We found that δ13CAA patterns in contrast to bulk δ13C values distinguished between carbon derived from algae, seagrass, terrestrial plants, bacteria and fungi. Furthermore, we showed for two aquatic producers that their δ13CAA patterns were largely unaffected by different environmental conditions despite substantial shifts in bulk δ13C values. The potential of assessing the major carbon sources at the base of the food web was demonstrated for freshwater, pelagic, and estuarine consumers; consumer δ13C patterns of essential amino acids largely matched those of the dominant primary producers in each system. Since amino acids make up about half of organismal carbon, source diagnostic isotope fingerprints can be used as a new complementary approach to overcome some of the limitations of variable source bulk isotope values commonly encountered in estuarine areas and other complex environments with mixed aquatic and terrestrial inputs.Tracing the origin of nutrients is a fundamental goal of food web research but methodological issues associated with current research techniques such as using stable isotope ratios of bulk tissue can lead to confounding results. We investigated whether naturally occurring δ Analyses of total organic matter (“bulk”) have become widespread due to the relative ease and low cost of sample preparation and analysis. However, the potential of variable environmental conditions to influence carbon isotopic ratios at the base of the food web . In the field, we only collected fresh and newly emerged thallus and leaves. For microbial reference samples we obtained axenically cultured bacteria and fungi. For testing the potential for δ13CAA source information to be independent of variation in bulk δ13C values among aquatic producers, we acquired multiple samples of the seagrass Posidonia oceanica and the giant kelp Macrocystis pyrifera collected from a range of different environments within their natural habitats. For P. oceanica, the maximum photosynthetically active radiation above their canopies ranged from 50 to 310 mmol m−2 s−1 between the sampling locations and the coverage of leaf necrosis ranged from 0 to 37.5%. M. pyrifera samples were collected either in spring or late fall, periods of contrasting ocean conditions that result in widely divergent bulk isotopic values 13CAA patterns from greenhouse plants and those collected in boreal and mangrove ecosystems. Finally, to assess the practical relevance of using δ13CAA patterns as diagnostic biomarkers in actual ecosystems we analyzed consumers from three well-studied ecosystems: from a marine pelagic ecosystem in the central North Pacific Ocean the carnivorous fish species opah (Lampris guttatus), common dolphinfish (Coryphaena hippurus) and broadbill swordfish (Xiphias gladius), from a littoral marine system (estuarine) the California mussel , and from oligotrophic arctic lakes the water flea Daphinia sp. and seston.To test whether freshwater, marine and terrestrial primary producers have different δM. pyrifera (see below) were collected on state tidelands for which collection without a permit is allowed for less than ten pounds fresh weight. The seagrass samples were provided under a permit to Joseph M. Long Marine Laboratory, Santa Cruz. Subsamples of mussels were obtained from recreationally harvested mussels under a permit to Natasha Vokhshoori. The three pelagic fish samples were collected as in Choy et al. Daphnia. The Alaskan samples were collected on land owned by the Alaskan state or Bureau of Land Management where no permission was required because we sampled on day trips by foot or rafts leaving no permanent marks; soil samples were collected between 2 and 8 cm below the soil surface and amounted to <50 g dry weight for each location; each plant sample amounted <1 g dry weight. No permit was required for sacrificing Daphnia, which are not protected or endangered; they were sampled with a zooplankton net (150–200 µm) and kept alive until sorting at Toolik Field Station. The terrestrial plant collection was supplemented with two species from public owned land in Denmark where no permission was required for sampling. We obtained five P. oceanica samples from the Catalonian shore in the Mediterranean Sea under a permit to Teresa Alcoverro by the Catalan Water Agency, and the five M. pyrifera samples from the Californian shore were sampled under a permit to Melissa M. Foley Daphnia and muscle tissue for mussels and fish. All samples were dried at 50°C except for animal, seston and soil samples, which were freeze-dried. After drying the samples were homogenized with a mortar.A detailed list of all our field samples and their locations is provided in 2 tissue culture flasks with vented cap in brackish water (13.9 psu) collected from Kiel Fjord or seawater (31.2 psu) collected at Multimar-Wattforum by the German North Sea. Water mixed with added nutrients was sterile filtered (Whatman celluloseacetate 0.2 µm filter)  –1) × 1000‰, where R is the ratio of heavy to light isotope; and the standard is Vienna Pee Dee Belemnite (VPDB) for carbon and air for nitrogen.Elemental content and bulk isotope ratios of plants, bacteria, fungi, macroalgae and animals were measured at the UCSC Stable Light Isotope Facility. Approximately 1 mg of sample was pelletized into tin capsules and analyzed on a Carlo Erba 1108 linked to a Thermo Finningan DeltaPlus XP mass spectrometer with an analytical standard deviation of typically<±0.15‰ (n = 3). Elemental content and bulk isotope ratios of microalgae were determined on 2 mg samples pelletized into tin capsules at the UC Davis Stable Isotope Facility using a PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20–20 isotope ratio mass spectrometer . Isotope data are expressed in delta (δ) notation as  we transferred between 1.5 and 7 mg of sample to Pyrex culture tubes (13×100 mm). Samples were flushed with N2 gas, sealed, and hydrolyzed in 1–2 ml 6 N HCl  at 110°C in a heating block for 20 h. After hydrolysis, samples collected on GF/C filters and coralline algae were purified with Dowex 50WX8 cation exchange resin according to Amelung & Zhang n-hexane/DCM  to the Pyrex tubes that were flushed shortly with N2 gas and sealed before vortexing for 30 s. The aqueous phase was then filtered through a Pasteur pipette lined with glass wool that had been pretreated at 450°C. All samples were transferred into 4 ml dram vials before evaporating the samples to dryness under a steam of N2 gas for 30 minutes at 110°C in a heating block. The samples were stored at −18°C. To volatize the AAs, we followed the derivatization procedure by Corr et al N-acetyl methyl ester derivatives). As a precautionary measure to reduce oxidation of amino acids during derivatization, we flushed and sealed reaction vials with N2 gas prior to the methylation and acetylation reactions. To account for carbon added during derivatization 13C values. Nor-leucine was used as an internal standard. Amino acid derivatives were injected with an autosampler into an Agilent Single Taper Ultra Inert Liner (#5190-2293) that was held at 280°C for 2 min. The compounds were separated on a Thermo TraceGOLD TG-200MS GC column (60 m×0.32 mm×0.25 um) installed on an Agilent 6890N gas chromatograph (GC). The oven temperature of the GC started at 50°C and heated at 15°C min−1 to 140°C, followed by 3°C min−1 to 152°C and held for 4 min, then 10°C min−1 to 245°C and held for 10 min, and finally 5°C min−1 to 290°C and held for 5 min. The GC was interfaced with a MAT 253 isotope ratio mass spectrometer (IRMS) via a GC-III combustion (C) interface (Thermo-Finnigan Corporation). All samples were analyzed in triplicate. The average reproducibility for the internal standard nor-leucine (Nle) was ±0.4‰ (n = 3) and the amino acid standards ranged from ±0.1‰ for Phe to ±0.6‰ for Thr (n = 12–15 for each batch). Of the amino acids we were able to analyze  that best explained differences between the categorical variables (i.e. the groups defined by the PCA and one-way ANOVA tests) and to construct models for predicting membership of unknown samples, we performed linear discriminant function analysis . Relative contributions of EAAs from diets to consumers was estimated in the software FRUITS  All statistical analyses except the mixing modeling were performed in R version 2.12.1 13CAA patterns we first carried out a PCA with δ13CAAn values that in contrast to a LDA does not require categorical variables. With this analysis we found that the samples clustered according to their major phylogenetic associations: algae, bacteria, fungi and vascular plants  were most important for distinguishing between algae, bacteria, fungi and terrestrial plants. The seagrass samples were omitted from this analysis due to a small number of samples (the number of samples did not exceed the number of EAAs). Bacterial, fungal, and terrestrial plant samples classified with 99.1±2.6% posterior probability within their own groups . All brown algae samples (Phaeophyceae) classified with 100% probability as algae, and eight out of nine red algae (Rhodophyta) classified with 98.6±3.8% probability as algae. The ninth red algae, Osmundea spectabilis, classified as a bacterium (54% probability) rather than an alga (46% probability). The most important linear discriminants for separating the four categorical variables were Lys, Phe, Leu and Val. We created a second LDA model based on the five most informative EAAs  to assess to what extent it would be possible to separate the three algal groups  and seagrass samples from each other. Of all 27 microalgal samples, 24 samples classified as microalgae, 10 out of 12 brown algae classified as brown algae, 7 out of 9 red algae classified as red algae, and all 7 seagrass samples classified as seagrass  and giant kelp samples (M. pyrifera) 13C values was five- to ten-fold greater for bulk than δ13CAAn values  from the central Pacific. The three categorical variables  were distinctly different and the δ13CEAA patterns of all three fish samples matched those of microalgae with 100% probability . The LDA model classified all the categorical variables correctly, and the δ13CEAA patterns of the two mussels resembled microalgae with ≥99.7% probability and brown algae with 0.3% probability  in the food sources Finally, we investigated how δbability . The sesbability . In the bability . We used13CAA patterns can be used as powerful and ubiquitous tracers for discerning carbon origins in both terrestrial and marine settings . In every habitat, the results were consistent with the hypothesis that δ13CEAA source patterns based are conserved in passage through food webs. In the freshwater ecosystem, the resemblance of Daphnia and seston AAs with microalgae rather than bacteria and terrestrial plants agrees with limnological food web studies based on bulk isotope data Daphnia nor seston were projected directly on top of the algal samples pointing to a possible influence of microbial reworking or allochtonous input, as has been previously suggested in Arctic lakes 13CAA patterns would be expected to align with algal sources in oceanic consumers. The δ13CEAA patterns exactly followed this prediction . A similar case of greater 13C enrichment in algae than plants was found by Chikaraishi and Naraoka n-alkanes that like most other lipids have pyruvate as a precursor 13C depleted in algae relative to plants. From a carbon mass balance point of view it is possible that 13C enrichment of pyruvate AAs in algae lead to depletion of the aromatic AAs because they are less coupled to lipid synthesis by having phosphoenolpyruvate and erythrose-4-phosphate as precursors. The aromatic AAs also serve as precursors for the synthesis of numerous primary and secondary metabolites such as alkaloids and lignins 13CAA fractionation of the aromatic AAs. For deepening our understanding of the biochemical processes leading to the 13CAA fractionation patterns further inquiry is needed into the overall carbon mass balance between the most abundant hydrocarbon groups and AAs.If algae and vascular plants share similar amino acid biosynthetic pathways 13CAA patterns can transcend bulk isotope analyses across diverse ecological environments, and be used to understand carbon sources and transfer in ecological research as exemplified with our classification and mixing modeling approaches. The strong diagnostic potential for algae and plants may be particularly powerful for applications in complex estuarine and coastal systems, where mixed aquatic and terrestrial inputs occur, or in freshwater environments with strong allochtonous influence. While our findings indicate that source-specificity of δ13CEAA patterns is conserved across environmental gradients, further controlled physiological studies are also warranted to better understand under what circumstances these patterns may be altered. For a broad understanding of food web cycling of nutrients, we also stress that it is also important to consider other major biochemical classes, such as lipids and carbohydrates. Finally, in addition to investigating ecosystem level transfer of carbon and nitrogen, we suggest that this fingerprinting method can help assessing symbiotic contributions of AAs from bacteria to animal hosts. There is mounting evidence that protein-nitrogen assimilation is possible in the lower gut of some animals during digestion 13CEAA patterns may offer a direct way to assess the importance of such microbial AA contributions, not only in the specific animals where this may occur, but more broadly up food chains.Taken together, our results show that δFigure S1GC-C-IRMS chromatogram.(PDF)Click here for additional data file.Table S1Field sample characteristics.(PDF)Click here for additional data file.Table S2Laboratory sample characteristics.(PDF)Click here for additional data file.Table S313C values of individual amino acids.δ(PDF)Click here for additional data file.Table S4Principal component analysis output for algal, bacterial, fungal and plant samples .(PDF)Click here for additional data file.Table S513CAAn values.ANOVA comparison of δ(PDF)Click here for additional data file.Table S6Linear discriminant analysis output for algal, bacterial, fungal and terrestrial plant samples .(PDF)Click here for additional data file.Table S7Linear discriminant function analysis output of aquatic primary producers .(PDF)Click here for additional data file.Table S8Linear discriminant function analysis output for amino acid origins of consumers .(PDF)Click here for additional data file.Appendix S1FRUITS mixing model data .(PDF)Click here for additional data file.


Congenital central hypoventilation syndrome (CCHS) is a rare neuro-respiratory disorder associated with mutations of the PHOX2B gene. Patients with this disease experience severe hypoventilation during sleep and are consequently ventilator-dependent. However, they breathe almost normally while awake, indicating the existence of cortical mechanisms compensating for the deficient brainstem generation of automatic breathing. Current evidence indicates that the supplementary motor area plays an important role in modulating ventilation in awake normal humans. We hypothesized that the wake-related maintenance of spontaneous breathing in patients with CCHS could involve supplementary motor area.−2) during resting breathing and during exposure to carbon dioxide and inspiratory mechanical constraints. They were compared with 8 healthy individuals. Segments of electroencephalographic tracings were selected according to ventilatory flow signal, from 2.5 seconds to 1.5 seconds after the onset of inspiration. After artefact rejection, 80 or more such segments were ensemble averaged. A slow upward shift of the EEG signal starting between 2 and 0.5 s before inspiration  was considered suggestive of supplementary motor area activation.We studied 7 CCHS patients . In CCHS patients, pre-inspiratory potentials were systematically identified in all study conditions, including resting breathing. They were therefore significantly more frequent than in controls.This study provides a neurophysiological substrate to the wakefulness drive to breathe that is characteristic of CCHS and suggests that the supplementary motor area contributes to this phenomenon. Whether or not this “cortical breathing” can be taken advantage of therapeutically, or has clinical consequences  remains to be determined. Congenital central hypoventilation syndrome (CCHS) is a rare neuro-respiratory disorder associated with several possible mutations of the PHOX2B gene Ventilation of the lungs results from phasic contractions of respiratory muscles in response to a so-called “neural drive to breathe” that is relayed to the muscles by spinal respiratory motoneurons. The net output of spinal respiratory motoneurons depends on the various inputs that they receive not only from automatic brainstem central pattern generators but also from various suprapontine circuits that are responsible for emotional breathing modulations, voluntary breathing control and the interplay between respiration and nonrespiratory processes of cortical origin, primarily speech and voluntary movements In humans, the contribution of cortical processes to the overall drive to breathe is phenomenologically illustrated by certain particularities of breathing control. For example, decreasing carbon dioxide partial pressure in the blood by passive hyperventilation (ventilator-induced hypocapnia) is associated with apnoea during sleep, but rarely while awake Therefore, we hypothesized that resting ventilatory activity in awake CCHS patients would be associated with respiratory-related EEG signs of SMA activity, i.e. pre-inspiratory potentials. Such a respiratory-related cortical activity is normally not observed in healthy individuals.post hoc in order to limit bias. The study was approved by the appropriate local legal and ethics authority .The study was conducted according to the principles expressed in the Declaration of Helsinki. Participants were informed about the general study procedure, the methods used and the absence of any associated risks, and gave their written consent to participate. The exact study objectives were only revealed to the subjects 2) while breathing room air less than 65 mmHg; partial pressure of carbon dioxide in arterial blood (PaCO2) greater than 55 mmHg; daytime dependence on mechanical ventilation; significant restrictive or obstructive ventilatory defect on pulmonary function testing; use of any kind of neurotropic medication. According to these criteria, an index group of 7 patients was constituted . Of note, at the time of study, this index group represented the majority of the known French adult patients with CCHS. Two patients were tracheotomized. In one case, the tracheotomy orifice was blocked during the experiments and the patient breathed through the natural airway; in the other case, the measurements were performed with the patient breathing via the tracheotomy with the cuff inflated. One of the patients received oral contraception with desogestrel and was subsequently found to have recovered some degree of carbon dioxide chemosensitivity CCHS patients with documented PHOX2B mutations, followed at the adult section of the French national CCHS reference centre, were systematically considered for inclusion. Exclusion criteria were: partial pressure of oxygen in arterial blood (PaO−2). They were all non-smokers and totally naive to respiratory physiology experiments.The above index group was compared to a control group of healthy volunteers free of any cardiac, pulmonary or neuromuscular disorder  involved breathing through an inspiratory threshold load with a 23 cmH2O load for 10 minutes. “Control 3” (condition #5) was identical to “Control 2”. Heart rate (HR) and transcutaneous pulse oximetry (SpO2) were monitored continuously as a safety measure.The experimental protocol comprised 5 study conditions. “Control 1” (condition #1) consisted of resting ventilation with minimal constraints, during which the participants only wore a respiratory inductance plethysmography vest (see below). “Control 2” (condition #2) also consisted of resting ventilation, but the participants breathed through a respiratory measurement apparatus (see below). “Chemostimulation” (condition #3) involved breathing a gas mixture of either 5–7% COIP, for “inductance plethysmography”), as previously described Rib cage and abdomen displacements were recorded by two coils placed in a sleeveless jacket, only allowing circumferential stretching of the wires . The signal was digitised at 40 Hz and processed to provide a measurement of ventilatory flow (V'2 pressure (PetCO2). This was achieved with an infrared gas analyzer connected to the mask . PetCO2 was only used to monitor the stability of the hypercapnic stimulations.During “Control 2”, “Chemostimulation”, “Loading” and “Control 3”, the subjects wore a facial mask  that gave access to expired air sampling for the measurement of end-tidal partial COIP signal were created. The EEG signal was filtered between 0.01 and 5 Hz. EEG epochs with clear artefacts  or EEG consistent with eye movements were discarded and the remaining epochs were ensemble averaged. Averaged tracings were examined for the presence of an inspiratory premotor activity in the form of a slow upward shift of the EEG signal starting between 2 and 0.5 s before inspiration. When this activity was observed, a first-order least-squares regression equation was fitted to the corresponding segment of EEG. A pre-inspiratory potential was considered to be present when and only when the slope of this equation was positive and significantly different from zero according to the F-test for equality of variance. The latency of the pre-inspiratory potential, if present, was defined as the interval in ms between the negativity and the start of inspiration. The amplitude of the pre-inspiratory potential corresponded to the potential measured in µV between baseline and the “zero flow” point.Surface scalp electroencephalographic signal (EEG) was recorded using a 12-electrode cap installed after rubbing and cleaning with alcohol and application of a conductive gel . Active electrodes were placed in equidistant positions  according to the conventional “10–20” topographic system. The earth electrode was positioned at AFz. The EEG signal was digitised at 2000 Hz and recorded using V-Amp® software  for subsequent processing, which was performed according to the method previously described 95). Between group comparisons of dichotomous variables (presence or absence of a PIP) were performed using Fisher's exact test. Latencies and amplitudes were compared between groups only in the “Load” condition in which all subjects of both groups exhibited a PIP . This comparison was performed using a Mann-Whitney test. Differences were considered significant when the probability P of a type I error was less than 5%.All statistics were performed using Prism 4® software . The normality of the distribution of the results was tested using a Shapiro-Wilk test. As the hypothesis of normality was not verified for all variables, continuous variables are expressed as their median and 95% confidence intervals  and during “Chemostimulation”, as one subject exhibited a PIP during “Control 1”, 3 during “Control 2”, 1 during “Control 3”. PIPs were also inconsistent during “Chemostimulation” (2 out of 8 subjects). In contrast, PIPs were systematically present during “Loading” (8 out of 8 subjects). PIPs were clearly visible in the Cz derivation and none of the other ones. 2-induced headache. One patient only participated in “Control 1”, “Chemostimulation” and “Load”. All patients exhibited PIPs in all study conditions in which they participated. PIPs were clearly visible in the Cz derivation and none of the other ones. Two patients dropped out of the study following “Chemostimulation” due to COP = 0.001; “Control 2”, P = 0.031; “Control 3”, P = 0.01) and during “Chemostimulation” (P = 0.006). No significant difference was observed during “Loading” (P = 1.000).Consequently, PIPs were significantly more frequent in patients than healthy control subjects during all control conditions . The corresponding values for median amplitude were 5.9 µV [2.1–14] and 3.7 µV [2.1–8.9], respectively . Of note, although statistical comparison of amplitudes and latencies was not possible in the other conditions due to the very small sample sizes, the amplitudes of PIPs inconsistently observed in controls were always much lower (5- to 10-fold) than those of the PIPs observed in patients. PIP latencies were also much shorter (2- to 4-fold) in controls.This study shows that awake, spontaneously breathing adult CCHS patients exhibit a respiratory-related cortical activity that is normally absent during resting ventilation in healthy subjects.2-stimulated breathing in normal individuals; they also predict that PIPs should consistently be present in response to inspiratory threshold loading 2-stimulated breathing. This incidence was higher in the CCHS patients than in the normal individuals, and the levels of significance of the control-CCHS differences were at their highest in the two conditions where the comparison was the most important, namely the “control 1” condition  and the “CO2 stimulated breathing” condition (p = 0.006). When PIPs were present in the normal subjects, they were several folds smaller than in the CCHS subjects. We therefore do not think that the small size of the control group (and the fact that the two groups were not exactly gender matched) compromises the physiological validity of our observations.Available data predict that PIPs should generally not be detected during resting breathing, not be detected during exercise and not be detected during CO2 stimulation also strongly facilitates the response of the diaphragm to corticospinal inputs generated by transcranial magnetic stimulation Studies using transcranial magnetic stimulation have demonstrated a direct connection between the SMA and phrenic motoneurones A respiratory-related motor cortical activity does not necessarily mean that “each breath is taken voluntarily”. There are numerous examples of automatized voluntary motor actions that can be performed “without attention being clearly directed toward the details of the movement” Irrespective of the previous observations, all made in children, our findings raise the question of putative “competition” for cortical resources in adult CCHS patients. In other words, can their respiratory-related cortical activity have an impact on their motor and/or cognitive performances? Voluntary breathing control disturbs simple motor tasks In conclusion, we have evidenced that pre-inspiratory potentials are present during resting ventilatory activity in awake CCHS patients, which suggests that SMA activation is required for maintenance of breathing in this setting. It is likely that this activation facilitates the response of spinal motoneurones to the residual bulbospinal drive to breathe. Further studies are needed to fully characterize the mechanisms of “cortical breathing” in CCHS, and to determine the clinical implications of these findings. Whether or not inducing spinal facilitatory plasticity through pharmacological or nonpharmacological interventions could have therapeutic benefits will also have to be evaluated.


The [P2W18O62]6− polyoxidometalate anion has site symmetry 1 and contains two structurally distinct types of W atoms: viz. six W atoms on vertical pseudo-mirror planes grouped in two sets of three, and 12 equatorial W atoms that do not lie in the pseudo-mirror planes grouped in two sets of six. In each [Co(en)2(ox)]+ cation, the CoIII ion is coordinated by four N atoms from two en ligands and two O atoms from the ox ligands, completing a distorted octahedral structure. The sodium cation lies on an inversion centre and additionally links the complex cations and anions. In the crystal, the various units are linked by N—H⋯O and O—H⋯O hydrogen bonds, which together with C—H⋯O hydrogen bonds form a three-dimensional structure. The contribution of a region of disordered electron density, possibly highly disordered solvent water molecules, to the scattering was removed with the SQUEEZE option of PLATON . To equilibrate the charges five H+ ions have been added to the polyoxidometalate. These H+ ions and the disordered solvent contribution were not included in the reported molecular weight and density.The title compound, Na(CSpek 2009. Acta Cr DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


The primary motor cortex (M1) is an effective target of non-invasive cortical stimulation (NICS) for pain threshold modulation. It has been suggested that the initial level of cortical excitability of M1 plays a key role in the plastic effects of NICS.Here we investigate whether transcranial direct current stimulation (tDCS) primed 1 Hz repetitive transcranial magnetic stimulation (rTMS) modulates experimental pressure pain thresholds and if this is related to observed alterations in cortical excitability.15 healthy, male participants received 10 min 1 mA anodal, cathodal and sham tDCS to the left M1 before 15 min 1 Hz rTMS in separate sessions over a period of 3 weeks. Motor cortical excitability was recorded at baseline, post-tDCS priming and post-rTMS through recording motor evoked potentials (MEPs) from right FDI muscle. Pressure pain thresholds were determined by quantitative sensory testing (QST) through a computerized algometer, on the palmar thenar of the right hand pre- and post-stimulation.p<0.001) with a parallel increase in pressure pain thresholds (p<0.01). In contrast, anodal tDCS-primed 1 Hz-rTMS resulted in a corresponding decrease in cortical excitability (p<0.05), with no significant effect on pressure pain.Cathodal tDCS-primed 1 Hz-rTMS was found to reverse the expected suppressive effect of 1 Hz rTMS on cortical excitability; leading to an overall increase in activity (This study demonstrates that priming the M1 before stimulation of 1 Hz-rTMS modulates experimental pressure pain thresholds in a safe and controlled manner, producing a form of analgesia. Pain perception and modulation have become crucial topics of exploration within the scientific community due to increasing numbers seeking treatment for pain and the current inadequacy of available therapies. Chronic, persistent pain is a complex condition that has been identified as one of the most disabling and costly afflictions in North America, Europe, and Australia with a lifetime prevalence of up to 55.2% in the adult population In particular, musculoskeletal pain is a cause of long term pain and disability associated with common disorders such as rheumatoid arthritis, osteoarthritis and fibromyalgia. Algometry is used for the assessment of pressure pain thresholds in these patients with chronic musculoskeletal pain. Measurement of these pressure pain thresholds and assessment of the defined 18 tender points forms part of the American College of Rheumatology (ACR) guidelines for the diagnosis of fibromyalgia One method of therapy has been realized in the form of non-invasive cortical stimulation (NICS) with the primary motor cortex (M1) being the most effective target for the modulation of both experimental and chronic pain In this study we investigate the physiological mechanism of a novel NICS protocol in healthy adults. Pressure pain thresholds were used as an index of pain sensitivity so as to aid translation of our findings to a musculoskeletal pain population in future studies. There are currently very few studies assessing the efficacy of NICS on this clinically useful pain modality, which needs to be addressed There are two main methods of NICS; transcranial direct current stimulation (tDCS) and repetitive transcranial magnetic stimulation (rTMS) which are safe, easy to use and associated with relatively negligible side effects et al., demonstrated how a combined paradigm using tDCS to “prime” or “precondition” motor cortex can successfully shape the conditioning effects of subsequent rTMS It has been suggested that the initial state of the motor cortex may play a key role in the plastic effects of rTMS. Siebner Previous work revealed that tDCS-primed 1 Hz-rTMS can be applied to enhance the modulation of experimental thermal pain thresholds In our present study the technique of tDCS-primed 1 Hz rTMS is applied to pressure pain thresholds. Pressure pain has been proposed as a particularly useful modality, in that it is more directly related to a variety of clinical outcomes in a diverse range of pain patients Here, this preconditioning protocol is used to investigate the modulation of both cortical excitability and mechanical pressure pain which may prove useful in the translation of NICS effects between experimental and chronic pain groups.The study was performed in accordance with the Declaration of Helsinki and approved by the Faculty of Health Sciences Research Ethics Committee, Trinity College Dublin. All participants provided written informed consent to participate in the study.Fifteen healthy male volunteers  were recruited to take part in the research study, by advertisement. Participants were approved to partake in the project once they completed a medical questionnaire which was deemed suitable by a qualified medical professional. Subjects with any previous or concomitant psychiatric or neurological disease, any conditions associated with acute or chronic pain or with somatosensory abnormalities were excluded. All participants were able to understand the QST protocol instructions and gave their written informed consent. All were completely naïve to the purpose of the experimental procedures.2 rubber plunger tip with a digital display of force in increments of 0.01 KPa. Real-time, visual and auditory feedback allowed the investigator to control and monitor applied pressure rates. The computerized algometer applied pressure stimuli according to the method of limits algorithm within the Pathway software and simultaneously recorded thresholds via a push-button response unit (In order to determine pressure pain thresholds (PPT), a QST protocol was conducted using a standard digital algometer  computer-controlled through The Medoc Pathway System . The algometer comprised of a pressure gauge and a 1 cmnse unit .PPT was defined as the amount of force required to elicit a sensation of pain distinct from pressure or discomfort or in other terms; the point at which pressure transitioned to discomfort or pain 2 stimulator  connected to a figure of eight double coil (70 mm). The coil was placed tangentially to the scalp, with the handle pointing backward and laterally at a 45° angle to the sagittal plane, inducing a posteroanterior current in the brain Motor evoked potentials (MEPs) were recorded using surface electromyography (EMG) from the right first dorsal interosseous (FDI) muscle using conductive adhesive Ag/AgCl electrodes  in a belly tendon montage. EMG signals were amplified, band-pass filtered (10–50 Hz) and sampled at 1,000 Hz using an Octal BioAmp . The peak-to-peak amplitudes of the MEPs were then analysed off-line using custom-written software. The optimal placement for stimulation was defined as the site where single pulse TMS resulted in the largest MEP in a consistent fashion. These biphasic pulses were provided using a Magstim RapidTo detect current-driven changes of excitability, motor-evoked potentials (MEPs) of the right FDI following stimulation of its motor-cortical representational field were recorded. A train of ∼20 TMS-evoked MEPs were recorded at 3.5 s intervals at an intensity of 120% of each individual's RMT. This train of MEPs was carried out prior to (PRE), in between (INTER) and immediately after stimulation (POST).2) with the active electrode placed over C3 and the reference electrode positioned contralaterally above the right orbit. These electrodes were held in place with use of the MindCap . Each of the four experimental conditions involved the continuous application of a current of 1 mA intensity for a duration of 10 minutes, as these parameters proved successful in priming the motor cortex in previous experiments The application of tDCS was performed to the hand area of the left motor cortex; C3 according to the international 10–20 EEG system, through the use of a HDC kit . Direct currents were applied through a pair of saline soaked (0.89%NaCl) synthetic buckskin electrodes  at the designated 90% of a participant's individual RMT. This sub threshold intensity was chosen for two reasons: First, the intensity is likely to activate the corticospinal neurons trans-synaptically avoiding direct activation of these neurons A sham-controlled, single-blind study was conducted, consisting of four separate experiments, to determine how priming the motor cortex using tDCS may enhance the effects of rTMS on the modulation of pressure pain thresholds in healthy volunteers. The four conditions consisted of;Sham tDCS – Sham rTMSSham tDCS – Real rTMSCathodal tDCS – Real rTMSAnodal tDCS – Real rTMSEach treatment was performed on the same day, at the same time over a period of 3 weeks in order to avoid any potential issues regarding a variation of pain thresholds due to circadian rhythms. Each experiment was performed by the same, trained female examiner and at the same time of day. Sessions lasted approximately 2 hours for each condition, with participants kept conscious and alert by being addressed by the examiner during breaks from stimulation. An interval of one week was required between active testing conditions to avoid any interference that could be caused by the after-effects of stimulation. Treatment 1 and 2 acted as control sessions and were therefore suitable to be performed on the same day without risk of interference. Sham, cathodal and anodal tDCS-primed – 1 Hz rTMS interventions were presented in a pseudo randomized sequence and participants were unaware of the presented condition.Collected data was first tested for normality of distribution using the Shapiro-Wilk's test within the SPSS statistical package . MEP data required transformation to square root in order to address moderate positive skew. All analyses were carried out using normally distributed data.2, 1). This statistic was used a measure of internal consistency and reproducibility across all conditions.Test-retest reliability of PPT baseline measurements for each condition was calculated using SPSS based on a single rating, absolute agreement, 2-way mixed effect model (ICCA two-way repeated measure (4×2) ANOVA was carried out to test the effects of Condition  and Time (Pre and Post) on the PPT data. A separate two-way repeated measure (4×3) ANOVA was used to assess the effects of Condition  and Time  on MEP values. Planned contrasts were employed to assess statistical significance within each model. Values are presented as the mean of each participant's mean ± the standard error of the mean (SEM). A confidence level of 0.05 was set for all statistical tests.Fifteen healthy male participants completed all protocols designed to determine if preconditioning the left M1 using tDCS  can modulate baseline cortical excitability and thereby shape the subsequent effects of low frequency rTMS (1 Hz) on pressure pain thresholds. None of the participants experienced any adverse side effects during or after the neurostimulation sessions.Pressure pain threshold testing was found to be reliable on a test-retest basis across all conditions with an average baseline of 360±3.7 KPa, an intraclass correlation coefficient (ICC) value of 0.9 and a 95% confidence interval of 0.798–0.960. This indicates test-retest reliability to be highly consistent, allowing for greater statistical power in further analysis.F = 3.42, p<0.05, 2pη = 0.196) and Time  = 5.02, p<0.05, 2pη = 0.264), however the respective interactions were not statistically significant  = 2.17, p = 0.105, 2pη = 0.135). Planned contrasts demonstrated cathodal tDCS-primed 1 Hz-rTMS produced a significant increase in PPT from a baseline threshold of 365.73±90.92 KPa to 405.07±97.44 KPa post neurostimulation in regards to both the effects of Condition  = 10.68, p<0.01, 2pη = 0.433) and Time   = 12.06, p<0.001, 2pη = 0.463) showing that this priming protocol did indeed have an effect on cortical excitability. However no significance was evident for the individual main effects of Condition  = 1.07, p = 0.35, 2pη = 0.071) or Time  = 1.03, p = 0.371, 2pη = 0.068).Within subject contrasts indicated a significant initial increase in MEP amplitude from a baseline of 0.6±0.34 mA (PRE) to 0.75±0.45 mA (INTER) after anodal tDCS priming  = 17.64, p<0.001, η2p = 0.558) and alternatively induced a significant decrease in amplitude ranging from 0.75±0.45 mA (INTER) to 0.6±0.47 mA (POST) due to the subsequent effects of 1 Hz rTMS  = 8.66, p<0.05, 2pη = 0.382). Cathodal tDCS-primed 1 Hz-rTMS resulted in an opposite response of cortical activity as displayed in F = 61.96, p<0.001, 2pη = 0.816) and an equally significant increase in excitability from 0.39±0.28 mA (INTER) to 0.68±0.44 mA (POST) immediately after 1 Hz rTMS  = 25.93, p<0.001, 2pη = 0.649). In comparing controls (Control 1: Sham tDCS-Sham rTMS vs Control 2: Sham tDCS-1 Hz rTMS) significant change was only seen INTER vs POST with a decrease in MEP size from 0.62±0.3 mA to 0.55±0.25 mA  = 9.56, p<0.01, 2pη = 0.406).The ANOVA revealed a significant main effect for the interaction of the factors Condition x Time  and final measurements (POST). Contrastingly, anodal tDCS-primed 1 Hz-rTMS produced a significant decrease in cortical excitability INTER vs POST with no effect on pain thresholds. These changes in excitability may provide a physiological basis for the observed pain modulatory effect.Prior to our exploration of homeostatic plasticity and pain threshold modulation, 1 Hz-rTMS had been found ineffective in pain relief and unworthy of further research in the field Our findings suggest that rTMS-induced plastic changes depend on the functional state of the motor cortex, which can be modified using tDCS, consistent with previous research Current preconditioning techniques seek to manipulate these homeostatic mechanisms. In effect, tDCS is used to augment background motor cortical excitability and thus enhance the cortical plastic changes induced by subsequent low-frequency rTMS. In the present study; we used the mean MEP amplitude as an index of the changes in primary motor cortex excitability. Of particular importance, participants displayed an increase in cortical activity in response to 1 Hz rTMS when previously inhibited by cathodal tDCS, successfully reversing the expected suppressive effects of 1 Hz rTMS. Contrastingly, the same 1 Hz rTMS protocol decreased the excitability of the motor cortex back to baseline levels after cortical excitability had been enhanced by a preceding anodal tDCS.The level of homeostatic regulation appears to be dependent on the intensity of the stimulation applied. A study using tDCS to precondition the motor cortex before subsequent stimulation via high frequency 5 Hz-rTMS reported effects in agreement with our findings Conversely, the same 5 Hz-rTMS protocol induced a rise in cortical excitability to return to baseline values after the motor cortex had been inhibited by a preceding session of cathodal tDCS The present study reinforces the results of our previous work How these forms of neurostimulation act on the M1 to modulate pain thresholds is also not yet fully understood. However many brain imaging studies have been carried out in relation to motor cortex stimulation (MCS), a technique comprised of an invasive, surgical procedure whereby electrodes are implanted onto the surface of the motor cortex. Results reveal activity changes in all neural structures involved in pain processing, such as the thalamus, anterior cingulate cortex (ACC), orbitofrontal cortex, insula, second somatosensory cortex (S2) or the periaqueductal grey matter (PAG) The molecular mechanisms underlying NICS-induced pain modulation also remain speculative. Lefaucheur et al., hypothesized that these analgesic effects may be mediated by the inhibitory GABAergic system We hypothesize that in altering the functional state of the M1 using cathodal tDCS before subsequent stimulation via 1 Hz rTMS, the resulting increase of pain thresholds mirror those of high frequency rTMS. This suggests the need for further research into the state dependent effects of rTMS in both healthy and patient populations with the aim of determining optimal protocols for clinical integration.However the strength of our conclusions may be limited by our small, gender-specific sample size. Previous work in NICS and pain modulation have mainly used male participants due to stable hormone levels throughout testing periods with the aim of reducing inter-subject variability. A recent report outlined the presence of this strong male-orientated bias in experimental subject choice to date and issued a call for all experiments to be performed on both sexes Future studies relating to clinical integration of these techniques must take into consideration the disease state of the individual. Pathophysiology is likely to alter the functional state of the cortex and lead to a different response pattern provoked by rTMS compared with healthy control subjects Our study has found clear evidence to support the use of this tDCS-primed 1 Hz rTMS technique as a novel method in the modulation of pain. In line with current literature on metaplasticity, our results have shown cathodal tDCS-primed 1 Hz rTMS can increase cortical excitability and further, lead to a parallel increase of pressure pain thresholds, successfully producing a form of analgesia. These combined neurophysiological treatments have the potential to be beneficial in both experimental and clinical settings as a safe, non-invasive and innovative approach to pain modulation.


Cytological observations suggest that initial steps of recombination are largely normal in a majority of −/−Hfm1 spermatocytes. Intermediate and late stages of recombination appear aberrant, as chromosomal localization of MSH4 is altered and formation of MLH1foci is drastically reduced. In agreement, chiasma formation is reduced, and cells arrest with subsequent apoptosis at diakinesis. Our results indicate that deletion of Hfm1 leads to the elimination of a major fraction but not all COs. Formation of chromosome axial elements and homologous pairing is apparently normal, and −/−Hfm1 spermatocytes progress to the end of prophase I without apparent developmental delay or apoptosis. However, synapsis is altered with components of the central region of the synaptonemal complex frequently failing to extend the full length of the chromosome axes. We propose that initial steps of recombination are sufficient to support homology recognition, pairing, and initial chromosome synapsis and that HFM1 is required to form normal numbers of COs and to complete synapsis.Faithful chromosome segregation during meiosis requires that homologous chromosomes associate and recombine. Chiasmata, the cytological manifestation of recombination, provide the physical link that holds the homologs together as a pair, facilitating their orientation on the spindle at meiosis I. Formation of most crossover (CO) events requires the assistance of a group of proteins collectively known as ZMM. HFM1/Mer3 is in this group of proteins and is required for normal progression of homologous recombination and proper synapsis between homologous chromosomes in a number of model organisms. Our work is the first study in mammals showing the  Meiosis is a specialized type of cell division during which haploid gametes are generated to offset doubling of the chromosome number that occurs at fertilization. Prior to segregation in the first meiotic division, meiotic chromosomes pair and recombine. Chiasmata, products of recombination, provide the temporary physical link between the chromosomes of each pair. This association is required to prepare the chromosomes for segregation to the opposite poles of the spindle at the first meiotic division, so that ultimately each gamete receives only one copy of each chromosome. For this reason, errors in recombination are commonly linked to human infertility, spontaneous abortions, and severe aneuploid-based birth defects. We observed that the absence of HFM1/Mer3 results in deficient recombination and in incomplete meiosis-specific modification of chromosome structure. Our findings thus indicate that the mouse HFM1 protein is required for normal progression of important aspects of meiosis that act to insure that the correct numbers of chromosomes are transferred to the next generation. Meiosis is a reductional type of cell division in which two rounds of chromosome segregation follow one round of DNA replication. This is a highly regulated and essential process to generate gametes containing the correct haploid chromosome complement. The first segregation, meiosis I, is reductional because homologous chromosomes, instead of sister chromatids, migrate to the opposite poles of the spindle. Recombination in meiosis I prophase prepares homologous chromosomes for segregation. Chiasmata, the cytological manifestation of recombination, provide physical links that hold the homologs in pairs and ensure the proper segregation of each chromosome of a pair to opposite poles of the spindle so that ultimately each gamete receives one copy of each chromosome The initial steps of homologous recombination involve introduction of double-strand breaks (DSBs) by the topoisomerase-like protein SPO11 Based on sequence homology with proteins known to be DNA helicases, mouse and human HFM1 and other Mer3 homologs are predicted to have seven motifs characteristic of the DExH box type of DNA/RNA helicases Coprinus cinereus and Sordaria macrospora demonstrate the importance of Mer3 in the normal progression of several critical aspects of meiosis. Molecular analysis of intermediates of recombination in yeast Mer3 mutants reveals that DSBs are formed normally but that repair is delayed, single-end invasions are reduced (reaching a maximum of 15% of wild-type levels) and formation of meiotic recombination-dependent DNA synthesis is rare Hfm1/mer3 mutants display severe deficiencies in chiasma formation, high levels of chromosome missegregation at meiosis I and partial or total infertility C. Cinereus and S. macrospora Mer3 mutants, development of axial elements is apparent normal and, except for S. macrospora, where homolog alignment is inefficient, the onset of chromosome pairing shows no differences with respect to wild-type Hfm1/mer3 mutants. However, there is a wide variation in the extent of synapsis failure across species. Budding yeast and C. cinereus show the most severe synaptic phenotype, where the onset of synaptonemal complex formation is delayed S. macrospora where it persists among late pachytene cells and is accompanied by a high level of interwoven chromosomes, the latter being more severe after chromosome alignment S. macrospora Mer3 mutants, the leptotene-pachytene transition is delayed, accompanied by defects in bouquet dynamics C. cinereus and Arabidopsis Mer3 mutants are apparently normal. However, in C. cinereus the majority of cells do not progress beyond meiosis I and became apoptotic Mutants that inactivate HFM1/Mer3 orthologs in yeast, Arabidopsis, rice, −/−Hfm1 spermatocytes the number of initial recombination events appears comparable to that observed in wild-type mice, but recombination is defective and the number of bivalents at diakinesis is reduced to ∼20%. These results are consistent with current models of recombination in which HFM1 participates in the major CO pathway. Although CO formation is severely defective in −/−Hfm1 spermatocytes, chromosomes pair and initial synapsis is apparently normal. Our results suggest that early recombination transactions are sufficient for homologous recognition, pairing and initial synapsis. However, synapsis is incomplete for all but a small number of chromosomes in −/−Hfm1 spermatocytes. We propose that completion of synapsis generally depends on an HFM1 activity that leads to the formation of COs.In this study we provide the first analysis of meiotic progression in HFM1/Mer3 null mice. We show that mutants of both sexes are infertile and that males show meiotic arrest at the end of prophase I. In a majority of −/−Hfm1 mice. The gene trap vector utilized to disrupt Hfm1 contains a viral long terminal repeat (LTR) followed by a splice acceptor sequence (SA) and a promoter-less selectable marker neomycin (Neo) with a polyadenylation signal (pA). The vector also contains the PGK promoter followed by the first exon of the Bruton's Tyrosine Kinase (Btk) gene upstream of a splice donor (SD). The Btk exon contains termination codons in all reading frames to prevent translation of downstream fusion transcripts . RT-PCR analysis of −/−/Flp+Hfm1 mice detected no Hfm1 transcripts for any of the predicted alternative splicing forms of Hfm1 are homozygous −/−Hfm1, a number that is close to a normal Mendelian distribution, and suggests that Hfm1 is not an essential gene  had significantly smaller testes than wild-type  littermates .Both male and female homozygous mutant mice were sterile due to severe blocks of spermatogenesis and oogenesis. Tissue analysis indicates that eiosis I , top rowequently . Matings−/−Hfm1 mice. Chromosomes appeared to be normally condensed at metaphase I in −/−Hfm1 cells but, in contrast to wild-type cells, the chromosomes often failed to form a metaphase plate and remained scattered throughout the nucleus  of homologous chromosomes are closely coaligned (synapsed), and sex chromosome axes appear in a typical pachytene conformation as previously defined by Dresser, et. al. −/−Hfm1 spermatocytes were subdivided into two categories by the state of X and Y chromosome pairing (XY = tightly associated and X-Y = loosely associated). We note that in wild-type cells at pachytene and diplotene, immunostaining with γH2AX marks only the sex body (n = 100). For a fraction of −/−Hfm1 spermatocytes, however, some γH2AX persists not only in the sex body but also in one or several patches along autosomes in pachytene-like spermatocytes with either tightly  or loosely associated  X and Y chromosomes. Remnants of γH2AX can also be observed in a fraction of diplotene −/−Hfm1 spermatocytes  (As described in the n = 240) .−/−Hfm1 mice. RAD51 foci decrease from late zygotene  to pachytene  in wild-type spermatocytes but were elevated in −/−Hfm1 spermatocytes in late zygotene  and persisted at particularly high levels in 31% of pachytene-like nuclei   . Persist−/Hfm1− spermatocytes, and that for a major fraction of −/Hfm1− cells the numbers and kinetics of γH2AX and RAD51 immunosignals are similar to wild-type.In sum, our results suggest that DSBs are formed essentially at wild-type levels in −/Hfm1− spermatocytes showed a reduced number of MSH4 foci at zygotene , early pachytene , mid pachytene  and late pachytene   . Thus, H−/−Hfm1 spermatocytes   , suggest−/−Hfm1 spermatocytes. Chiasmata join homologs to make stable bivalents, so we first scored the number of bivalent chromosomes per cell in air-dried diakinesis spreads. We found an average of 20±0.5 (n = 100) bivalents per wild-type spermatocyte and only 3.7±2.2 (n = 100) bivalents per −/−Hfm1 spermatocyte  . The reduction in the number of chiasmata in knockout spermatocytes indicates a requirement for HFM1 in the formation of the major fraction of COs in mice.To confirm that deletion of HFM1 reduces CO formation, we analysed chiasma frequency in wild-type and matocyte . The drahiasmata . We agai−/−Hfm1 spermatocytes. However, the X and Y chromosome PAR regions are unsynapsed and separated in 43.5% of −/−Hfm1 cells (n = 161) versus only 5% of wild-type cells  . Synapsis at PAR is very sensitive to reduced rates of recombination and perturbations in synapsis, and thus can be used to detect defects in these processes ct test) . It is pi. e., synaptonemal complex formation) are codependent processes −/−Hfm1 spermatocytes. In addition, absence of splitting of the axes indicates that sister chromatid cohesion is not affected by the absence of HFM1 (21±0.7 centromeres for wild-type (n = 100) and 21±1.2 centromeres for −/−Hfm1 (n = 100) spermatocytes at pachytene)  and diakinesis  and a decrease in pachytene  in −/−Hfm1 spermatocytes relative to wild-type. However, no significant differences were observed when the distribution of spermatocytes for the entire population of spermatocytes (from leptotene to diakinesis) from wild-type and −/−Hfm1 were compared .Given that meiotic recombination and synapsis (chytene) . Spermat−/− mice . When in−/−Hfm1 spermatocytes but synapsis is frequently defective.Notably, synaptic defects are evident early in pachytene spermatocytes . We analmer3 mutant of S. macrospora−/−Hfm1 spermatocytes but entangled chromosomes appeared in a fraction of −/−Hfm1 diplotene nuclei   . Whether−/−Hfm1 spermatocytes is the substantially reduced immunostaining for SYCP1  and components of the synaptonemal complex central element TEX12  and SYCE1  compared with wild-type pachytene cells, in which all chromosomes show strong, uninterrupted immunolabeling    are significantly different . However, no significant difference was observed when wild-type was compared with the fraction of −/−Hfm1 bivalents with higher levels of SYCP1 staining  (A novel characteristic of chromosomes in n = 608) . In Hfm1y spaced . Intrigu n = 67) . The ave t test) .−/−Hfm1 spermatocytes, (1) for all but a small fraction of chromosomes, synapsis is incomplete and characterized by subnormal amounts of central region components of the synaptonemal complex and by regions of complete asynapsis, (2) the X and Y chromosomes are frequently unsynapsed even though they colocalze in the sex body, suggesting premature desynapsis, though failure to initiate synapsis cannot be ruled out, and (3) chromosome entanglement becomes visible at diplotene.In summary, efficient homologous recognition and synaptic initiation occur in the absence of HFM1. However, in C. cinereus and S. macrospora−/−Hfm1 spermatocytes is severely reduced but not eliminated  with zip1, zip3 or mer3. In these mutants, interhomolog associations occur at many more “axial association” sites than the ones giving rise to COs and generate such uniform and close alignment of homologous axes that they appear “pseudosynapsed” A simple explanation for the largely normal pairing and early synapsis in mouse Hme pairs . The ide−/−Hfm1 spermatocytes. This suggests that completion of synapsis is dependent on later steps of recombination that lead to CO formation or to CO formation per se. Alternatively, the HFM1 contribution to completion of synapsis may be unrelated to its function in recombination.Although initial steps of recombination and chromosome pairing apparently are not affected by the absence of HFM1 and the concomitant reduction in the numbers of COs, incomplete synaptonemal complex formation is observed in −/−Dmc1 and −/−Hop2 spermatocytes, homologous pairing and synapsis are absent −/−Mlh1 and −/−Mlh3 spermatocytes, mature, complete synaptonemal complexes form −/−Hfm1 spermatocytes and the link to recombination may be extended to other model organisms with similar HFM1/Mer3-dependencies, as in Arabidopsis, rice and, to a lesser extent, S. macrospora. The more extensive synapsis defects observed in yeast and C. cinereus Mer3 mutants presumably indicate different dependencies in these organisms in the mechanisms directing and/or coordinating synaptonemal complex formation with recombination.The synaptic phenotypes of mouse mutants for proteins that apparently act before HFM1 (DMC1 and HOP2) and after HFM1 (MLH1 and MLH3) are consistent with the idea that HFM1 provides an “intermediate activity” in recombination that is required for completion of synapsis. In −/−Hfm1 spermatocytes progress through prophase I with no apparent sign of checkpoint activation. For a major fraction of −/−Hfm1 spermatocytes, we assume that most DSBs are repaired so that no intermediates remain that could trigger a DNA repair checkpoint. In this case, despite incomplete synaptonemal complex and partial defects, mutant spermatocytes are still able to satisfy the surveillance mechanisms. In this scenario, however, it is not clear to us why the fraction of −/−Hfm1 spermatocytes that exhibit persistent γH2AX and RAD51 immunosignals do not show apparent prophase I delay or a cell response to an overly long delay/arrest such as apoptosis. A possible explanation is that persistent γH2AX and RAD51 foci are a consequence of inefficient removal of these proteins following abnormal (but completed) DNA repair. Alternatively, a low level of apoptosis during prophase I might not be detected by our assay. It also is possible that HFM1/Mer3 itself is required for a checkpoint, so that in the absence of Mer3 the checkpoint is inactivated. Regardless of the details, the requirements for HFM1/Mer3 for cell-cycle progression in mouse are different from those in budding yeast or C.cinereus, where Mer3 deletion results in mid prophase I arrest, or in S. macrospora, where Mer3 mutants show a delay in the leptotene-zygotene transition. These differences among species indicate that the biological functions that require HFM1/Mer3 and/or the responses to its absence have diverged along with other features of meiotic chromosome metabolism in these organisms.To avoid deleterious outcomes in recombination and synaptic defects, checkpoints survey meiotic errors and ultimately eliminate cells containing unresolved defects. In budding yeast and mice, among other species, meiocytes with defects in DSB repair and/or chromosome synapsis trigger meiotic arrest during prophase I Mice used in this study were wild-type (C57BL/6), transgenic Flpe B6.Cg-Tg (ACT FLPe) 9205Dym and HFM1 knockout. Experiments conformed to relevant regulatory standards and were approved by the IACUC .Gt(ost347241)Lex containing a gene trap insertion in the second intron of the Hfm1 gene Lex. The PCR product was sequenced revealing that the insertion site was 1510 bp into intron 2. The Hfm1Gt (ost347241) Lex embryonic stem cells were injected into C57BL/6 blastocysts to create chimeric mice, which were bred with C57BL/6 to generate the first progeny of +/−Hfm1 heterozygous mice genotyped by PCR analysis (Hfm1 transcript by removing the PGK/Btk/SD cassette was accomplished by crossing heterozygous Hfm1+/− mice with a mouse line expressing FLPe recombinase (B6.Cg-Tg (ACT FLPe) 9205Dym/J) (The mouse embryonic stem (ES) cell line Hfm1fm1 gene  was obtaanalysis . Elimina05Dym/J) .Flpe was assessed by genotyping using the primers (#10 and #11) under the same PCR conditions.The genotyping was carried out by PCR using oligonucleotide #1 (F1) and #2 (R1) to amplify the wild-type allele (a 340 bp fragment) and primers #1(F1) and #3 (R2)  to ampliHfm1 were amplified using Power Syber Green PCR Master Mix (Applied biosystems) with the following primers: #4 and #5, #6 and #7 and #8 and #9, respectively. The cycling conditions were: 95°C 10 min; 95°C 10 sec, 60°C 30 sec, for 40 cycles. The melt curve was of 95°C 10 sec and 65°C to 95°C performed in increments of 0.5°C.Total RNA was isolated from adult testis with the RNeasy Mini Kit (Qiagen). 2.0 µg of RNA was oligo dT primed and reverse-transcribed with the High capacity RNA-to-cDNA kit (Applied Biosystems). Three different exon boundaries of Testes and ovaries for histological examination were removed and fixed overnight in 10% neutral-buffered formalin (Sigma). Serial sections from either testes or ovaries were positioned on microscope slides and analyzed using either hematoxylin and eosin staining or a TUNEL assay (Roche).We employed established experimental approaches for the visualization of chromosomes in chromosome surface spreads Spermatocyte chromosome spreads for electron microscopy analysis was performed as previously described Table S1Summary of the oligonucleotide sequences used in this work.(DOCX)Click here for additional data file.


ICE2 gene in maintaining ER zinc homeostasis. Thus, functional profiling has provided many new insights into genes and processes that are needed for cells to thrive under the stress of zinc deficiency.Zinc is an essential nutrient because it is a required cofactor for many enzymes and transcription factors. To discover genes and processes in yeast that are required for growth when zinc is limiting, we used genome-wide functional profiling. Mixed pools of ∼4,600 deletion mutants were inoculated into zinc-replete and zinc-limiting media. These cells were grown for several generations, and the prevalence of each mutant in the pool was then determined by microarray analysis. As a result, we identified more than 400 different genes required for optimal growth under zinc-limiting conditions. Among these were several targets of the Zap1 zinc-responsive transcription factor. Their importance is consistent with their up-regulation by Zap1 in low zinc. We also identified genes that implicate Zap1-independent processes as important. These include endoplasmic reticulum function, oxidative stress resistance, vesicular trafficking, peroxisome biogenesis, and chromatin modification. Our studies also indicated the critical role of macroautophagy in low zinc growth. Finally, as a result of our analysis, we discovered a previously unknown role for the  Zinc is needed for the growth of all organisms because it acts as a required cofactor for many different proteins. Zinc deficiency is a common problem faced by free-living microbes, as well as plants and animals including humans. Among bacterial and fungal pathogens, zinc deficiency is also a key problem they can encounter during pathogenesis. To identify genes and processes that are important for growth when zinc is scarce, we used genome-wide functional profiling. In this approach, a collection of ∼4,600 mutant yeast strains, each lacking the function of a different gene, was tested to determine which genes are needed for optimal growth in low zinc. More than 400 genes were identified as being important. The identity of these genes implicates a large number of different processes as critical for low zinc growth. These included genes that are targets of the zinc-regulated Zap1 transcription factor as well as genes involved in secretory pathway function, oxidative stress resistance, vesicular trafficking, peroxisome biogenesis, and chromatin modification. As a result, we now know of many processes that might be good targets for the development of new antifungal drugs. Among bacterial and fungal pathogens, zinc deficiency is also a key problem they can encounter during pathogenesis. The innate immune system is an important first line of defense against infection. One strategy of innate immunity has been referred to as “nutritional immunity”, i.e. the withholding of essential nutrients such as iron from the invading pathogen ZAP1 gene itself. Up-regulation of its own transcription is likely to enhance the overall transcriptional response, especially for genes that have low affinity Zap1 binding sites in their promoters ZRT1, ZRT2, and FET4 genes that encode zinc transporters needed for zinc uptake ZRT3 expression because this gene encodes a transporter responsible for mobilizing zinc stored in the yeast vacuole TSA1 gene. Tsa1, the major peroxiredoxin in the cytosol of cells, degrades hydrogen peroxide To address the question of what genes and processes are important for growth of a zinc-limited yeast cell, we and others have used genome-wide transcription profiling to identify genes with altered expression in response to changes in zinc status As these discoveries demonstrate, transcriptomics analysis of gene expression has told us many things about how cells respond and adapt to zinc-limiting conditions. While useful, however, this approach is limited by the fact that it requires a gene to change in expression in order to be identified as potentially important. Many proteins needed for low zinc growth are likely to be constitutively expressed or regulated at post-transcriptional levels. A complementary approach that does not have this limitation is genome-wide functional profiling 2) or zinc-replete (LZM+100 µM ZnCl2) media. LZM allows for control of the zinc available to cells due to the high concentrations of EDTA (1 mM) and citrate (20 mM) serving as a metal buffers in that medium. Wild-type cells grew markedly better in LZM+100 µM ZnCl2 [area under curve (AUC) value = 34.7] than did cells grown in LZM+1 µM ZnCl2 (AUC = 21.0)  . Thus, cTo perform genome-wide functional profiling, mixed populations of 4607 different viable deletion mutants were inoculated into zinc-limiting and zinc-replete media. The cells were grown for either five or fifteen generations, genomic DNA was isolated from the populations, and the unique “barcode” oligonucleotides that identify each mutant in the deletion collection were amplified by PCR. These PCR products were then labeled and hybridized to Affymetrix TAG4 microarrays containing oligonucleotides complementary to the barcode sequences see . Four reFET3, FTR1), copper  or manganese (e.g. SMF1) did not show a growth defect in low zinc relative to zinc-replete conditions confirming that the growth medium used in these experiments was specifically controlling zinc availability and the availability of other metals was not significantly affected. As shown in icy2Δ, tsa1Δ) or, alternatively, initially slow growing strains may have accelerated growth in later generations.Mutations in many genes were found to affect low zinc growth. After five generations, 110 mutants showed a reproducible growth defect in low zinc and were designated “low zinc sensitive”. Another 163 mutants were apparently resistant to low zinc and showed greater prevalence in the low zinc cultures than they did in the zinc-replete medium . After fgal2Δ mutant. GAL2 encodes galactose permease and is not predicted to be required for growth in the medium used here where glucose is the carbon source. No growth defect was observed  was transformed with an integrating plasmid vector that expressed GFP from a strong promoter observed , Table 1es 0.6%, , Table 1tsa1Δ, uth1Δ, scj1Δ, erd1Δ) showed decreased competitive fitness relative to wild type in zinc-replete media but that growth defect was exacerbated by low zinc conditions. Independent confirmation of the low zinc growth defects of these strains indicated that the functional profiling results were very reliable.Using this method, we confirmed the results for fifteen of the mutants that showed poor low zinc growth in the functional profiling analysis . SeveralWe analyzed the low zinc sensitive set of 433 genes identified in the five and fifteen generation experiments  for enriS. cerevisiae functional interaction data set. Fitness scores for each mutant, i.e. the difference in the mean of the log2 hybridization signal between LZM+1 µM ZnCl2 and LZM+100 µM ZnCl2, were then used to identify a sub-network within the BioGRID network enriched for genes that have sensitive fitness data associated with them, and so may identify cellular processes or protein complexes affected by zinc limitation. Mutants sensitive to low zinc were then assessed for significant overrepresentation of GO categories, which can be visualized in many different ways  required for full Yap1 activity were also identified as growing poorly in low zinc We had also previously shown that zinc-limited yeast cells produce more reactive oxygen species than do replete cells MSC2, which encodes a zinc transporter protein that, along with Zrg17 (see above), transports zinc into the ER. HAC1 and IRE1, which encode the transcription factor and its controlling sensor that are active in the ER unfolded protein response (UPR), molecular co-chaperones , and several genes involved in ER protein modification and trafficking were identified as growing poorly in low zinc. These results suggested that disruption of ER function in many different ways impairs the ability of cells to grow in low zinc. To test this hypothesis further, we examined the effects of tunicamycin, an inducer of ER stress 2) or zinc-replete conditions (LZM+100 µM ZnCl2) with a range of tunicamycin concentrations. As shown in Many genes related to endoplasmic reticulum function were also identified . These iSurprisingly, thirteen mutant strains disrupted for peroxisome biogenesis genes were defective for low zinc growth . The perSIF2, SIN3, SET3, HOS2, SNT1, PHO23, SAP30, HOS4, HDA2, and HDA3) indicates that chromatin structure plays an important role in low zinc.The functional enrichment of genes encoding histone deacetylase subunits  were confirmed by flow cytometry to have a low zinc growth defect  had little or no effect on low zinc growth. We conclude from these genetic studies that macroautophagy is required for low zinc growth but the cargo-specific pathways of CVT, pexophagy, and mitophagy are not.Autophagy, the process whereby cells degrade cytosolic proteins and organelles to recycle their components h defect . Mutatioatg3Δ, atg8Δ, atg15Δ, and atg16Δ) showed little or no growth defect in low iron or low copper. In fact, these four mutants showed improved growth in low iron. As a positive control for a low iron growth defect, a fet3Δ mutant grew poorly on BPS but not on BCS. FET3 encodes a component of the high affinity iron uptake system. As a positive control for a defect in low copper, an irc21Δ mutant was used and found to grow poorly on BCS but not BPS. IRC21 encodes a putative cytochrome oxidoreductase and the low copper growth defect of the irc21Δ was observed in our previous studies . When tested in minimal media, the greater importance of autophagy to low zinc growth relative to iron- or copper-limiting conditions was confirmed . This result indicated that Ice2 is required for optimal growth under severe zinc-limiting conditions but not under more moderate conditions. Zinc-deficient wild-type cells have induced expression of the ER-stress induced unfolded protein response (UPR) and this induction is exacerbated in mutants disrupted for MSC2 or ZRG17ice2Δ disrupting ER zinc homeostasis, we found that a UPRE-lacZ reporter was also hyper-induced in a zinc-limited ice2Δ mutant relative to wild-type cells and this hyper-induction was suppressed by supplementing zinc at 10 µM or above . Loss of Ice2 function in this quadruple mutant background caused still greater induction of the UPR when zinc was supplied at a concentration of 3 µM but the UPR was still suppressible with higher concentrations of zinc . Thus it does not appear that Ice2 transports zinc directly but may transport another substrate that is required for optimal ER zinc trafficking.In control experiments, we confirmed that zinc supplements do not inhibit UPR induction in response to other ER stressors such as tunicamycin . Thereforsm27Δ, rpl2bΔ, rps7aΔ, and rpl6bΔ mutants grown in zinc-replete conditions grew very poorly relative to wild-type cells. These mutants also grew less well than wild type in zinc-limiting conditions but were better able to compete when the wild-type strain is growth limited. Thus, a likely explanation for the functional profiling results is that in low zinc, growth of the wild-type strain is slowed by zinc deficiency to a rate closer to that of the mutant. In zinc-replete media, the wild-type strain grows at a much faster rate while the mutant growth is still impeded by the mutation and competes less well with wild type. Direct analysis of rpl2bΔ growth supported this hypothesis . Therefore, these mutants are likely to show apparent low zinc resistance by the same mechanism as was found for the ribosomal subunit mutants. We assayed growth of the remaining mutants with no known fitness defect and found that seven of these did indeed confer a growth advantage relative to wild-type cells in low zinc  were found to have growth defects in low zinc. Surprisingly, ∼90% of Zap1 target genes (72/80) were not found to have growth defects in low zinc. These mutants may have a defect too subtle to be detected even after fifteen generations but could have a fitness disadvantage relevant to natural selection. Alternatively, these genes may be important under conditions where low zinc is combined with some other stress such as high temperature ZRC1. Zrc1 is a vacuolar zinc transporter that moves zinc into the vacuole for storage and detoxification. Surprisingly, ZRC1 is a Zap1 target gene and its expression is induced in low zinc Previous transcriptome analysis identified many genes induced in low zinc including ∼80 that are likely direct targets of the Zap1 transcription factor. We predicted that Zap1-regulated genes would be important for zinc-limited growth and several such mutants  grew normally in low zinc. ADH4 encodes an alcohol dehydrogenase isozyme that is up-regulated in zinc-limited cells at the same time that expression of two other isozymes, Adh1 and Adh3, are repressed by Zap1 adh4Δ mutant may reflect the decreased overall ADH activity due to Zap1-mediated repression of ADH1 and ADH3. HNT1 encodes an adenosine phosphoramidase and may serve as a possible positive regulator of Kin28 HNT1 by Zap1 may enhance or help maintain transcription of genes in zinc-limited cells. Finally, ICY2 is a gene of unknown function but its importance specifically to low zinc growth is clear from our results. icy2Δ mutant cells grew as well or even better than wild type cells in zinc-replete media, but were severely defective for growth in low zinc. This growth defect appears specific for zinc; icy2Δ mutants showed no growth defect in low iron or low copper media (data not shown).Almost half of the 15 genes with both altered expression and a detectable low zinc growth defect are Zap1 target genes. Among these are S. cerevisiae was suggested by the studies of Carman and colleagues In addition to identifying key Zap1 target genes, functional profiling highlighted a large number of other processes that are also important for low zinc growth. Of particular interest was the discovery of the need for autophagy in zinc-limited cells. Autophagy is a process whereby cells degrade cytosolic proteins and organelles to recycle their components Aspergillus fumigatusatg1 mutants, which are defective for all of these pathways of autophagy in S. cerevisiae, were defective for low zinc growth. By examining the effects of various mutants affecting different autophagy pathways, we found here that macroautophagy is required for low zinc growth of S. cerevisiae and that the cargo-specific pathways are less important.Autophagy can be subdivided into several different pathways including macroautophagy, CVT, mitophagy, and pexophagy. Richie et al. previously showed that some process of autophagy was required for zinc-limited growth of S. cerevisiae to high zinc indicating the need for autophagy at both ends of the range of zinc status What role does autophagy play in low zinc growth? An appealing hypothesis is that its purpose is to degrade zinc-containing proteins and organelles to release that zinc for other purposes. Alternatively, the stress of low zinc could damage proteins and organellar membranes and those damaged components may need to be turned over. ER stress induces autophagy yap1Δ and skn7Δ mutants is consistent with the increased oxidative stress found in zinc-limited cells and the importance of Yap1/Skn7-mediated transcription. Also consistent with this hypothesis, we found that hyr1Δ, ybp1Δ, and ybp2Δ mutants are also defective for low zinc growth. These mutations disrupt accessory factors required for Yap1 activity A large number of transcriptional regulators were identified as important in low zinc including Ace2, Crz1, Dal82, Gat1, Opi1, Rox1, and Ume6. These observations suggest that target genes of these factors are somehow needed under these conditions. Two other transcription factors that were found to be required for optimal growth in low zinc are Yap1 and Skn7. Both of these factors control the transcriptional response to oxidative stress CTA1 did not confer sensitivity to low zinc indicating that the importance of the peroxisome in low zinc is not in combating oxidative stress . A role of the peroxisome in fatty acid β–oxidation in zinc-limited cells would be surprising given that the cells were supplied with abundant glucose and not given exogenous fatty acids as carbon sources. It is conceivable that zinc-limited cells may have a defect in glucose utilization and catabolize lipid stores as a source of energy. This switch occurs in response to glucose deprivation FAT1 encoding a peroxisomal fatty acid transporter and POT1, encoding 3-ketoacyl-CoA thiolase caused some growth defect in low zinc , glycosylation , ER Ca homeostasis (SPF1), ER-to-Golgi trafficking , and other ER processes were also sensitive to low zinc. One property shared by most if not all of the ER-related mutants we identified is that they cause ER stress or reduce the ability of the cells to respond to that stress hac1Δ or ire1Δ mutants in either low iron or low copper minimal media that were zinc replete  . JSY5 has the genotype of MATα his3Δ leu2Δ trp1Δ ura3Δ msc2Δ::kanMX4 zrg17Δ::natMX4 zrc1Δ::HIS3 cot1Δ::URA3. Cells were grown in a rich medium  or low zinc medium (LZM) prepared as previously described 2. An iron-limiting medium was generated by adding the iron chelator bathophenanthroline disulfonate  and copper limitation was imposed by adding the copper chelator bathocuproine disulfonate  to YPD medium. Also, LZM was made iron-limiting by leaving out the iron supplement and adding 100 µM ZnCl2 to make it zinc replete. Iron was then supplemented to either 1 (low iron) or 100 (replete iron) µM FeCl3. LZM was modified to control copper availability by leaving out the copper supplement and replacing the EDTA with 200 µM BCS. Copper was then supplemented to either 0.1 (low copper) or 10 (replete copper) µM CuCl2. Preliminary growth experiments confirmed that these conditions were limiting or replete for the respective metal.Diploid yeast deletion strains used for functional profiling and for analysis of individual strains were of the BY4743 background  of 0.0165 in either LZM+1 µM ZnCl2 or LZM+100 µM ZnCl2, and dispensed in triplicate into different wells of a 48-well plate . Plates were incubated in a GENios microplate reader  set to 30°C with intermittent shaking. OD595 measurements were taken at 15-minute intervals for a period of 24 hours. Raw absorbance data were averaged for all replicates, background corrected, and plotted as a function of time. The area under the curve (AUC) as a measure of growth was calculated with Excel 2008 .To assess the effect of limiting zinc on growth, yeast strains were pre-grown to mid-log phase in LZM+100 µM ZnCl600 of 0.04 (6×105 cells per ml). The cultures were then incubated with aeration at 30°C and grown to a final OD600 of 1.2 (1.8×107 cells per ml). For the fifteen-generation experiments, the cells were inoculated at a starting OD600 of 0.005 (7.5×104 cells per ml) and grown to an OD600 of 1.2 (eight generations), reinoculated into fresh media at a starting OD600 of 0.01 (1.5×105 cells per ml) and grown to a final OD600 of 1.2 (seven generations). The initial inocula are equal to 0.75–6×107 total cells. Thus, the cultures were inoculated with ∼1,600–13,000 cells of each mutant genotype. Four independent replicate cultures were used for each condition of zinc and generation number. After the desired generation number was reached, the cells were chilled, centrifuged at 1000×g for 6 min at 4°C, and the pellets resuspended in 2 ml dH20. The resuspensions were divided equally among three microfuge tubes, centrifuged at 1000×g for 5 min, the supernatant was removed and the pellets frozen at −80°C.For the analysis of fitness over five generations, cells from frozen aliquots of the deletion collection pools were inoculated into 100 ml culture volumes in 250 ml metal-free polycarbonate Erlenmeyer flasks to a starting ODhttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37254).Extraction of genomic DNA from cultured cells, PCR amplification of barcodes, and Affymetrix TAG4 array hybridization were performed as previously described 2{Y|X = LZM1})-ave(log2{Y|X = LZM100}), the difference in the mean of the log2 hybridization signal between LZM+1 µM ZnCl2 and LZM+100 µM ZnCl2) was calculated for each deletion strain, to identify those differentially sensitive. This fitness score is an indicator of the effect of low zinc on the growth of the deletion strain. A negative fitness score indicates the deletion strain is sensitive to zinc limitation.Raw microarray data were processed and significant strains identified as previously described Significant genes (strains) were statistically inferred using an exact binomial test, assuming that the outcomes for each gene in all effective treatment-control pairs were independent binary variables with the same probability of success (p = 0.5) for all trials  http://funspec.med.utoronto.ca/) Data sets from DSSA were verified for enrichment of any particular biological attribute by identifying significantly overrepresented Gene Ontology (GO) categories S. cerevisiae BioGRID interaction dataset Fitness data for all significantly sensitive strains identified from this study were mapped onto the TEF2 promoter HIS3 gene that is already mutant in the parental strain. The GFP-tagged wild-type strain and untagged isogenic mutant strains were grown overnight to exponential phase (OD600 = 0.5) in YPD, mixed in approximately equal numbers, and then inoculated into LZM+1 or 100 µM ZnCl2. Cells were first inoculated into 5 ml cultures with a starting OD600 of 0.00375 and grown to an OD600 of 1.0. These cells were then inoculated into fresh cultures of the same media to an OD600 of 0.0075 and grown to a final OD600 of 1.0 for a total of fifteen generations. Approximately 20,000 total cells per culture were analyzed by flow cytometry using a FACSCalibur flow cytometer (BD Biosciences) and an air-cooled 488 nm argon ion laser. A 530/30 filter was used for measuring GFP fluorescence and a 585/42 filter was used to detect autofluorescence exhibited by both tagged and untagged cells. GFP-expressing wild type cells were readily distinguished from untagged mutant cells. In control experiments, wild-type cells that had lost GFP expression were found to be rare  and therefore contribute little variability to mutant cell counts. Means of three independent cultures are reported with standard deviations and statistically significant differences between the low zinc and zinc-replete cultures were determined using Student's t-test.The BY4743 wild-type strain was transformed with an integrating vector that expresses GFP from the strong Figure S1rpl2bΔ mutant grows as poorly in LZM+1 µM ZnCl2 (LZM1) as in LZM+100 µM ZnCl2 (LZM100). Because the wild-type strain grows less well in low zinc relative to high zinc, the proportion of the mutant in the low zinc culture is greater than in the high zinc culture so the mutant appears to be resistant to low zinc.Analysis of mutants with apparent resistance to low zinc growth. Mutations that disrupt the cytosolic (A) and mitochondrial (B) ribosomal subunits show apparent resistance to low zinc. C) An (PDF)Click here for additional data file.Table S1Yeast gene deletion mutants with increased sensitivity to low zinc. Deletion mutants identified in the functional profiling analysis as sensitive to low zinc are listed.(PDF)Click here for additional data file.Table S2Yeast gene deletion mutants with increased tolerance to low zinc. Deletion mutants identified in the functional profiling analysis as resistant to low zinc are listed.(PDF)Click here for additional data file.Table S3Yeast gene deletion mutants unaffected in low zinc. Deletion mutants tested in the functional profiling analysis but not identified as sensitive or resistant to low zinc are listed.(PDF)Click here for additional data file.Table S4Autophagy is not greatly required for low iron or low copper growth. Various autophagy mutants showing growth defects in low zinc were cultured in metal replete (YPD) and low iron (YPD+100 µM BPS) or low copper (YPD+200 µM BCS) and assayed for growth as described for (PDF)Click here for additional data file.Table S52 to make it zinc replete. Iron was then supplemented to either 1 (low iron) or 100 (replete iron) µM FeCl3. LZM was modified to control copper availability by leaving out the copper supplement and replacing the EDTA with 200 µM BCS. Copper was then supplemented to either 0.1 (low copper) or 10 (replete copper) µM CuCl2.Effects of mutations in autophagy and ER stress response on growth of iron- or copper-limited cells that are zinc replete. Various autophagy mutants and mutants disrupting ER function were cultured in metal replete, low iron, and low copper conditions and assayed for growth as described for (PDF)Click here for additional data file.Table S6Enrichment among low zinc-resistant mutants for molecular function, biological processes, and other categories.(PDF)Click here for additional data file.Table S7Zinc-regulated genes whose mutants were sensitive to low zinc. Deletion mutants identified in the functional profiling analysis as sensitive to low zinc and also known from other studies to be induced in low zinc are listed.(PDF)Click here for additional data file.


N = 2,417) from the European Prospective Investigation of Cancer (EPIC)-Norfolk study, a region in FTO was found to have stronger association (rs7204609∼rs9939881 at the first intron P = 4.29×10−7) than single SNP analysis  and a latent quantitative phenotype was obtained using a subset sample of EPIC-Norfolk . We believe our method is appropriate for assessment of regional association and polygenic effect on a single or multiple traits.Genetic association studies are now routinely used to identify single nucleotide polymorphisms (SNPs) linked with human diseases or traits through single SNP-single trait tests. Here we introduced partial least squares path modeling (PLSPM) for association between single or multiple SNPs and a latent trait that can involve single or multiple correlated measurement(s). Furthermore, the framework naturally provides estimators of polygenic effect by appropriately weighting trait-attributing alleles. We conducted computer simulations to assess the performance via multiple SNPs and human obesity-related traits as measured by body mass index (BMI), waist and hip circumferences. Our results showed that the associate statistics had type I error rates close to nominal level and were powerful for a range of effect and sample sizes. When applied to 12 candidate regions in data ( Current genetic association studies in humans, including genome-wide association studies (GWASs) We set to exploit association between multiple SNPs and multiple traits through a latent variable partial least squares path modeling (PLSPM) N = 2,566) was a random sample of the study cohort at baseline and cases were part of the remaining individuals with BMI≥30 kg/m2 . A total of 2,417 individuals in the subcohort and 1,135 cases with 446,861 SNPs passed quality control and in silico genotypes were obtained according to HapMap (http://www.hapmap.org) NEGR1 (rs3101336), SEC16B (rs10913469), TMEM18 (rs6548238), ETV15 (rs7647305), GNPDA2 (rs10938397), BDNF (rs925646), MTCH2 (rs10838738), SH2B1 (rs7498665), FAIM2 (rs7132908), FTO (rs1121980), MC4R (rs17782313), and KCTD15 (rs369784).Participants in the EPIC-Norfolk study were men and women aged between 45 and 74 from Norwich and the surrounding towns and rural areas http://en.wikipedia.org/wiki/Body_shape.The influence of body fat distribution has been linked with body shape named crudely after the fruits and vegetable(s) they resemble most As hinted earlier, our framework resembles structural equation modeling (SEM) with three types of parameters defined: (1) Latent variable scores  SNP set .CEU data at the brain-derived neurotrophic factor (BDNF) region (Chr 11∶27633610..27692970 with 24 SNPs) were used to generate the simulated genotypic data; (2) Based on (1), a large sample of 500,000 individuals was obtained via software gs 2.0 th SNP being the causal variant; (3) Quantitative genetic data was generated according to a trivariate normal distribution . Assume that the causal SNP had no effect on body shape , and quantitative genetics models with the given δ were created by the R mvtnorm package. The window size had 10 SNPs from the 3th to the 12th SNP. Under H0, 10,000 simulations given various sample sizes were conducted to assess the type I error. Under H1, for each model and a given δ, 10,000 simulations were conducted under various sample sizes to assess power. The procedures were implemented with Linux and the R plspm package. Both mvtnorm and plspm packages are available from CRAN. (http://cran.r-project.org/)Simulations were conducted as follows: (1) HapMap phase II α-level was defined as Scan statistics were built through the subcohort for association between the 12 regions and body shape, and to contrast with a SNP-wise single trait test performed by linear regressions (δ), or nominal level (α) (2c–2f). Even with a very small α, for effect size greater than 0.15 and the sample size of up to 4,500, the scan statistics remained to have >80% power .As shown in The model provided the usual association results for single trait adjusted for sex and age including effect size estimate, proportion of variance explained and statistical significance. −4 level according to single-SNP –single-trait tests nor according to sliding window sizes of 1–4 SNPs at the 10−5 level, but smaller p values were obtained for window sizes of 5–11 and 12–15 SNPs. Of particular interest was rs7204609∼rs9939881 at the first intron of FTO with P = 4.29×10−7 for a sliding window of size 10; its model structure is shown in As shown in SNP⋅21β) (95%CI) per allele on body shape was 0.08 . The proportion of variance explained was 0.8% by PRS. All genetic variants showed associations with body shape, though some loadings of the SNPs were not significant at α = 0.05 (FTO) and rs925646 (BDNF) for all the four traits, followed by rs6538238 (TMEM18), rs17782313 (MC4R) for BMI and hip; rs17782313 (MC4R), rs7132908 (FAIM2) for waist and body shape. Non-standardized effect sizes were largest with rs1121980 (FTO) (0.39), but smallest with rs7647305 (ETV5) (0.05)  of 2.28 (7.91×10−24).Shown in χ2 = 1556.8, P<.0001), especially in types 4, 5, 6, 9, 2, 3; (2) for both men and women, along with the risk of obesity, body shape score was seen to be monotonically increasing from types 1 to 9 . Linear regression between the scores and types had good fit for men  and women   , suggest = 0.74) ; (4) The = 0.74)  in both A latent variable PLSPM framework is outlined for association of multiple SNPs with multiple traits, the behavior of such an association was investigated by simulation study through type I error rate and power. Meanwhile, a polygenic statistic was developed for quantification of a polygenic effect by appropriately weighting trait-attributing alleles. These methods were applied to the study of obesity-related variables in the EPIC-Norfolk study for which a latent score was obtained. Below we compare these with available methods, discuss implications of our findings as with other issues involved and indicate some further work.Compared to SEM, PLSPM is robust to multicollinearity commonly encountered in GWAS data (such as strong linkage disequilibrium between SNPs and high correlation between traits). It is a “soft modelling” approach requiring very few distributional assumptions, variables can be numerical, ordinal or nominal, and no need for normality assumptions, while covariance-based SEM is a “hard modeling” with heavy distributional assumptions FTO gene is of interest, as with some of the reported obesity-susceptibility SNPs near or in the 12 genes Analysis of the EPIC-Norfolk discovery sample involving 12 gene regions suggested that the scan statistics are more powerful than single SNP – single trait tests with the size 10 providing the strongest evidence. In particular, the region (rs7204609∼rs9939811) within the first intron 1 of A reviewer has indicated previous work on multiple linked quantitative trait loci (QTLs) A reviewer has questioned the adequacy of body shape as with PLS with a view that body share should be supported by various other measurements such as limb lengths, shoulder widths, etc. However, our interest lies more in utilizing the anthropometric traits from a population study for investigation of health risks. Indeed our results showed that BSS is approximately normal  and servhttp://www.broadinstitute.org/collaboration/giant/index.php/Main_Page) teleconference, a colleague instantly questioned the feasibility across the whole consortium. Secondly, the scan statistics seemed slightly anticonservative and a parametric counterpart is preferable. Thirdly, it will be desirable to catch both linear and nonlinear effects between genome region and latent quantitative trait.There will be several lines of further research. Firstly, there is an important need to examine the precise nature of regional or polygenic effect on a single trait or a collection of traits, as it may involve both polygenic and pleiotropic effects. This is also the case with GWAS. Long before this work when we reported work using SEM to differentiate pleiotropic effect on obesity-related traits in a GIANT consortium  as with distribution of their PRS and cumulative effects of these variants .(TIF)Click here for additional data file.Figure S2The distribution of body shape score (BSS).(TIF)Click here for additional data file.Table S1Nine types of human body shape defined by BMI combination with WHR.(DOC)Click here for additional data file.Table S2Loadings, P values, indirect and overall effects of 12 SNPs and PRS on BMI, waist and hip with adjustment for sex and age.(DOC)Click here for additional data file.Information S1Some theoretical results.(DOC)Click here for additional data file.Information S2Single trait results from the EPIC-Norfolk replication sample.(DOC)Click here for additional data file.