In vitro experiments were conducted with preadipocytes isolated from VAT and SAT biopsies. The expression of 23 out of 33 PCP genes was enriched in VAT compared to SAT. Strong positive expression correlations of individual PCP genes were observed in VAT. WNT5A expression in VAT, but not in SAT, correlated with indexes of JNK signaling activity, IL6, waist-to-hip ratio and hsCRP. In vitro, WNT5A promoted the expression of IL6 in human preadipocytes. In conclusion, elevated non-canonical WNT5A signaling in VAT contributes to the exacerbated IL-6 production in this depot and the low-grade systemic inflammation typically associated with visceral adiposity.The accumulation of visceral adiposity is strongly associated with systemic inflammation and increased cardiometabolic risk. WNT5A, a non-canonical WNT ligand, has been shown to promote adipose tissue inflammation and insulin resistance in animal studies. Among other non-canonical pathways, WNT5A activates planar cell polarity (PCP) signaling. The current study investigated the potential contribution of non-canonical WNT5A/PCP signaling to visceral adipose tissue (VAT) inflammation and associated metabolic dysfunction in individuals with obesity. VAT and subcutaneous adipose tissue (SAT) samples obtained from subjects undergoing bariatric surgery were analyzed by qRT-PCR for expression of WNT/PCP genes.  However, a wealth of evidence has now confirmed that the regional distribution of adipose tissue is as important, if not more important, than overall adiposity in determining cardiometabolic risk1. It is widely accepted that accumulation of intra-abdominal visceral fat is a major contributor to systemic metabolic dysfunction and cardiovascular risk. In this regard, an increasing body of evidence supports the notion that visceral (VAT) and subcutaneous (SAT) adipose tissue exhibit different intrinsic properties, which make VAT a more pathogenic depot2. More specifically, the increased cardiometabolic risk associated with visceral adiposity has been linked with an overproduction of pro-inflammatory and pro-atherogenic cytokines, which contribute to establish a low-grade systemic inflammatory state that drives cardiometabolic disease8. However, the specific signaling pathways in VAT that account for this exacerbated inflammatory response remain largely unexplored.Obesity, defined as a body mass index (BMI) ≥30 kg/m11. Wingless-type MMTV integration site family (WNT) proteins are secreted signaling molecules that play fundamental roles during embryonic development and have been implicated in numerous critical aspects of physiology and disease in the adult. There are 19 WNT family members in mammals, which typically act in an autocrine/paracrine fashion and activate a number of different signaling pathways, typically classified as either canonical (β-catenin-dependent) or non-canonical (β-catenin-independent). While the role of canonical WNT signaling in the regulation of adipose tissue expansion is generally accepted12, the potential effects of non-canonical WNT signaling activation in this tissue and its contribution to fat depot heterogeneity in humans remain poorly defined. We recently reported that WNT5A, a WNT ligand that predominantly activates non-canonical WNT signaling contributes to obesity-induced adipose tissue inflammation and systemic insulin resistance in obese mice14. Although WNT5A is able to activate various non-canonical signaling pathways, its roles in embryo development are mostly mediated by activation of the WNT/planar cell polarity (PCP) pathway, which frequently leads to activation of JUN N-terminal kinase (JNK) signaling16. In adults, WNT/PCP signaling has been shown to contribute to cancer development in different settings, but its specific contribution to other disease processes has just recently begun to be explored22. The present study provides evidence supporting that WNT5A/PCP signaling is overactivated in VAT compared to SAT and contributes to the increased local and systemic inflammation associated with visceral adiposity.Emerging evidence suggests that developmental genes play central roles in fat distribution and the different properties of specific adipose tissue depots23, there was a marked upregulation (14.72-fold) of WNT5A expression in VAT compared with SAT , but it was greater in men  and in subjects with diabetes .To investigate the role of WNT5A and downstream signaling in obesity-associated adipose tissue dysfunction in humans, we used quantitative real-time PCR (qRT-PCR) to evaluate transcript expression of 33 genes involved in WNT/PCP signaling in visceral  and subcutaneous fat obtained from 44 individuals at the time of bariatric surgery. The clinical characteristics of this human population are summarized in Supplementary Table SAT Fig. . The difWNT5A in the PCP pathway were markedly and uniformly overrepresented in VAT. FRIZZLED (FZD) proteins are the main receptors for WNT ligands, and many FZD genes, including FZD1, 2, 3 and 7, were overrepresented 2- or more-fold in VAT compared with SAT  , Dishevelled (DSH) 1, 2 and 3, and PRICKLE1  Fig. , one of VAT Fig. . SimilarWNT5A, the increased expression of PCP genes in VAT was not affected by gender or diabetes status (data not shown). However, there were strong positive correlations between the expressions of individual WNT/PCP receptor components in VAT. Expression levels of ROR2, FZD7, VANGL2 and PRICKLE1 correlated with each other . Consistent with a previous report30, we found increased levels of both phosphorylated and total JNK1/2 in VAT  expression in experimental modelsipt Fig.  and protipt Fig.  level. NVAT Fig. , but notVAT Fig. . FurtherVAT Fig. . IL6 traWNT5A knockdown affect IL6 transcript levels in human primary preadipocytes. Supporting the validity of this cell culture model, we observed that transcript levels of both WNT5A and IL6 are higher in omental versus subcutaneous preadipocytes, consistent with their higher expression in VAT  and this did not have a statistically significant effect on IL6 transcript levels  levels was observed after exclusion of patients on anti-inflammatory drugs (NSAIDs and immunosuppressive drugs)  Fig. . This cogs) Fig. . In contgs) Fig. . Similar11. In this study we analyzed gene expression patterns in visceral and subcutaneous adipose tissue to gain insight into the potential role of non-canonical WNT5A/PCP signaling in adipose tissue inflammation in the setting of obesity. Our data show an over-activation of this signaling pathway in VAT under obesity conditions, which may contribute to the exacerbated local and systemic inflammation associated with visceral adiposity and its metabolic and cardiovascular complications.The differential expression of developmental genes has been suggested to contribute to the qualitative differences between visceral and subcutaneous adipose tissue and to the different cardiometabolic risk associated with the expansion of these depots in the context of obesity23, a finding that we corroborated in the current study. However, the expression of the many signaling genes that modulate WNT5A-mediated PCP signaling had remained largely unexplored. In this regard, our study adds insight into the role of non-canonical WNT signaling in adipose tissue heterogeneity by demonstrating that most of the downstream components of the non-canonical WNT/PCP signaling pathway  are markedly overrepresented in VAT compared to SAT. These data suggest that the over-activation of this signaling pathway extends beyond the increased expression of the WNT5A ligand in this depot. It is noteworthy that many of the WNT/PCP receptor components that we found most highly upregulated in VAT  have been shown to be essential for WNT5A signaling in different experimental settings33. We are unaware of any other signaling pathway that has been reported to be so uniformly and robustly upregulated in visceral fat. These findings suggest that PCP signaling modulators represent unique nodal drivers of VAT dysfunction in the setting of obesity.We and others have previously reported that WNT5A is expressed at higher levels in VAT than SAT in the setting of obesity34, although not to the extent that is observed for some WNT/PCP genes. On the other hand, we have previously reported that myeloid cells are a significant source of Wnt5a in mouse adipose tissue14, and pro-inflammatory TLR signaling has been reported to increase WNT5A expression in human monocyte/macrophages35. Thus, the differential expression of WNT5A in VAT and SAT could also reflect differences in macrophage infiltration and/or activation. Regardless of the underlying mechanism, these data suggest that not only VAT does produce more WNT5A, but also that this fat depot is likely to be more responsive to its actions, given its higher expression of WNT/PCP receptors and other WNT signaling intermediates. Consistent with this notion, we find that WNT5A is associated with increased JNK signaling in VAT, but not in SAT. Although WNT5A is able to activate a plethora of signaling pathways in a context-dependent manner, JNK is the main signaling kinase activated by the WNT/PCP pathway in mammals16, and it frequently mediates the effects of WNT5A. Notably, experimental studies suggest that JNK signaling is particularly relevant in the setting of obesity-induced inflammation and associated metabolic dysfunction29.Our data also reveals a remarkable correlation among the expression of different PCP genes in VAT, particularly in subjects with diabetes, suggesting the existence of common regulatory mechanisms that coordinate their expression. The regulators of PCP gene expression in omental fat are probably multiple. Omental and subcutaneous fat have different embryological origins, and this could contribute to the differential expression of WNT/PCP genes. In this regard, the greater expression of WNT5A observed in omental versus subcutaneous preadipocytes is consistent with this possibility. Several developmental genes have been reported to be differentially expressed in the different adipose tissue depots36. The pro-inflammatory cytokine IL-6 is generally believed to be a particularly relevant contributor to the high cardiometabolic risk associated with intra-abdominal fat accumulation. It is produced at higher levels in VAT compared to SAT7 and circulating IL-6 levels exhibit a stronger association with visceral adiposity than other pro-inflammatory cytokines, such as TNF31. Despite of these findings, the specific mechanisms that account for the increased production of IL-6 in VAT remain poorly defined. In this regard, we recently reported that Wnt5a promotes adipose tissue inflammation and systemic insulin resistance in mouse models of obesity, at least in part through an increased production of IL-6 in adipose tissue14. However, the extrapolation of these mouse results to human obesity remained challenging due to the many differences between adipose tissue depots in mice and humans37. While omental fat is the prototypical visceral fat depot in humans, this depot is essentially absent in mice. Furthermore, inflammatory responses in mice and humans are known to differ significantly38, and the biology of IL-6 is likely to exhibit many differences between species, as suggested by the low amino acid sequence identity between human and mouse IL-6 proteins (~41%)39. The results of the current studies with subcutaneous and omental preadipocytes in culture and the in vivo association between the expression of WNT5A and IL6 in VAT presented herein supports our previous mouse studies14, and provides evidence that WNT5A/PCP signaling is a clinically-relevant contributor to the elevated IL-6 production in VAT. The correlation observed in VAT between WNT5A and the pJNK/JNK ratio, an index of JNK signaling, also supports this possibility. JNK signaling is known to promote IL-6 expression in adipose tissue29, and our previous mouse studies suggest that JNK signaling is a main mechanism linking WNT5A to IL-6 production14. Further supporting this WNT5A/IL-6 connection, we also observed a significant association between WNT5A expression in VAT and circulating levels of CRP, a marker of systemic inflammation. The connection between WNT5A expression in VAT and IL-6/CRP levels is of particular relevance, given the large body of clinical evidence that links increased IL-6 signaling and/or increased circulating CRP levels to insulin resistance, diabetes and atherosclerotic CVD43. Some of our data suggest that the contribution of non-canonical WNT5A signaling to IL-6 production and inflammation may be particularly relevant in subjects with diabetes, which typically exhibit high levels of systemic inflammation. However, the differences between subjects with and without diabetes observed in our study need to be interpreted with caution, given our limited sample size and the substantial differences in clinical characteristics between subjects with and without diabetes. Future studies with greater sample size will be needed to assess whether a direct connection between diabetes and WNT5A expression in VAT exists in humans, as suggested by our previous mouse studies14.Systemic low-grade inflammation is one of the main mechanisms that link obesity with insulin resistance, diabetes and cardiovascular disease. While conflicting data have been reported on the contribution of subcutaneous fat, a consistent body of evidence suggests a strong association between the accumulation of intra-abdominal visceral fat and systemic inflammation2. The analysis of such population may preclude the detection of pathophysiologically relevant associations that might be clearer in more diverse populations. Another limitation of our study is that most patients were on treatment with a number of drugs, including thiazolidinediones, anti-inflammatory drugs and statins, which may have confounded our results. Indeed, the association between WNT5A expression in VAT and circulating CRP levels only reached statistical significance after exclusion of subjects on anti-inflammatory drugs. Similarly, we cannot discard that the low calorie diet and associated body weight loss that is required prior to bariatric surgery may have affected gene expression or systemic inflammation in the subjects included in our study. Overall, these limitations highlight the need for more extensive studies that include the analysis of PCP gene expression in VAT and/or SAT of individuals across different BMI groups and before and after bariatric surgery. Future studies are also warranted to evaluate the potential association between WNT5A/PCP signaling in adipose tissue and atherosclerotic CVD, given the major role that IL-6 signaling plays in this condition as revealed by large Mendelian randomization studies45.Our study is limited by its cross-sectional nature and the specific characteristics of the studied patient population, which included mostly patients with BMI ≥ 35 kg/mIn summary, the present study reveals a remarkable overactivation of non-canonical WNT5A/PCP signaling in visceral fat under obesity conditions, which contributes to the higher IL-6 production and systemic inflammation typically associated with visceral adiposity, in particular in individuals with diabetes. Future studies are warranted to evaluate the mechanisms underlying PCP signaling activation in visceral adipose tissue and the therapeutic or prognostic potential of this pathway in the setting of obesity and associated cardiometabolic disease.2, age >18 years) who were referred to undergo bariatric weight loss surgery at Boston Medical Center were recruited for this study. Subjects with unstable medical conditions such as active coronary syndromes, congestive heart failure, systemic infection, acute illness, malignancy or pregnancy were excluded from participation. Anthropometric measures and blood draws were obtained during a single baseline pre-surgical visit, which occurred within 1–2 weeks before their planned surgery and adipose tissue collection. Clinical characteristics including blood pressure, heart rate, height, weight, BMI, and waist circumference were recorded by established methods. Subjects were in a fasting state and their medications held in the morning when blood samples were drawn for biochemical analyses including glucose and insulin levels at the time of the baseline pre-surgical visit. Subjects were diagnosed with diabetes if they had fasting plasma glucose ≥126 mg/dl and/or HbA1C ≥6.5%, were taking glucose lowering medications or had history of diabetes. Study population characteristics are summarized in Supplementary Table Consecutive men and women with obesity  and stored at −80 °C. mRNA of whole adipose tissue samples was isolated using RNeasy Lipid Tissue Mini Kit (Qiagen) and RNA quality was confirmed by gel electrophoresis. Reverse transcription to cDNA was performed with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and a pre-amplification step of selected target genes was conducted using TaqMan PreAmp Master Mix and specific TaqMan gene expression assays (Applied Biosystems). Real-time PCR was performed in a Viia7 thermal cycler (Applied Biosystems) using TaqMan gene expression assays and TaqMan Gene Expression Master Mix. Expression data for all target genes was normalized to Protein extracts from human fat samples were obtained by tissue homogenization in a TissueLyser II disruptor (Qiagen) in the presence of ice-cold lysis buffer  supplemented with protease/phosphatase inhibitors (Roche Applied Science). Commercially available ELISA kits were used to quantify relative levels of phosphorylated and total JNK1/2  and IL-6 (R&D Systems) following manufacturer’s instructions.in vitro experiments. Cells on passage 4 were treated with recombinant human recombinant WNT5A protein (R&D) or vehicle as control, and siRNA against WNT5A or scrambled siRNA as control . RNA from cells was isolated using RNeasy Micro Kit (Qiagen). cDNA synthesis and RT-PCR to asses WNT5A and IL6 mRNA levels were performed as described above (without pre-amplification).Preadipocytes isolated from subcutaneous and omental adipose tissue biopsies obtained from individuals with obesity during bariatric surgery were purchased from the Boston Nutrition and Obesity Research Center (BNORC). Cells isolated from 5 patients were used for the t tests. Pearson’s correlation coefficients (r) were used to analyze the association between variables. A two-way ANOVA was used for the rWNT5A in vitro study. All statistical tests were performed using GraphPad Prism software (GraphPad Software Inc.).Data are shown as mean ± SEM unless otherwise stated. Statistical significance of differences between gene expression in omental and subcutaneous fat were assessed by paired Student’s The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Information


It has recently been found that anti-Stokes photoluminescence can be observed in degenerately n-doped indium phosphide nanowires, when exciting directly into the electron gas. This anti-Stokes mechanism has not been observed before and allows the study of carrier relaxation and recombination using standard photoluminescence techniques. It is important to know if this anti-Stokes photoluminescence also occurs in bulk semiconductors as well as its relation to carrier recombination and relaxation. Here we show that similar anti-Stokes photoluminescence can indeed be observed in degenerately doped bulk indium phosphide and gallium arsenide and is caused by minority carriers scattering to high momenta by phonons. We find in addition that the radiative electron-hole recombination is highly momentum-conserving and that photogenerated minority carriers recombine before relaxing to the band edge at low temperatures. These observations challenge the use of models assuming thermalization of minority carriers in the analysis of highly doped devices. Anti-Stokes luminescence - the emission of photons with higher energy than those absorbed – in nanomaterials is widely used for optoelectronic applications. Here the authors observe it in degenerately doped bulk InP and GaAs, indicating it as a more general property of direct bandgap semiconductors. For biological imaging, anti-Stokes photoluminescence allows for improved sensitivity with reduced photo damage7. Lasers using this effect have been demonstrated8. Anti-Stokes photoluminescence enables enhanced detection of infrared photons3, which can be used for white light generation9 and could improve the efficiency of solar cells5.Anti-Stokes photoluminescence, where photoluminescence occurs at energies higher than that of the excitation photons, can occur through different physical mechanisms11 in two-dimensional systems such as type II heterojunctions12, CdS nanoribbons13, and GaAs quantum wells;14 and in high band gap bulk materials such as GaN15 and ZnO16.Anti-Stokes luminescence has, in semiconducting materials, been observed in zero-dimensional systems such as quantum dots;18 or two-photon absorption (photon upconversion)20. Two other anti-Stokes mechanisms involve phonons. In one, the simultaneous absorption of photons and phonons enables absorption below the band gap22 and can lead to upconverted band edge luminescence. Alternatively, photogenerated charge carriers can be excited to higher states through phonon absorption, which can lead to recombination involving these higher energetic excited states23.Most reported anti-Stokes photoluminescence in semiconductors requires states below the band edge and excitation by Auger processes25, where the anti-Stokes mechanism relies on absorption of photons with energies less than the Fermi energy of the degenerately doped material, followed by phonon scattering of the photo-excited hole to higher k-values (where k is the charge carrier momentum). Such anti-Stokes photoluminescence has so far not been reported for bulk semiconductors.For efficient anti-Stokes photoluminescence, two-step processes with an intermediate state are advantageous, due to the  lower probability of processes involving interaction between a larger number of particles. In bulk semiconductors, two-step anti-Stokes photoluminescence requires defect states within the band gap. We recently reported on anti-Stokes photoluminescence in degenerately n-doped InP nanowiresk-conservation in radiative recombination as well as thermalization of photogenerated minority carriers. We find that the radiative recombination in our samples is strongly dominated by k-conserving vertical transitions, despite high doping. In contrast, in the literature it is often assumed that the k-selection rule is violated in strongly doped semiconductors. We will argue that the photoluminescence lineshape is determined by the distribution of holes recombining vertically (conserving k) with the Fermi sea of electrons. In addition, we find that the charge carriers recombine before the holes have thermalized to the valence band maximum. This means that the photoluminescence lineshape is not determined by holes that have relaxed to the valence band maximum before recombining, which would lead to photoluminescence peaked at the band gap.In this work we demonstrate that such phonon assisted anti-Stokes photoluminescence is not limited to nanoscale materials, but is a more general property of direct band gap semiconductors. During this experimental demonstration of anti-Stokes photoluminescence, we also identified important aspects of 25. It was interpreted as a nanowire-specific attribute due to the enhanced coupling of charge carriers to optical phonons in nanoscale materials13 and efficient heating of the electron gas by the exciting laser. However, the results presented in Fig. 26 and the Fermi energy of the sample was estimated to be 1.50 eV. For excitation with 1.494 eV photons at room temperature, we observed luminescence on both sides of the laser energy. Similar results as for n-doped InP were also found for n-doped and p-doped GaAs indicating that our results are quite general. Despite an extensive literature search, we have not found any previous reports on this anti-Stokes effect, which is slightly surprising since the experiment can be performed on commercially available samples.Anti-Stokes photoluminescence in highly doped semiconductors was first observed in sulfur-doped InP nanowiresEF) since there are no available empty electron states. For example at 4 K, 5 meV below the Fermi energy less than a fraction of 10−6 of the states are unoccupied, and 98% of the states are occupied at 4kBT = 1.4 meV below the Fermi energy. Figure kBT = 103 meV). A high electron gas temperature can be the result of a high sample temperature or due to direct heating of the electron gas by the photo-exciting laser25.A schematic image of our model of the anti-Stokes photoluminescence mechanism in degenerately n-doped direct band semiconductors is shown in Fig. k-conserving, and due to this k-conservation, anti-Stokes photoluminescence requires scattering of the photo-excited hole to higher k-values. The strong temperature dependence of most hole scattering processes27 leads to negligible k-increasing scattering rates at low temperatures. This means that at low temperature, Fig. k-values and thus, no observed anti-Stokes photoluminescence. In Fig. k-values and anti-Stokes photoluminescence can be detected. Hole scattering to lower k-values leads to Stokes photoluminescence, which may be detected at both temperatures shown in Fig. We show, later in this work, strong evidence that the radiative recombination in our samples is strongly In order to get further information on the processes behind the anti-Stokes photoluminescence, we measured the excitation energy dependence and the temperature dependence of the anti-Stokes photoluminescence in degenerately n-doped InP. The results at 4 K, 100 K, and room temperature are presented as photon energy contour plots in Fig. Eg,intrinsic, is 1.424 eV at 4 K). For laser photon energies lower than 1.48 eV, we do not observe any Stokes or anti-Stokes PL. We conclude from this lack of PL signal that for laser energy < 1.48 eV, the band-to-band absorption is negligible. Note that for n-doped InP nanowires, the situation was different and absorption was possible into the electron gas also at a sample temperature of 4 K24 giving rise to Stokes PL. The low-energy absorption in nanowires was attributed to laser-induced heating of the electron gas.For the measurements at 4 K Fig.  luminescFor measurements at 100 K, both anti-Stokes and Stokes PL is visible for laser energies between about 1.44 and 1.48 eV Fig. , indicatAn established method to study absorption in weakly absorbing semiconductors is PLE, where the dependence of the PL intensity on excitation energy is measured. Figure The shift of the on-set of the PLE spectra with temperature confirms that high temperatures allow excitation deep below the Fermi energy. It is important to remember that, at the selected detection energies, PLE measures only inter-band absorption that leads to the creation of holes in the valence band. Inter-subband absorption, free-carrier absorption, and other similar absorption processes are not detected. The gray spectra in Fig. 24.In Fig. k-vector28. The relaxation of the k-selection rule is then caused either by crystal imperfections introduced by dopants or by momentum taken up by the electron gas29. The photoluminescence lineshape is then determined by the occupancy of the electron states and the transition matrix elements between holes at the top of the valence band and the electrons29.It has been discussed in some cases that in degenerately n-doped semiconductors, photo-excited holes relax/thermalize to the top of the valence band and then recombine with an electron from the electron gas with a possible change in k-selection rule is strongly obeyed for radiative recombination. We know this since at low temperatures, 4 K, there is no anti-Stokes PL that we can measure when we excite below the Fermi energy, see Fig. k electrons available, and recombination with such electrons should lead to recombination with emission of photons with an energy > 1.49 eV. However, we find no such luminescence in the 1.49 eV excitation spectrum, and hence conclude that k-non-conserving recombination, which would lead to anti-Stokes PL, is not significant).In our samples the situation is very different. The k-selection rule is obeyed. We thus know that there is scattering of holes to smaller k-values, also at low temperatures.Next, we note that there is Stokes PL and, as argued above, the k-selection rule is obeyed. We also noted that holes can relax toward the top of the valence band. In that case, if the holes scatter all the way to the top of the valence band before recombining, we would expect the photoluminescence to be peaked at the band gap energy. This is not the case in our measurements  the recombination occurs before the holes relax/thermalize to the valence band maximum.Summarizing, our results show that (1) in degenerately n-doped bulk InP, the majority of radiative recombination of photo-excited holes occurs through vertical, Our interpretation of the observed anti-Stokes PL suggests similar processes in other degenerately doped direct band gap semiconductors. Figure k-conserving recombinations of hot minority charge carriers.According to the reasoning for n-doped InP, an asymmetric spectral change relative to the laser energy indicates that the radiative recombination is dominated by k-values. We argue that the radiative electron-hole recombination is k-conserving and that the recombination rate is faster than the k-changing scattering rate. Thus, we believe that the spectral PL shape is not determined by indirect (non k-conserving) radiative recombinations of Γ-point minority carriers (as commonly assumed), but by the minority charge carrier energy relaxation rate relative to the recombination rate. The similarities of the results of n-type InP, n-type GaAs, and p-type GaAs suggest that our model may be general for all direct band gap III–V semiconductors.In summary, we have demonstrated that anti-Stokes PL can be achieved in degenerately doped InP and GaAs, and that the underlying process is scattering of photo-excited minority carriers to higher Our findings allow a more accurate description of radiative recombination in semiconductors and show that anti-Stokes PL may influence the behavior of opto-electronic semiconductor devices. Future experiments should investigate the time-dependence of the Stokes and anti-Stokes PL to yield additional insight into the underlying minority carrier scattering and recombination dynamics. Comparing detailed modeling with the PLE and absorption experiments should give important information on the hole scattering dynamics.+n+-junction-based solar cells. Although our results pertain only to the physics of highly doped bulk InP and GaAs, we note that upconversion has been studied for its device relevance. For instance Auger-assisted upconverted photocurrent has been studied for intermediate band solar cells30 and photochemical upconversion for other photovoltaic devices31.Finally, we would like to highlight that we saw in our steady-state experiment a strong indication of a lack of thermalization of photogenerated minority carriers. Such an observation challenges the use of simplified models, based on the thermalization assumption, for describing carrier dynamics in the analysis of semiconductor devices with highly doped regions, such as degenerately doped p32. The studied InP sample was degenerately sulfur doped, that is, n-type, with a doping concentration of about 1019 cm−3. The studied GaAs samples were silicon-doped n-type (about 4 × 1018 cm−3), zinc-doped p-type (about 1 × 1019 cm−3), and nominally undoped GaAs.We studied a widely used commercially available single crystalline bulk substrate grown by the vertical gradient freeze method (VGF), which is a modification of the Bridgman technique−2. The photoluminescence was collected by a ×50 microscope objective lens, dispersed by a single monochromator, and detected by a scientific Andor Neo CMOS-camera. To allow measurements close to the laser line energy, a cross-polarized dark field PL setup was used33, where the polarization state of the excitation light is orthogonal to the polarization of the detection. In our setup, we reached a suppression of the backscattered laser line by a factor of about 105 allowing us to measure the laser line simultaneously with the photoluminescence.For photoluminescence measurements, the samples were mounted onto the cold finger of a continuous flow liquid helium cryostat system. The samples were optically excited by a continuous wave (cw) tunable optically pumped Ti:Sapphire laser. The laser light was focused on the sample and the optical power density was kept constant around 40 kW cmThe data that support the findings of this study are available from the corresponding author upon request.Peer Review File


In a multiple logistic regression, breastfeeding ≥12 months, higher maternal PVL and ≥2 previous HTLV-1-infected children remained independently associated with the outcome. Thus, high maternal PVL and breastfeeding beyond 12 months were independently associated with MTCT of the HTLV-1 infection. Our results reinforce the need for both prenatal HTLV screening in endemic areas and for advising mothers on breastfeeding.HTLV-1 is transmitted primarily either through sexual intercourse or from mother to child. The mother/child pairs were classified as seroconcordant or serodiscordant. We analyzed mother to child transmission (MTCT) according to sociodemographic, clinical and epidemiological characteristics of the mother, child’s gender and duration of breastfeeding. Between June 2006 and August 2016 we followed 192 mothers with HTLV-1 infection (mean age 41 years old), resulting in 499 exposed offspring, 288 (57.7%) of whom were tested for HTLV-1, making up the final sample for the study, along with their 134 respective mothers. Among the tested mother/child pairs, 41 (14.2%) were HTLV-1 positive, highlighted that seven of 134 family clusters concentrated 48.8% of positive cases. Variables associated with a positive child: breastfeeding duration ≥12 months, maternal PVL ≥100 copies/10 This infection is endemic in many parts of the world, including southwestern Japan, South America, some of the Caribbean islands, western and central Africa, Australo-Melanesia and some areas of Middle-East, such as regions of Iran5. There are at least an estimated five to 10 million HTLV-1 carriers worldwide3, but screening of the population for the presence of the virus is performed for only one third of the endemic areas. Brazil is most likely the country with the highest absolute number of seropositive individuals, with almost one million infected people6.Human T-cell lymphotropic virus type 1 (HTLV-1), a virus infecting humans since ancient times, is the causative agent of a lymphoproliferative malignancy named Adult T-cell Leukemia/Lymphoma (ATLL), as well as of the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP); HTLV-1 has also been associated with uveitis, infective dermatitis, and other inflammatory disorders7. Concerning mother-to-child-transmission (MTCT) transmission, a review8 pointed out that in infants breastfed, the MTCT occurs at rates varying from 7.4%9 to 32%10, compared with a rate of less than 5% among bottle-fed children, some of them tested during adulthood14. Although prolonged breastfeeding seems to play a central role in the spread and maintenance of endemicity14, the relative importance of HTLV-1 main routes of transmission in Brazil is still a matter of discussion16. Preventive measures, such as prenatal screening and abstinence from breastfeeding by carrier mothers might reduce the prevalence of HTLV carriers, such as in Nagasaki population, in which a decline from 20–25% to 4% in the prevalence of HTLV carriers was observed. In Brazil, unfortunately, prenatal screening for HTLV has not yet been implemented throughout the country. In addition, there are no specific studies establishing the rate of MTCT transmission of the virus in the country, or the role of the HTLV-1 maternal proviral load as a risk factor for mother-to-child transmission. Analyses accounting simultaneously for possible epidemiological, clinical and biomolecular markers for vertical transmission are thus needed to assess any independent effect and may increase our understanding of the determinants of MTCT.HTLV-1 transmission primarily occurs through the following routes: 1) vertically (from mother to child), including a long time of breastfeeding (8); 2) sexual contact, primarily from men to women; and 3) parenterally through the transfusion of contaminated blood and blood components or through contaminated needlesA cross-sectional study involving mothers with HTLV-1 infection at the Institute of Infectology “Emílio Ribas” in São Paulo from June 2006 to August 2016 and their exposed child tested for HTLV infection. Mothers were excluded if they did not have any child tested or if their documented seroconversion occurred after the birth of their children. We classified the mother-child pairs into two groups: a) seropositive mother and her seropositive child tested and b) seropositive mother with her seronegative child tested.17.The serological tests were carried out in the clinical laboratory at the Instituto de Infectologia Emilio Ribas, at its immunology section, employing the GOLD ELISA HTLV-1/2 (Immunoenzyme Test) and Western Blot Test (WB) (MP Diagnostics (MPD) HTLV Blot 2.4), for HTLV screening and confirmation, respectively. All WB positive cases were also submitted to a qualitative polymerase chain reaction (Nested-PCR) testpol gene: SK110 , and SK111 . The internal HTLV-1 TaqMan probe (5′-CTTTACTGACAAACCCGACCTACCCATGGA-3′) was selected using the Oligo  and Primer Express  software programs and checked through a search of GenBank. The probe was located between positions 4829 and 4858 of the HTLV-1 genome and carried a 5′ reporter dye FAM (6-carboxy fluorescein) and a 3′ quencher dye TAMRA (6-carboxy tetramethylrhodamine). For quantification of the human albumin gene, the primers Alb-S (5′-GCTGTCATCTCTTGTGGGCTGT-3′) and Alb-AS (5′-AAACTCATGGGAGCTGCTGGTT-3′) and the albumin TaqMan probe (5′-FAMCCTGTCATGCCCACACAAATCTCTCCTAMRA-3′) were used as described previously19. Albumin DNA was quantified jointly in all samples in order to determine the amount of DNA used as an endogenous reference to normalize variations due to differences in PBMC counts or DNA extraction. The 25-μl PCR mixture for HTLV-1 or albumin DNA amplifications consisted of 5 μl DNA extract, primers SK110 and SK11 or Alb-S and Alb-AS (10 nM of each), 10 nM HTLV-1 or albumin TaqMan probe, TaqMan® Universal Master Mix II . For both the HTLV-1 and albumin DNA amplifications, after one cycle at 50 °C for 2 min and one cycle at 95 °C for 10 min, a two-step PAC procedure was used consisting in 15 s at 95 °C and 1 min at 60 °C for 45 cycles. Amplifications were carried out using the ABI 7300Fast Real-Time PCR System . The HTLV-1 copy number in each clinical sample was estimated by interpolation from the plasmid regression curve. To determine the proviral load, the HTLV-1 DNA copy number was normalized to the amount of the cellular albumin of the clinical sample, which was quantified in parallel. All samples were run in duplicates. Results were expressed as HTLV-1 DNA copies/104 PBMCs, as described elsewhere13. Based on the median of asymptomatic individuals, 200 copies/104 PBMCs of PVL was the value used as a cut off to discriminate from HAM/TSP subjects20.The HTLV-1 proviral load was quantified by real-time PCR, using primers and probes targeting the 4 PBMC were considered high; for breastfeeding duration, which averaged 11 months, the cut-off point adopted was 12 months.The categorical variables included in the univariate analysis were “having HTLV-1 infected mother or sibling”, HAM/TSP, ATL, race/ethnicity, place of birth, hepatitis C virus infection (HCV) status, history of blood transfusion and educational level. We transformed numerical variables into categorical variables for the analysis of mother-to-child transmission, with their mean or median serving as reference; for PVL, ≥100 copies/10Stata: Release 10. Statistical Software. College Station, TX). We extracted all analyzed variables from our REDCap database  and used the statistical software Stata/IC 13.1 for the statistical analysis. P values < 0.05 were considered statistically significant. Variables reaching a significance level of p < 0.20 in the univariate analysis were included in a multiple logistic regression analysis, which we used to determine those that were independently and significantly associated with the outcome.Statistical analysis was conducted using Student’s t-test for parametric data, and the chi-square test for proportions. Possible differences in patient characteristics or laboratory values among the groups were evaluated with two-way Mann-Whitney’s test. Bivariate logistic analysis was performed to identify independent variables associated with HAM/TSP. Variables associated with the outcome at a significance level of p < 0.20 (HAM/TSP) in the bivariate analysis were included in a multiple logistic regression model; the only exception to this procedure was the inclusion of the variable gender, which was included regardless of its statistical significance in the bivariate model. Logistic analysis was performed with the aid of Stata 10 software ; the number of children by mother ranged from 1 to 10, with a mean of 3.4 (±1.5)  attended our invitation and were tested for HTLV-1, 41 of whom were positive for the virus, resulting in a transmission rate of 14.2%. Mothers’ age at delivery ranged from 14 to 48 years, with an average of 26.1 (±6.4) years. Maternal PVL ranged from 0 to 1221 copies/10Odds Ratio (OR) for HTLV-1 seropositivity among 288 children exposed to vertical transmission according to the child’s gender, duration of breastfeeding, and maternal clinical and epidemiological characteristics.From 288 tested children, 253 had been breastfed, and the duration of breastfeeding ranged from less than two weeks to 60 months (mean 10.7 months). Co-infections were detected in 19 mothers. We identified a tendency to familial aggregation of cases, where among 134 tested mother/child pairs, 41 (14.2%) were HTLV-1 positive, highlighted that seven of 134 family clusters concentrated 48.8% of positive cases. Table p < 0.001), having two or more HTLV-1 positive children(p < 0.001), mother’s PVL (≥100 copies/104 PBMC) (p = 0.014), mother with an epidemiological history suggestive of virus acquisition by mother-to-child transmission (her mother and/or sibling with HTLV-1 infection) (p = 0.016), age (p = 0.005), Asian maternal race (p = 0.02) and divorced marital status (p = 0.016). The odds ratio of possible protection conferred by cesarean delivery did not reach statistical significance (Table 4 PBMC (p = 0.005), breastfeeding for 12 months or more (p < 0.001), and having two or more HTLV-1-infected children (p < 0.001) (Table Variables associated with an HTLV-1-seropositive offspring were: breastfeeding for 12 months or more (1) Table .Table 3M22, Peru (18%)23, Iran (16.6%)24, Gabon (17.5%)25, Martinique (27%)26, and Japan between 1986 to 199127 before implementing screening policies among pregnant women. In contrast, it was higher than in French Guiana (9.7%)28, and in Japan by the end of the 1990s (3.9%)27. This variability of transmission rates to the offspring between different populations, and the lower rates in countries that have implemented control measures, strongly suggest the predominant role of variables related to breastfeeding in HTLV-1 transmission28.For the first time in Brazil, in this study we simultaneously analyzed epidemiological, clinical markers, and maternal proviral load, for mother-to-child HTLV-1 transmission. The prevalence of concordant transmisson to the child was 14.2%, which was lower than found in countries such as Jamaica (18% to 22%)21, and in the present study women with PVL >100 copies/104 PBMC presented a higher transmission risk.We addressed several risk factors for mother/child positive pairs, including maternal load of circulating HTLV-1. The risk of this kind of transmission increases exponentially in the presence of higher PVLs10. In those children, the median time of HTLV-1 infection was estimated at 12 months29. Among children of Peruvian women with HTLV-1 the risk was 15.1 times greater for those who were breastfed during 12 to 24 months compared to less than six months30. One possible explanation would be that passive transfer of maternal antibodies during pregnancy may inhibit HTLV-1. However, those antibodies decline and disappear six to 12 months after birth31.In agreement with our results a similar study done in Jamaica showed an association between prolonged breastfeeding and MTCT. Breastfeeding over 12 months was also associated with a risk of 32% transmission compared with only 9% for a shorter duration of breastfeeding4 PBMC. In fact, both the high mother PVL and prolonged breastfeeding were independently associated with transmission to the offspring. Divorced marital status was another possible confounding factor, which also did not remain in the multivariate analysis. Unfortunately, the small sample of this specific subgroup is not strong evidence and deserves further investigation of divorce as a risk for vertical transmission.Overall, the mother/child positivity rate was 14.2%, reaching 50% for infected Asian-descendant mothers. However, the positive association between Asian ethnicity and seropositive child on the univariate analysis was not confirmed on the multivariate analysis. This higher transmission probably occurred because of the longer breastfeeding time in this group (24–36 months). The only one of the six positive children who had been breastfed for less than 24 months (six months) had a mother with a proviral load of 488 copies/1032, for whom sexual transmission is more efficient33. This can also be due to the time between the beginning of the relationship with an infected partner and seroconversion. The seropositivity of the husband apparently did not influence child transmission, but most of the studied women were diagnosed after becoming mothers and information on the partner or ex-partner serological status was unknown for 40% of them. Thus, this possibility could not be totally ruled out in this study. In turn, the higher number of children per mother, which could be related to age, did not influence the transmission rate.Mother’s age (>26 years) at the time of delivery was associated with seropositivity in the child, and one of the explanations could be the increase in the prevalence of HTLV-1 infection with increasing age, observed mainly among women2 and to MTCT35. Transmission occurred regardless of the sex of the child, but this data is controversial, since some studies have associated a higher risk of transmission to males23 or females22 exposed children, whereas others found a similar transmission risk for boys and girls37. However, the small sample size is a limitation to influence the child infection from other factors, such as history of blood transfusion, cesarean delivery or maternal coinfection with HIV, HTLV-2, HBV or an association of HIV/HCV.Mother’s schooling, as a prediction for mother income, tended to correlate inversely with the risk of seropositivity of the child, but probably the sample size was not large enough to identify this possible protecting factor. In fact, several studies reported that indicators of lower socioeconomic status, such as lower educational level and low income, are associated with HTLV-1 transmissionThe main limitation of this study was that most of the participants were 15 years old or older when tested. Therefore, we cannot assure that all our patients were infected through the vertical route. However, it is unlikely that all patients were infected by the sexual route at such young age. This possibility may not invalidate the major findings of our study. On the other hand, we cannot prospectively test such hypothesis due to obvious ethical issues.4 PBMC), prolonged breastfeeding, and familial aggregation with more than one infected child in the same offspring. In fact, there is a genetic susceptibility for HTLV-1 infection38, but this possibility must be addressed further in another study. Thus, PVL was a predictive marker for a positive child, even when performed several years after the putative transmission from the mother. Finally, screening for HTLV-1 among pregnant women and a non-breastfeeding policy by infected mothers continue to be the main measures to prevent MTCT where this infection is endemic.It is important to highlight some independent risk factors identified in this cohort, such as a higher maternal proviral load (>100 copies/10


E. coli and its pathovars, this study probed for the potential of an E. coli MG1655 ΔrpoS mutant to acquire HHP resistance by directed evolution. Despite the higher initial HHP sensitivity of the ΔrpoS mutant compared to the wild-type strain, evolved lineages of the former readily managed to restore or even succeed wild-type levels of resistance. A number of these ΔrpoS derivatives were affected in cAMP/CRP regulation, and this could be causally related to their HHP resistance. Subsequent inspection revealed that some of previously isolated HHP-resistant mutants derived from the wild-type strain also incurred a causal decrease in cAMP/CRP regulation. cAMP/CRP attenuated HHP-resistant mutants also exhibited higher resistance to fosfomycin, a preferred treatment for STEC infections. As such, this study reveals attenuation of cAMP/CRP regulation as a relevant and RpoS-independent evolutionary route towards HHP resistance in E. coli that coincides with fosfomycin resistance.High hydrostatic pressure (HHP) processing is an attractive non-thermal alternative to food pasteurization. Nevertheless, the large inter- and intra-species variations in HHP resistance among foodborne pathogens and the ease by which they can acquire extreme resistance are an issue of increasing concern. Since RpoS activity has been considered as a central determinant in the HHP resistance of  In particular, a substantial proportion of Shiga-toxigenic Escherichia coli (STEC) isolates appears to be resistant to exposures of 600 MPa5. Furthermore, extreme HHP resistance is a trait that can reproducibly be genetically acquired by E. coli when iteratively exposed to HHP shocks6.High hydrostatic pressure (HHP) treatment is a promising alternative to conventional thermal pasteurization of foods as it is able to improve the microbial stability and safety of foods without compromising its fresh-like attributes. However, while commercial HHP treatment ranging from 400 to 600 MPa can achieve a significant level of reduction of most vegetative spoilage and pathogenic foodborne bacteria9. In particular, the general stress response governed by the RpoS sigma factor strongly contributes to HHP resistance in E. coli, which in part could be traced back to the RpoS mediated upregulation of cyclopropane fatty acid synthase, outer membrane lipoproteins and trehalose-6-phosphate synthase11. Indeed, stationary phase induction of this general stress response accounts for the increased HHP resistance of stationary over exponential phase cells10. Moreover, also the large variability in pressure resistance among natural STEC isolates has been related to polymorphisms in their rpoS allele and the corresponding variations in cellular RpoS activity11.The mechanism underlying bacterial inactivation by HHP treatment is still not well understood; however, some cellular targets and stress responses involved in pressure survival have been identifiedE. coli populations to their particular ecological niches13. In fact, strains with attenuated RpoS activity tend to display an improved use of secondary carbon sources like succinate, fumarate and acetate at the expense of self-preservation under stress14, while strains with a high basal RpoS activity display increased stress resistance at the expense of nutritional competence15. Furthermore, given the extensive regulatory network surrounding RpoS activity16, modulatory mutations affecting this activity are likely to occur at a relative high frequency, enabling lineages to balance between self-preservation (with high RpoS activity) and nutritional competence (with low RpoS activity) 17. As a result, modulation of the RpoS response seems to be an important evolutionary strategy in E. coli to improve nutritional competence on the one hand or stress tolerance and cross-resistance on the other.These polymorphisms are thought to reflect the evolutionary adaptation of different E. coli O157:H7 (ATCC 43888) to a limited number of progressively intensifying HHP shocks rapidly selected for mutants that were cross-resistant to mild pressure (id est 300 MPa) and heat, and that showed signs of increased RpoS activity15. A random transposon knock-out screen subsequently confirmed the role of RpoS activity in HHP resistance of ATCC 43888, as a disruption of the rssB gene (encoding an anti-RpoS factor that binds to RpoS and quenches its activity16) yielded a similar HHP and heat cross-resistance18. Interestingly, the same transposon screen also revealed that disruption of either the cyaA (encoding the adenylate cyclase or cAMP synthetase) or crp (encoding the cAMP Receptor Protein) gene could confer HHP resistance as well. Even though lack of cAMP/CRP regulation has been described to increase rpoS expression, it remains unclear whether or not the underlying mechanism is dependent on RpoS19. Nevertheless, given the growth defect imposed by cyaA or crp mutations20, we anticipated that mutants spontaneously compromised in these loci could not readily enrich in populations evolving towards HHP resistance18.The latter also became clear when exposure of E. coli to develop HHP resistance in the absence of this sigma factor, in order to elucidate potential alternative evolutionary pathways that are truly independent of RpoS upregulation.Given the central and perhaps also dominant role of the RpoS global regulator in HHP resistance and resistance development, this study aimed to investigate the remaining potential of E. coli MG1655 lacking RpoS to develop HHP resistance, an MG1655 ΔrpoS derivative was constructed and five independent cultures of this mutant strain were subjected to successive cycles of increasingly severe HHP shock  with intermittent outgrowth of survivors. Figure rpoS lineages during the selection regimen in comparison to the inactivation of the original ΔrpoS and WT parental strains. As anticipated, deprivation of the rpoS gene sensitized the ΔrpoS strain to HHP treatment, resulting in a circa 1,000-fold higher (P ≤ 0.05) inactivation at 470 MPa compared to the WT strain.To examine the intrinsic ability of rpoS lineages managed to develop piezotolerance, with the single clones subsequently isolated from each of the five lineages (designated MT1 to MT5) being on average 2.8 log10 cycles more resistant than the parental ∆rpoS background at 470 MPa (data not shown). Further phenotypic analysis of these five clones revealed varying levels of HHP resistance, with three clones even displaying a HHP resistance equal  or significantly (P ≤ 0.05) higher (i.e. MT1 and MT4) than the MG1655 WT (i.e. rpoS proficient) parent at 500 MPa despite lacking RpoS activity  more sensitive to a 57 °C shock than its ∆rpoS parent  actually significantly (P ≤ 0.05) increased by 0.7 log10 cycle on average (data not shown), indicating that serial passaging itself did not select for pressure resistance.Importantly, when three independent lineages of the ancestral ∆E. coli O157:H7 (strain ATCC 43888) had revealed that disruption of cAMP/CRP homeostasis could confer HHP resistance18, we examined whether cAMP/CRP regulation was compromised in the evolved ΔrpoS mutants. As lactose metabolism in E. coli requires a functional cAMP/CRP complex, we first examined the evolved ΔrpoS mutants for their lactose fermentation phenotype on MacConkey agar  was visibly reduced in comparison to the ΔrpoS parent. Moreover, this phenotype persisted regardless of the addition of cAMP to the MacConkey medium. Sequencing subsequently revealed that MT1 incurred an IS2 insertion of ca. 1.3 kb length in the crp promoter region, while the crp gene of MT4 incurred a point mutation resulting in an V184M amino acid substitution in the DNA-binding domain of CRP21 (Table Since a random transposon knock-out screen performed earlier in ar Table . InteresP > 0.05) reduced β-galactosidase activity compared to its ∆rpoS parent as did MT1 and MT4  and exposed to pressure (500 MPa). Please note that the HHP resistance of the ∆rpoS parent and some of its derivatives (i.e. MT2 and MT5) significantly (P ≤ 0.05) decreased by ca. 1.1 log10 cycles in the presence of the pACYC184 control vector  decreased the HHP survival of MT1 and MT4 by 4.2 log10 cycles on average. Moreover, in agreement with the lack of cAMP complementation already observed in the lactose fermentation assay (Table cyaA gene (using pACYC184-cyaA18) did not significantly (P > 0.05) attenuate the HHP resistance of MT1 and MT4. The inactivation of the ∆rpoS parent also decreased significantly (P ≤ 0.05) but to a lower extent (ca. 0.8 log10 cycle) when equipped with pACYC184-crp and pACYC184-cyaA at 400 MPa  affected the HHP resistance of MT2 and MT5. In the case of MT3, which displayed altered MacConkey phenotype without compromising lactose fermentation and harboring intact crp and cyaA alleles, complementation with both pACYC184-crp or pACYC184-cyaA appeared to significantly (P ≤ 0.05) attenuate HHP resistance by 1.7 log10 cycles on average, indicating that this mutant might for some other reason be more susceptible to overdosing CRP and CyaA.In order to confirm that a compromised CRP functionality is indeed causally linked to the HHP resistance phenotype of MT1 and MT4, the evolved Δor Figs.  and 3, lrpoS parent, a number of previously documented mutants that were evolved starting from a wild-type (i.e. RpoS proficient) MG1655 background to withstand exposure to 800 MPa and higher23 were evaluated on MacConkey agar, M9 lactose and β-galactosidase activity for their capacity to utilize lactose  attenuated by 3.1 log10 reductions on average when equipped with pACYC184-cyaA but not with pACYC184-crp  reduced by 3.0 log10 cycles in the presence of pACYC184-crp and to a lesser extent (ca. 1 log10 cycle) by pACYC184-cyaA.As such, it appeared that strain LMM1010, LMM1020 and DVL1 indeed displayed a clearly compromised lactose fermentation, and that the addition of cAMP to the MacConkey medium could completely alleviate this phenotype in LMM1010 and DVL1. Correspondingly, LMM1010 and DVL1 were subsequently found to harbor frameshift mutations in their crp and cyaA loci (Table P ≤ 0.05) reduction of 1.2 log10 cycles from the presence of pACYC184-crp. Similarly, the survival of the WT strain to 600 MPa also significantly (P ≤ 0.05) decreased by 0.9 log10 cycle when complemented with both plasmids  attenuated the HHP resistance of LMM1030 by 2.3 log10 reductions on average, suggesting again that this mutant is sensitized to cAMP/CRP upregulation by an alternative mechanism.Strain LMM1030 and DVL20 did not suffer compromised lactose fermentation nor mutations in their ci Table . The HHPi.e. not exposed to directed evolution), the crp or cyaA gene was synthetically deleted in the WT or ΔrpoS parent, after which HHP resistance of the resulting strains was examined. This unambiguously established that complete loss of either of the components giving rise to the cAMP/CRP complex could significantly (P ≤ 0.05) improve the HHP resistance in both a ΔrpoS  and WT MPa Fig.  and 800 y P ≤ 0.0 improve crp allele of MT4 (i.e. the most HHP resistant mutant obtained in the ∆rpoS parent) was chromosomally reconstructed in its parental strain (leading to ΔrpoS crpMT4), the HHP resistance of ∆rpoS strain increased ca. 600-fold (P ≤ 0.05) at 500 MPa  significantly (P ≤ 0.05) attenuated the HHP resistance of MT4 by 2.6 log10 cycles, while similarly replacing the crpMT4 allele with the non-functional Δcrp allele (leading to MT4 Δcrp) did not affect (P > 0.05) its HHP resistance  often still remained higher than the synthetically reconstructed strains, suggesting secondary determinants to contribute to HHP resistance as well. In particular, while the survival of MT1 to 500 MPa matched (P > 0.05) the inactivation of the ΔrpoS background lacking crp or cyaA locus, the HHP resistance of MT4 was ca. 1 log10 cycle significantly (P ≤ 0.05) higher than that of ΔrpoS Δcrp and ΔrpoS ΔcyaA strains  than the ∆rpoS parent  was also significantly (P ≤ 0.05) higher (from 0.9 to 1.9 log10 cycles) than that of the WT parent in the absence of crp or cyaA  improve heat resistance in an ΔrpoS or WT MG1655 background  in which the stimulated general stress response yielded cross-resistance to both HHP and heat  and cAMP/CRP downregulation  could further improve HHP resistance. However, it appeared that deletion of rssB did not significantly (P > 0.05) change the HHP resistance of a Δcrp or ΔcyaA mutant at 800 MPa  than the WT strain.In contrast to its effect on HHP resistance, deletion of und Fig. . This speat Fig. . Since Ry P > 0.0 improve 25, which has been regarded as one of the preferred antibiotic treatment options for STEC infections27, the spontaneous HHP resistant mutants raised in this study were examined for their fosfomycin resistance. Table crp and ∆cyaA strains as a control) increased from 2-fold (LMM1020 and DVL1) to 16-fold (MT4) compared to their parental strain, while mutants that did not incur cyaA or crp mutations  maintained the same fosfomycin resistance as their parental strain.As it was previously found that mutants with decreased cAMP/CRP activity display an increased resistance to fosfomycinE. coli, and mutations upregulating the activity of this sigma factor therefore highlight one evolutionary strategy towards HHP resistance development15. Surprisingly, however, an E. coli genetic background deprived of the RpoS protein and therefore hypersensitive to HHP shock readily managed to acquire wild-type (or even higher) levels of HHP resistance upon recurrent exposure to HHP shock. Further analysis subsequently revealed that compromised cAMP/CRP regulation was causally involved in HHP resistance development of two (i.e. MT1 and MT4) of the five independently evolved ΔrpoS mutants. Furthermore, mutations compromising cAMP/CRP regulation were also discovered to causally contribute to the phenotype of extremely HHP resistant (and RpoS proficient) E. coli mutants that were evolved in earlier studies23. To the best of our knowledge, this study is therefore the first to identify and phenotypically validate cyaA and crp mutations as causal HHP resistance conferring mutations arising in spontaneously evolved mutants of E. coli.RpoS activity has previously been established as a central determinant in stationary phase HHP resistance in Listeria monocytogenes (strains LO28 and ScottA), spontaneous mutations leading to HHP resistance have been documented earlier and found to compromise the ctsR gene, which encodes the class III heat shock repressor30. Disruption of the ctsR gene can lead to a defect in the repression of clpC family chaperones which results in transcription of these stress response genes and consequently in increased cross-resistance to HHP, heat, acidity and H2O228. In contrast, cAMP/CRP regulation has a more pleiotropic and metabolically oriented impact on E. coli physiology, and a large number of genes are responsive to cAMP/CRP activation or repression31. Intriguingly, mutations attenuating CyaA or CRP functionality have previously been found to affect osmotic32, acid33, heat34 and oxidative36 stress resistance of E. coli, although the latter phenotypes might indirectly stem from upregulation of the rpoS gene which is repressed by cAMP/CRP36. Moreover, while this report clearly underscores compromised cAMP/CRP activity to support HHP resistance in an RpoS independent manner, both transcriptional regulators oppositely affect a largely overlapping set of downstream genes38. As such, it could well be that (some of) the functions required to withstand or address HHP stress in the cell are independently but similarly directed by either the upregulation of RpoS or the downregulation of cAMP/CRP. However, the phenotypic constitution of mutants with increased RpoS activity or decreased cAMP/CRP activity is bound to differ, since RpoS upregulation (by deleting ΔrssB) supports both HHP and heat resistance while cAMP/CRP downregulation (by deleting cyaA or crp) only seems to support HHP resistance in E. coli MG1655.In crp or cyaA displayed 2- to 16-fold higher resistance to fosfomycin than the parental strains. Fosfomycin is an antibiotic that targets the UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) that is involved in the first step of peptidoglycan synthesis39, and the observed resistance is likely due to decreased expression of the glycerol-3-phosphate transporter (GlpT) and the hexose phosphate transporter (UhpT) in the absence of cAMP/CRP complex25. These transporters are responsible for the uptake of fosfomycin in the cell due to its similar structure to phosphorylated sugars39. Besides being an alternative drug for urinary and systemic infections caused by multidrug-resistant Enterobacteriaceae40, fosfomycin is actually proposed as one of the antibiotic therapies of choice for STEC infections27. Indeed, in contrast to DNA damaging antibiotics such as fluoroquinolones and trimethoprim-sulfamethoxazole that tend to activate the lytic cycle of the Shigatoxin (Stx) encoding prophages in STEC and thereby improve the production and release of Stx, fosfomycin has no effect on Stx expression and might therefore reduce the risk of haemolytic uremic syndrome development caused by Stx43. The potential of HHP treatment to select for cAMP/CRP compromised HHP resistant E. coli mutants with a concomitantly decreased susceptibility to fosfomycin can therefore pose a concern for food safety that should be further investigated.Importantly, we also demonstrated that HHP resistant clones with mutations in rpoS mutants developed HHP resistance without apparent cyaA or crp mutations or lactose metabolism defects , suggesting still alternative RpoS (and likely cAMP/CRP) independent evolutionary routes towards HHP resistance development exist. Nevertheless, these mutants might still have acquired mutations that directly affect the same downstream effector genes that are otherwise governed by RpoS upregulation or cAMP/CRP downregulation.Finally, it should be noted that some ∆E. coli, and (ii) draws attention to the decreased fosfomycin susceptibility that tends to coincide with this route of HHP resistance development.In summary, this study (i) highlights attenuation of cAMP/CRP regulation as a novel and valid evolutionary route and an RpoS independent mechanism towards HHP resistance development in E. coli K-12 MG165544 and its derivatives listed in Supplementary Table rpoS knock-out of MG1655 was constructed by transferring the kanamycin resistance cassette replacing the rpoS locus of E. coli JW543745 by P1-transduction46. Further deletions of rssB, crp and cyaA loci were performed according to the method of Datsenko and Wanner47, using the cells of interest equipped with the plasmid pKD46 (encoding λ red recombinase genes behind the araBAD promoter) and an amplicon prepared on pKD13 (containing the kanamycin resistance cassette) using the primers listed in Baba et al.45. The antibiotic marker was flanked by FRT sites to be further excised by transiently equipping the strain with the plasmid pCP20 (expressing the Flp site-specific recombinase48), resulting in the desired deletion mutant.crp alleles were reconstructed by the dual counter-selection system described by Li et al.49. Firstly, the crp gene was replaced by an amplicon containing the tetA-sacB marker prepared on E. coli XTL298 using the primers: 5′-CTACCAGGTAACGCGCCACTCCGACGGGATTAACGAGTGCCGTAAACGACATCAAAGGGAAAACTGTCCATATGC-3′ and 5′-GGCGTTATCTGGCTCTGGAGAAAGCTTATAACAGAGGATAACCGCGCATGTCCTAATTTTTGTTGACACTCTATC-3′. In a second step, counter-selection was used to replace the tetA-sacB cassette with a crp amplicon obtained by the following primers: 5′-ACCCACTTCACTCGCGCTT-3′ and 5′-GGAACGAGGGAGAAGAGCAG-3′. Constructs were verified by PCR and sequencing using the locus specific primers described below.The crp (encoding the E. coli ATCC 43888 crp gene under the control of its native promoter18), pACYC184-cyaA (encoding the E. coli ATCC 43888 cyaA gene under the control of its native promoter18) or the corresponding backbone control plasmid (pACYC18450) by electroporation. Please note that while crp and cyaA sequences originated from E. coli ATCC 43888, their sequence identities with the corresponding sequences of E. coli MG1655 are 99.5% and 98.2%, respectively, and both pACYC184-crp and pACYC184-cyaA proved to specifically attenuate the HHP resistance of ∆crp and ∆cyaA strains, respectively, in both wild-type (WT) and ∆rpoS backgrounds , 20 μg/ml of tetracycline , 100 μg/ml of ampicillin  or 30 μg/ml of chloramphenicol (Sigma-Aldrich) was added to select for the presence of recombined amplicons49, pKD46 and pCP2048 or pACYC184-based vectors50, respectively. For inactivation experiments, stationary phase cultures were obtained by inoculating test tubes containing 4 ml of Tryptone Soy Broth  with a single colony of the required strain prior to incubating the culture aerobically with shaking (300 rpm) for 18 h at 37 °C. In pACYC184-based complementation assays, 30 μg/ml of chloramphenicol supplemented TSB was used to select for the presence of the plasmids50.For strain construction, Lysogeny Broth (LB) mediumca. 2 × 109 Colony Forming Units per milliliter (CFU/ml), were harvested by centrifugation  and resuspended in an equal volume of 0.85% KCl (Sigma-Aldrich). For HHP treatment, 200 μl of the suspension was heat sealed in a sterile polyethylene bag after exclusion of the air bubbles and subjected to pressure (200–800 MPa) for 15 min in an 8-ml pressure vessel , held at 20 °C with an external water jacket connected to a cryostat. Both the slow pressure increase (100 MPa/min) and the external water jacket attenuated adiabatic heating during pressure build-up. Finally, decompression was almost instantaneous. For heat treatment, on the other hand, three sterile PCR tubes were aseptically filled with a 75 μl portion of resuspended cells and subjected to 57.0 °C for 15 min using a PCR apparatus . The intensity of HHP and heat treatment was chosen to inactivate ≥ 5 log10 reductions of the WT (800 MPa) or ∆rpoS  parental strain for a 15 min treatment in order to clearly observe levels of HHP and heat resistance development in the mutants and plasmid complementation effects. After HHP or heat treatment, samples were aseptically retrieved from the polyethylene bags or PCR tubes, respectively, and survival was determined as described below.Cells from a stationary phase culture, containing E. coli MG1655 ΔrpoS mutants with enhanced pressure resistance, five independent cultures of this strain were reiteratively exposed to HHP shocks (15 min), progressively increasing pressure by 30 MPa each cycle (from 200 MPa to 470 MPa). After each HHP shock, an aliquot of the treated sample was inoculated 1/100 into fresh, prewarmed TSB and regrown for 23 h at 37 °C prior to the next round of pressurization. After ten cycles of selection, two surviving clones from each of the evolved cultures were purified on TSA and rechallenged to the last HHP treatment (470 MPa) in order to select a clone that represented the HHP resistance of the evolved culture. Simultaneously, in order to determine the effect of the serial passage itself in the selection of E. coli HHP resistance, three independent lineages of the parental ΔrpoS strain were daily subcultured in the absence of HHP stress, which corresponded to ca. 6.6 ( = log2 100) generations per day.To obtain 52. After 24 h of incubation at 37 °C, spots containing between 5–50 colonies were counted, so that the quantification limit was 1,000 CFU/ml. The logarithmic reduction factor was calculated as log10 (N0/N), in which N0 and N represent the number of survivors in CFU/ml prior and after treatment, respectively.Samples were serially diluted in 0.85% KCl supplemented with 0.1% bacteriological peptone water (Oxoid), and subsequently 5 μl-samples of each dilution were spotted onto Tryptone Soy Agar , as previously describedE. coli mutants was screened indirectly by their ability to ferment lactose in MacConkey agar No 3 . A spot of 20 µl of stationary phase cultures diluted 1/100 in 0.85% KCl was plated on the MacConkey agar supplemented with or without 5 mM of 3′,5′-cyclic adenosine monophosphate , and then incubated overnight at 37 °C. E. coli forms pink colonies and a hazy precipitate of bile salts because of acidification53. Strains lacking crp or cyaA locus, and therefore compromised in lactose fermentation, form yellowish colonies because of ammonia production19. The addition of exogenous cAMP to MacConkey agar reverts the phenotype of ΔcyaA strains to the WT strain  or D-lactose  and incubated at 37 °C for 48 h.To determine the ability of growth on glucose or lactose as the sole carbon source, stationary phase cultures were washed twice and diluted 1/100 in M9 salts51. Cells were grown in LB in the presence of 1 mM isopropyl β-D-1-thiogalactopyranoside  to mid-exponential phase. The β-galactosidase activity of permeabilized cells on ortho-nitrophenyl-β-galactoside  cleavage was measured in a Multiskan RC  and expressed in Miller units (MU).The β-galactosidase assay was carried out as previously described by MillerE. coli MG1655 rpoS, rssB, crp and cyaA loci was generated using the site-specific primers previously described18 and sequenced by Macrogen . The sequences were compared with those from the parental strain (WT or ∆rpoS background), which did not differ from the published genome of E. coli str. K-12 substr. MG1655 in the GenBank database44.A PCR amplicon encompassing the open reading frame and its upstream promoter region of the 54. Serial twofold dilutions  of fosfomycin (Sigma-Aldrich) in TSB were prepared in a 96-well plate and inoculated with 5 × 105 CFU/ml of the corresponding E. coli mutant. The inoculated plate was incubated at 37 °C overnight. The MIC of fosfomycin was defined as the lowest concentration that inhibited visible growth of the corresponding mutant. MIC value for each strain was obtained in triplicate.The minimal inhibitory concentration (MIC) for fosfomycin was determined by the broth microdilution method described by Jorgensen and FerraroP was ≤ 0.05. All microbial inactivation outcomes shown in figures correspond to averages and standard deviations calculated from three replicates performed in different working days. Miller units data correspond to averages and standard deviations obtained in three independent measurements.Statistical analyses, ANOVA and t-test, were carried out using the software GraphPad PRISM 5.0 , and differences were regarded as significant when Supplementary Information