environment created by an initial cellular stress. Using several models of retinal diseases, mounting evidence suggests that the retinal cannabinoid system might play a neuroprotective role in the retina. Oxidative stress is known to be a key mechanism in the pathological process of age-related macular degeneration (AMD) and diabetic retinopathy (DR) , 86. In According to the large distribution of endocannabinoids in the retina and considering the role of cannabinoids in modulating the retinal neurophysiology, it is now crucial to consider potential effects of cannabis, after both acute and regular use, on the structural and functional characteristics of the human retina , 31. The−2 (cds·s·m−2) [The fERG measures the electric biopotential evoked mainly by photoreceptor cells, namely, rods and cones, and the ON-bipolar and Müller cells complex, in response to a light stimulation. fERG recordings performed under photopic and scotopic conditions are called light- and dark-adapted fERG, respectively, according to the flash luminance intensity used, which is measured in candelas·s·ms·s·m−2) . Two mais·s·m−2) . Specifis·s·m−2) .The PERG records the central macular function of the retina, as well as the retinal ganglion cells response using reversal black and white checkerboards viewed with a central fixation . To inveThe EOG measures a variation of electrical potentials between skin electrodes located on the external and internal canthi . This vaThe neural retina is composed by three critical stages: the photoreceptor cells, called rods and cones, the bipolar cells, and the ganglion cells. The retina also contains interneuron cells: amacrine and horizontal cells and the retinal pigment epithelium. The functional properties of photoreceptors, bipolar cells, ganglion cells, and retinal pigment epithelium cells are evaluated by objective electrophysiological retinal techniques.Considering the large distribution of the cannabinoid system in the different retinal cell layers and its broad involvement in the regulation of retinal neurophysiology, these electrophysiological measures may be of benefit to the evaluation of the impact of cannabis on the human retinal function.To date, only one study, to our knowledge, has evaluated the impact of cannabis on the retinal function . In thisAlthough cannabis is one of the most prevalent drugs used worldwide, there is to date few studies that have evaluated the impact of cannabis on the human visual function. These studies were already reported in several reviews , 15. As This review outlines the presence of endocannabinoids in critical stages of the neural retina such as photoreceptor cells, bipolar cells, and ganglion cells. Endocannabinoids are also detected in interneuron cells, namely, amacrine and horizontal cells, as well as in Müller and retinal pigment epithelium cells. The presence of endocannabinoids in the retina supports a regulatory role of the cannabinoid system in the vertical transmission of visual information from photoreceptors to ganglion cells, the ultimate stage before the transmission of the visual information to the brain, mostly the visual cortex. This also supports a potential dysregulation induced by exogenous cannabinoids, such as THC and cannabidiol, the main constituents of cannabis. This review also described the localization of the cannabinoid system in the retinal pigment epithelium suggesting a role of this system in the renewal of the photoreceptor disks as well as in trophic functions. Another important finding of this review is the involvement of cannabinoids in retinal neurotransmission, neuroplasticity, and neuroprotection. Firstly, cannabinoids regulate the release of retinal neurotransmitters by acting on ionic channels or enzymatic activity and are thus able to alter the retinal signal. These results were recently supported by alterations of electroretinogram recordings in mice lacking cannabinoid receptors CB1 and CB2 . UnfortuThe retina constitutes a relevant and useful site to investigate neurotransmission signaling pathways as well as CNS processes. The retinal organization in mammals is well known  and the This review gives an overview of the distribution of the cannabinoid system in the retina together with its involvement in the regulation of retinal neurotransmission, neuroplasticity, and neuroprotection. These suggest potential alterations of structural and functional retinal properties by exogenous cannabinoids, especially THC and cannabidiol contained in joints. As cannabis is widely spread worldwide, it is now critical to explore the effects of cannabis on the human retina. Based on experimental studies in animals, this review also aims to provide several retinal methods to correlate the cellular and molecular changes induced by cannabinoids to potential functional retinal deficits in cannabis users. However, considering the neuroprotective role of the cannabinoid system in the retina, this review also argues for therapeutic uses of synthetic cannabinoids in the treatment and the prevention of retinal diseases.


The mitochondrial m.1555A>G mutation is associated with a high rate of permanent hearing loss, if aminoglycosides are given. Preterm infants have an increased risk of permanent hearing loss and are frequently treated with aminoglycoside antibiotics.We genotyped preterm infants with a birth weight below 1500 grams who were prospectively enrolled in a large cohort study for the m.1555A>G mutation. Treatment with aminoglycoside antibiotics in combination with mitochondrial m.1555A>G mutation was tested as a predictor for failed hearing screening at discharge in a multivariate logistic regression analysis.-10). 12 infants (0.2%) had the m.1555A>G-mutation. In a multivariable logistic regression analysis, the combination of aminoglycoside treatment with m.1555A>G-carrier status was associated with failed hearing screening (p = 0.0058). However, only 3 out of 10 preterm m.1555A>G-carriers who were exposed to aminoglycosides failed hearing screening. The m.1555A>G-mutation was detected in all mothers of m.1555A>G-positive children, but in none of 2993 maternal DNA-samples of m.1555A>G-negative infants.7056 infants were genotyped and analysed. Low birth weight was the most significant predictor of failed hearing screening (p = 7.3 × 10Antenatal screening for the m.1555A>G mutation by maternal genotyping of pregnant women with preterm labour might be a reasonable approach to identify infants who are at increased risk for permanent hearing loss. Additional studies are needed to estimate the relevance of cofactors like aminoglycoside plasma levels and birth weight and the amount of preterm m.1555A>G-carriers with permanent hearing loss. In 1993 a maternally transmitted non-syndromic deafness with susceptibility to aminoglycoside treatment was reported to be due to a single nucleotide (A to G) substitution at position 1555 of the mitochondrial genome . SubsequThe role of the m.1555A>G mutation has not been investigated in populations with high aminoglycoside treatment rates and routine hearing tests. We therefore screened a large cohort of preterm infants with a birth weight below 1500 grams for the m.1555A>G mutation to test, if established risk factors for congential hearing impairment and m.1555A>G in combination with aminoglycoside treatment are associated with a higher failure rate of newborn hearing screening. We furthermore screened a large number of mothers of these infants to determine if maternal genetic screening for m.1555A>G would be feasible in order to prevent postnatal aminoglycoside treatment of infants carrying the mutation.http://www.vlbw.de. We analysed infants with a gestational age below 37 + 0 weeks and a birth weight less than 1500 grams and their mothers who were enrolled from 46 German neonatal intensive care units between 2003 and 2012. Antenatal and postnatal clinical data were documented on standardised data sheets during the hospital course of the patients. Data quality was ensured by regular on-site-monitoring done by physicians of the central office of the German Neonatal Network at the University of Lübeck. Clinical data were coded and entered into a central database. Results of newborn hearing screening were collected during the whole study. Written informed consent for sample and data collection was given by parents of all participating infants. The study was approved by the ethics committee of the University of Lübeck and by ethics committees of all participating hospitals and universities.The German Neonatal Network (GNN) is a prospective multicentre cohort study of preterm infants with a birth weight below 1500 grams which is supported by the German Federal Ministry of Research and Education. Improvement of the long term outcome of preterm infants is the major aim of the GNN. Some additional information is given at According to a recent review on risk factors for hearing loss in very low birth weight infants  and a reDNA was collected by buccal swabs (mothers and infants) or umbilical cord tissue (infants). DNA was extracted by standard procedures  and stored at -20 degrees centigrade at the University of Lübeck. DNA-samples of infants who were born between 2003 and 2012 and were available at our lab on December 31st 2012 were genotyped for m.1555A>G. We used a custom assay  for allelic discrimination with a HT7900 thermocycler . Maternal samples were genotyped for infants carrying the m.1555A>G-genotype and additional maternal samples of infants who had no m.1555A>G-mutation.Statistical analysis was done with SPSS Version 20 , by using a logistic regression modell with failed newborn hearing screening as the dependent and birth weight (100 g – increments), any treatment with glycopeptide antibiotics, any treatment with furosemide, CMV-infection, bronchopulmonary dysplasia and aminoglycoside treatment × m.1555A>G-genotype as independent variables. The global significance level was set to 0.05. Adjustments for multiple testing were done by Bonferroni correction for multiple comparisons with 7 independent variables, and the nominal p-value thus was 0.007. All p-values are two-sided. Data are reported according to STROBE statement .DNA-samples of 8504 infants were available. Infants without neonatal hearing screening , or missing data for confounding variables  were excluded. 412 infants were excluded because of failed m.1555A>G-genotyping and one infant because of suspected DNA-contamination with a positive control . 7056 infants were finally included in our analysis.3554 infants were male (50.4%) and 2357 infants (33.4%) were multiples. Clinical data and results of the multivariate logistic regression analysis are given in Table 12 infants (0.2%) carried the m.1555A>G-mutation. Only 2 of these infants were not treated with aminoglycoside antibiotics like gentamicin or tobramycine. Both had normal newborn hearing screening. Three of ten m.1555A>G carriers, who were treated with aminoglycosides failed newborn hearing screening. The combination of aminoglycoside treatment × m.1555A>G-genotype was a significant predictor of failure . If aminoglycoside treatment and m.1555A>G-genotype were analysed as independent risk factors in the same multivariate logistic regression model, none of them was predictive for failed hearing screening (data not shown). Furosemide treatment was associated with failed hearing screening as well, whereas other factors such as vancomycin treatment, cytomegalovirus-infection and BPD were not predictive. To test if other potentially important confounders are associated with hearing loss, we added sepsis (confirmed by positive blood-culture) and abnormal head scans (intra-ventricular haemorrhage detected by ultrasound) to our multivariate logistic regression model. However, both variables were not predictive for failed hearing screening .Additional clinical data of infants carrying the m.1555A>G-mutation are given in Table Aminoglycoside treatment is standard of care for suspected sepsis in newborns. Analysis of large scale epidemiological data indicate, that substitution of aminoglycosides with broad spectrum antibiotics such as cephalosporins as first line treatment of newborn sepsis is associated with an increased rate of death ,11. TherOther risk factors which were included in our analysis were associated with an increased risk of failed neonatal hearing screening as well. Low birth weight itself was the most important predictor, but all other reported risk factors were associated with higher failure rates of newborn hearing screening, with significant p-values for treatment with furosemide. As reversible hearing loss after furosemide treatment is well described, this observation might be of minor importance for current treatment . HoweverAlthough we screened more than 7000 preterm infants, the total number of m.1555A>G carriers in our study was low, which limits the statistical power of our observation. The rate of failed hearing screening in our cohort was 11%, which is close to reported data in the literature . It is iHowever, family studies of m.1555A>G carriers indicate a very high penetrance of permanent hearing loss if aminoglycosides are given [5-year follow-up for GNN-infants who were enrolled in 2009 or later will start in 2014. Since measurement of evoked otoacoustic emissions are part of the follow-up, we will be able to analyse more data concerning the long-term effects of aminoglycoside treatment of preterm m.1555A>G carriers within the next years.In this large cohort study of preterm infants with a birth weight below 1500 grams, frequency of the m.1555A>G mutation was 0.2% 12 of 7056 infants). M.1555A>G was associated with failed hearing screening at discharge if carriers were treated with aminoglycoside antibiotics. We found the mutation in all mothers of m.1555A>G-carriers. It is reassuring that large cohort studies in children and adults report normal hearing of m.1555A>G-carriers if they were not treated with aminoglycosides  infants.. Our datBPD: Bronchopulmonary dysplasia; CI: Confidence interval; CMV: Cytomegalovirus; DNA: Deoxyribonucleic acid; OR: Odds ratio.The authors declare that they have no competing interests.WG conceived the study and drafted the manuscript, SB carried out the molecular genetic studies, MP and AZ performed the statistical analysis, HK, UFM, LG, MM, CH and EH participated in the design of the study, patient enrolment and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/14/210/prepub


Mucormycosis is a rare and potentially fatal fungal infection occurring primarily in severely immunosuppressed patients. Because it is so rare, reports in the literature are mainly limited to case reports or small case series. The aim of this study was to evaluate inpatient mortality, length of stay (LOS), and costs among a matched sample of high-risk patients with and without mucormycosis in a large nationally representative database.We conducted a retrospective analysis using the 2003–2010 Healthcare Cost and Utilization Project – Nationwide Inpatient Sample (HCUP-NIS). The NIS is a nationally representative 20% sample of hospitalizations from acute care United States (US) hospitals, with survey weights available to compute national estimates. We classified hospitalizations into four mutually exclusive risk categories for mucormycosis: A- severely immunocompromised, B- critically ill, C- mildly/moderately immunocompromised, D- major surgery or pneumonia. Mucormycosis hospitalizations (“cases”) were identified by ICD-9-CM code 117.7. Non-mucormycosis hospitalizations (“non-cases”) were propensity-score matched to cases 3:1. We examined demographics, clinical characteristics, and hospital outcomes . Weighted results were reported.From 319,366,817 total hospitalizations, 5,346 cases were matched to 15,999 non-cases. Cases and non-cases did not differ significantly in age (49.6 vs. 49.7 years), female sex (40.5% vs. 41.0%), White race (53.3% vs. 55.9%) or high-risk group . Cases experienced significantly higher mortality , with mean LOS and total costs more than 3-fold higher .In a national hospital database, hospitalizations with mucormycosis had significantly higher inpatient mortality, LOS, and hospital costs than matched hospitalizations without mucormycosis. Findings suggest that interventions to prevent or more effectively treat mucormycosis are needed. Mucormycosis (formerly zygomycosis) is a rare invasive fungal infection (IFI) associated with substantial morbidity and mortality. Immunosuppression is a common predisposing factor for mucormycosis, with the infection generally limited to patients with hematological malignancies or hematopoietic stem cell transplants, solid-organ transplants, and diabetes -5. A revWith advances in treatment, mortality rates among patients infected with mucormycosis have declined , but recTo date, mucormycosis has been studied in small and narrowly defined populations or as a subgroup within a larger sample of patients with IFIs, resulting in sample sizes of 200 cases or fewer ,11-13,15We conducted a cost-of-illness analysis of hospital discharge data from the Healthcare Cost and Utilization Project—Nationwide Inpatient Sample (HCUP-NIS) from 2003 through 2010. The HCUP-NIS, maintained by the Agency for Healthcare Research and Quality (AHRQ), is a 20% stratified representative sample of all US inpatient stays in acute-care non-federally funded institutions. It contains records on approximately 8 million hospitalizations each year from over 1000 US facilities.Core hospital stay files contain details on patient demographics , International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) diagnosis codes , Elixhauser comorbidities , length The HCUP-NIS data used in this study represent de-identified human subject data. The database does not contain data elements that would allow direct or indirect identification of specific individuals. All parties with access to the data were signatories of HCUP’s formal data use agreement (DUA), including the provision that no cell sizes less than 10 can be reported, and additionally completed the HCUP DUA Training. This provision is deemed by AHRQ to be an adequate safeguard against identification of individual patients.Hospitalizations of patients considered to be at risk for mucormycosis were identified based on the following hierarchy of high-risk categories for this infection:A. Severely immunocompromised B. Critically ill -stage renal disease/hemodialysis, lupus, malignant solid tumors, diabetic ketoacidosis, blood disorders, diabetes)C. Mildly/moderately immunocompromised  codes listed for the admission. Codes by date or time within the hospitalization are not available in the NIS database. Hospitalizations not falling into one of these categories were excluded from the analysis. Because the assignment to a risk group was hierarchical , the four groups were mutually exclusive. The categories were developed based on both the distribution of the observed comorbid conditions among all mucormycosis hospitalizations in a preliminary analysis and on the clinical understanding of traditional high risk factors (categories A and C).All four categories together comprised the analysis pool for hospitalizations at risk for mucormycosis. Among these high-risk groups, hospitalizations with mucormycosis were identified by the ICD-9-CM diagnosis code 117.7  and matched to high-risk hospitalizations without a mucormycosis diagnosis (non-cases) based on a propensity score (PS). Since the NIS is strictly an administrative database without any clinical data, we were unable to classify mucormycosis cases further as “proven”, “probable,” or “possible” as recommended by the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG). To minimize center effect, selection of non-cases was restricted to hospitals with at least one mucormycosis hospitalization in the same year.Patient and hospital characteristics were evaluated for all hospitalizations. We included demographic , clinical , length of stay (LOS), and total hospitalization charges and costs. Costs were derived by applying hospital-specific cost-to-charge ratios provided by HCUP to the charges for each individual hospitalization. All costs and charges were adjusted to 2011 USD using the medical care component of the US Consumer Price Index. Indirect  costs were not available in the database and thus excluded from analyses. The NIS data do not allow for the calculation of attributable costs.Propensity scores were computed by modeling the likelihood of having mucormycosis using a logistic regression. Predictors included patient , hospital , and admission  characteristics. Predictors were chosen on the basis of an expected or potential relationship to mucormycosis exposure. Data availability was also a factor in selecting predictors. Variables that were not consistently available across years or hospitals were not included to minimize the number of hospitalizations that would need to be excluded.Non-mucormycosis high-risk hospitalizations from hospitals with at least one mucormycosis hospitalization were matched 3:1 to mucormycosis patients using a greedy 8-to-1 digit matching algorithm . StratifAll study measures were compared between cases and non-cases before and after matching. Mean, standard deviation, median, interquartile range (IQR), minimum and maximum were reported for continuous measures, and proportions were reported for categorical variables. Significance was tested for proportions using the Rao-Scott Chi-Square Test, and for continuous measures using an independent sample t-test. An alpha level of 0.05 was used to assess statistical significance.Among the total of over 300 million hospitalizations in the US during the study period, 146,300,612 had at least one of the high-risk conditions, of whom 5,515 carried a diagnosis of mucormycosis , and one-fifth of cases and non-cases fell into the critically ill (high-risk group B) category. Approximately one quarter of both cases and non-cases were in high-risk groups B and D (major surgery and pneumonia), those considered non-traditional risk factors for mucormycosis. The most common comorbid conditions for cases and non-cases after matching included: hypertension , fluid and electrolyte disorders (42.5% and 27.5%), and diabetes with complications (26.9% and 22.3%).Approximately one third of matched hospitalizations were from hospitals in the West and South census regions. Three-quarters of these institutions were teaching hospitals and a slightly higher proportion were large . Cases had a higher risk than non-cases of being admitted urgently (26.0% versus 18.2% for non-cases).P < 0.001). Inpatient mortality was over five times higher among cases (22.1%) than among matched non-cases . Average total costs varied considerably, with $90,272  observed among cases and $25,746  among non-cases. This difference represents $64,526 in mean excess costs for mucormycosis cases . Differences between cases and non-cases for all three outcomes persisted over the 8-year timespan of the study, with no clear trend in the magnitude of the difference over time. For example, in-hospital mortality for mucormycosis cases was 25.2% in 2003 and 25.7% in 2009.Hospitalization outcomes are presented in Tables Critically ill patients (high-risk group B) demonstrated the largest differences in the outcomes between matched cases and non-cases ; mean excess costs: $101,388, ), followed by the severely immunocompromised patients . Patients considered only mildly or moderately immunocompromised (high-risk group C) or those with major surgery or pneumonia (high-risk group D) exhibited smaller differences in these outcomes .We confirm that mucormycosis remains a rare infection, present in less than 0.01% of all US hospitalizations between 2003 and 2010. Some degree of immune compromise, identified in three-quarters of admissions with this diagnosis, continues to represent the most common risk factor for this infection. The remainder, however, carried coexisting conditions less traditionally recognized as associated with mucormycosis, such as critical illness, pneumonia, and major surgery. Of the admissions over the eight years examined carrying a mucormycosis ICD-9-CM code, only 6% failed to fall into one of the four high-risk groups. Propensity-score matched analysis revealed a dramatic rise in hospital mortality (mean excess 17.7% [SE 1.3%]), LOS prolongation (mean excess days 16.5 [SE 2.2]) and costs  associated with a diagnosis mucormycosis.Prior studies examining outcomes among patients with mucormycosis have been limited by small numbers of patients with mucormycosis examined as a subset of those with IFIs. For example, a recent single center study by Dodds and colleagues  comparedMenzin and colleagues  examinedAlthough generalizable and internally valid, our study needs to be interpreted with some caution. One concern stems from the similarity of our mortality data to that observed in a specific population of patients with mucormycosis. Hammond and colleagues  studied Our study is subject to a number of additional limitations. First, it was cross-sectional, and thus did not allow us to explore hospital events in relation to the onset of mucormycosis. For this reason it is likely that at least some of the excess days and costs seen in mucormycosis group were incurred prior to the onset of mucormycosis. Therefore, we were unable to assess the degree to which this burden was specifically attributable to mucormycosis. The cross-sectional nature of the analysis (and database) also precluded the ability to factor the timing of concomitant diagnoses within the admission into the high-risk categorization process. Due to variability in data availability across years, not all hospitalization characteristics were included as predictors in the logistic regression to estimate propensity scores. Specifically, admission source (approximately 30% missing) and admission type (approximately 20% missing) were excluded.Furthermore, we relied on diagnosis codes to identify cases of mucormycosis. This predisposes our case identification to potential misclassification, including inadvertent exclusion of “probable” and “possible” mucormycosis cases. However, if this misclassification is non-differential, then the magnitude of group differences found in our study is actually likely to be biased toward the null. In other words, if such non-differential misclassification exists, then the actual between-group differences in the outcomes are potentially even greater than what we computed. Lack of clinical data also limited our ability to adjust for confounding clinical characteristics when assessing excess costs, such as the site of the mucormycosis infection. In addition, the cost analysis did not include costs of physician services, patient out-of-pocket expenses, indirect costs, costs post-discharge, or costs in the outpatient setting; therefore, this study may have underestimated the true burden of mucormycosis from a societal perspective.In summary, mucormycosis remains a rare but deadly infection among hospitalized patients. In addition to its clinical burden, it is associated with a vast increase in hospital resource use as measured by both LOS and costs. Finally, approximately one quarter of all admissions afflicted with mucormycosis have non-traditional risk factors for this infection such as critical illness, pneumonia, and major surgery. Although rare, by virtue of its dismal outcomes, mucormycosis demands more concentrated attention from the research and clinical communities.AHRQ: Agency for Healthcare Research and Quality; HCUP: Healthcare Cost and Utilization Project; ICD-9-CM: International Classification of Diseases, 9th Revision, Clinical Modification; IFI: Invasive fungal infection; IQR: Interquartile range; LOS: Length of stay; NIS: Nationwide Inpatient Sample; PS: Propensity score; SE: Standard error; SD: Standard deviation; TRANSNET: Transplant Associated Infections Surveillance Network; US: United States; USD: United States Dollars.HH, VF, and JM are employees of Boston Health Economics, Inc. and were funded by Astellas Scientific and Medical Affairs, Inc. for this study. PC is an employee of, and MZ and AS are consultants to the sponsor. MZ is also a consultant to Cubist Pharmaceuticals, Pfizer, and Optimer Pharmaceuticals. AS is also a consultant to Cubist Pharmaceuticals, Forest Pharmaceuticals, Inc., Bayer, Pfizer, Theravance, and Trius Therapeutics. The authors have no other financial or non-financial competing interests to disclose.MZ, AS and JM were involved in the conception and design of the study, the interpretation of data, and revised the manuscript critically. HH and VF contributed to the design of the study, the acquisition of data, the analysis and interpretation of data, and drafting the manuscript. PC contributed to the design of the study, the interpretation of data, and the critical revision of the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/310/prepub