Influenza genes evolve mostly via point mutations, and so knowing the effect of every amino-acid mutation provides information about evolutionary paths available to the virus. We and others have combined high-throughput mutagenesis with deep sequencing to estimate the effects of large numbers of mutations to influenza genes. However, these measurements have suffered from substantial experimental noise due to a variety of technical problems, the most prominent of which is bottlenecking during the generation of mutant viruses from plasmids. Here we describe advances that ameliorate these problems, enabling us to measure with greatly improved accuracy and reproducibility the effects of all amino-acid mutations to an H1 influenza hemagglutinin on viral replication in cell culture. The largest improvements come from using a helper virus to reduce bottlenecks when generating viruses from plasmids. Our measurements confirm at much higher resolution the results of previous studies suggesting that antigenic sites on the globular head of hemagglutinin are highly tolerant of mutations. We also show that other regions of hemagglutinin—including the stalk epitopes targeted by broadly neutralizing antibodies—have a much lower inherent capacity to tolerate point mutations. The ability to accurately measure the effects of all influenza mutations should enhance efforts to understand and predict viral evolution. Seasonal influenza is a recurrent threat to human health, largely because it rapidly accumulates amino-acid mutations in proteins targeted by the immune system . MeasuriThis experimental noise arises primarily from bottlenecking of mutant diversity during the generation of viruses from plasmids. The influenza genome consists of eight negative-sense RNA segments. During viral infection, gene expression from these segments is a highly regulated process ,11,12. GSeveral strategies have been used to overcome problems associated with bottlenecks during the generation of influenza from plasmids. One strategy is to generate and titer each viral variant individually, and then mix them ,17. A seTo overcome these limitations, we have developed a novel approach that uses a “helper virus” to generate virus libraries without strong bottlenecking. We have combined this approach with other technical improvements to perform deep mutational scanning of all amino-acid mutations to an H1 hemagglutinin (HA) with much higher accuracy and reproducibility than existing deep mutational scans of influenza genes. We use phylogenetic analyses to show that our measurements accurately reflect constraints on HA evolution in nature. We confirm that antigenic sites in the globular head of HA are highly tolerant of mutations, and identify other regions of the protein that are more constrained. These advances improve our understanding of HA’s inherent evolutionary capacity and can help inform evolutionary modeling and guide the development of vaccines targeting sites with a limited capacity for mutational escape.We reasoned that the process of generating viral libraries carrying HA mutants would be more efficient if transfected cells only needed to produce HA from plasmid, and the other gene segments and proteins were delivered by viral infection A. The PaGGC→CAT) as well as single-nucleotide  codon mutations. There are We cloned triplicate plasmid libraries of random codon mutants of the A/WSN/1933 HA gene. These libraries contain multi-nucleotide  with the goal of generating pre-formed viral ribonucleoprotein complexes carrying the HA segment. These transfected cells were then infected with the HA-deficient helper virus, and 24 h later, we determined the titer of fully competent virus in the supernatant. The highest titers  of 0.0075 TCIDet al. [et al. [et al. [To quantify selection on HA, we needed our deep sequencing to be sufficiently accurate to determine the frequency of each mutation pre- and post-selection. Standard Illumina sequencing has an error rate that is too high. In our previous deep mutational scanning of influenza ,5,6, we et al.  and firs [et al. . Sequenc . The results are quantified in terms of the preference of each site for each amino-acid; the set of all 20 preferences at a site can be thought of as representing the expected post-selection frequency of each amino acid at that site if all amino acids are initially present at equal frequencies.We next evaluated the reproducibility of our measurements of the effects of each amino-acid mutation. We estimated the effect of each mutation from its change in frequency in the mutant viruses relative to the original plasmid libraries, correcting for the site-specific error rates determined by sequencing unmutated virus and plasmid, and performing the analyses using the algorithms described in  and implphydms [http://jbloomlab.github.io/phydms/), which in turn uses Bio++ [We next tested whether our new measurements better describe the evolution of HA in nature. The accuracy with which experimental measurements of site-specific amino-acid preferences reflect the constraints shaping a protein’s evolution in nature can be quantified by comparing the phylogenetic fit of experimentally informed substitution models . We assephydms  , the experimentally measured preference for tyrosine is at odds with nature’s strong preference for serine . The labWe computed the inherent mutational tolerance of each site using the stringency-scaled amino-acid preferences from the combined datasets A. The muet al. [The H1 HA antigenic sites defined by Caton et al.  are signet al.  , and a vestigial esterase domain . We compsertions . TherefoWe have described new techniques that greatly improve the reproducibility of deep mutational scanning of influenza. The largest improvement appears to result from using a helper virus to generate virus mutant libraries without the bottlenecks that plague the creation of viruses purely from plasmids. We have used these techniques to more accurately measure the effects of all amino-acid mutations to HA. Our measurements confirm at greater precision and resolution the finding ,9 that HIn this study, we measured the effects of all mutations to the HA from a lab-adapted H1N1 strain. To what extent can these measurements be extrapolated to other HAs? Due to epistasis, the effects of mutations sometimes change as proteins evolve ,44. HoweOverall, our work demonstrates a method for making accurate large-scale measurements of the effects of mutations to influenza proteins. Our results offer insight into how protein-intrinsic mutational tolerance shapes influenza evolution, and provide a basis for using deep mutational scanning to improve quantitative models of viral evolution and understand virus-immune interactions.MDCK-SIAT1 cells  were engineered to constitutively express the HA protein of A/WSN/1933 (H1N1) under control of the EF1a promoter by lentiviral transduction. These newly created cells will be referred to as MDCK-SIAT1-EF1a-WSN-HA cells since they are MDCK-SIAT1 cells that we have engineered to express the WSN HA under an EF1a promoter. HA surface expression was validated by flow cytometry .not contain non-coding regions of the HA segment or a promoter for the transcription of negative-sense viral RNA). Transfection was performed with BioT transfection reagent  with each well receiving 250 ng of each plasmid. Twenty-two hours after transfection, we changed the media to WSN growth media   plus a pexpected . We collT1 cells .Codon mutagenesis was performed as described in  except tTo generate mutant virus libraries, we transfected 293T cells with a DNA mixture containing one of the three pHH21-MutantHA libraries (or the wild-type pHH21-WSN-HA control) and protein expression plasmids for the four proteins that compose the ribonucleoprotein complex, using plasmids HDM-Nan95-PA, HDM-Nan95-PB1, HDM-Nan95-PB2, and HDM-Aichi68-NP . SpecifiWe passaged these transfection supernatants to create a genotype-phenotype link and impose functional selection on HA. We passaged over β-mercaptoethanol, pipetting 30 times, transferring to an RNase-free microcentrifugefuge tube, adding 600 For each of the three replicate HA virus libraries and the wild-type HA virus, we extracted viral RNA by ultracentrifuging 24 mL of supernatant at 22,000 rpm in a Beckman Coulter SW28 rotor. RNA was extracted using the Qiagen RNeasy kit by resuspending the viral pellet in 400 We generated PCR amplicons of HA for each of the eight samples  using KOD Hot Start Master Mix  with the PCR reaction mixture and cycling conditions described in  and the These PCR amplicons were quantified using Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies) and used as the templates for the barcoded-subamplicon sequencing in We then mixed all six subamplicons from each experimental sample at equal concentrations and diluted these subamplicon pools such that the number of template molecules used in PCR 2 was less than the anticipated sequencing depth to ensure multiple reads per barcode. Specifically, we reduced the total amount of DNA for each experimental sample used as template in PCR 2 to We performed PCR 2 for each sample with the following reaction conditions: 20 uL 2X KOD Hot Start Master Mix, 4 dms_tools (http://jbloomlab.github.io/dms_tools/), version 1.1.12, to align subamplicon reads to a reference HA sequence, group barcodes to build consensus sequences, quantify mutation counts at every site in the gene for each experimental sample, and infer site-specific amino-acid preferences based on mutation frequencies pre- and post-selection using the algorithm described in [We used ribed in . The codphydms version 1.1.0 [http://jbloomlab.github.io/phydms/), which in turn uses Bio++ [We sub-sampled human and swine H1 sequences (1 sequence per host per year) from the set of sequences from , removedon 1.1.0   was performed with the same classifications as described in . AdditioSequencing data are available from the Sequence Read Archive under accession numbers SRR3113656 (mutant DNA library 1), SRR3113657 (mutant DNA library 2), SRR3113658 (mutant DNA library 3), SRR3113660 (mutant virus library 1), SRR3113661 (mutant virus library 2), SRR3113662 (mutant virus library 3), SRR3113655 (wild-type DNA control), and SRR3113659 (wild-type virus control). An iPython notebook (and a static HTML version of it) for all analyses is in


Light fractionation significantly increases the efficacy of 5-aminolevulinic acid (ALA) based photodynamic therapy (PDT) using the nano-emulsion based gel formulation BF-200. PDT using BF-200 ALA has recently been clinically approved and is under investigation in several phase III trials for the treatment of actinic keratosis. This study is the first to compare BF-200 ALA with ALA in preclinical models.In hairless mouse skin there is no difference in the temporal and spatial distribution of protoporphyrin IX determined by superficial imaging and fluorescence microscopy in frozen sections. In the skin-fold chamber model, BF-200 ALA leads to more PpIX fluorescence at depth in the skin compared to ALA suggesting an enhanced penetration of BF-200 ALA. Light fractionated PDT after BF-200 ALA application results in significantly more visual skin damage following PDT compared to a single illumination. Both ALA formulations show the same visual skin damage, rate of photobleaching and change in vascular volume immediately after PDT. Fluorescence immunohistochemical imaging shows loss of VE-cadherin in the vasculature at day 1 post PDT which is greater after BF-200 ALA compared to ALA and more profound after light fractionation compared to a single illumination.The present study illustrates the clinical potential of light fractionated PDT using BF-200 ALA for enhancing PDT efficacy in (pre-) malignant skin conditions such as basal cell carcinoma and vulval intraepithelial neoplasia and its application in other lesion such as cervical intraepithelial neoplasia and oral squamous cell carcinoma where current approaches have limited efficacy. Photodynamic therapy (PDT) using topically applied 5-aminolevulinic acid (ALA) or other protoporphyrin IX (PpIX) precursors is used as a treatment modality for various (pre-) malignant skin lesions –2. It isAlthough ALA-PDT is effective, it is predominantly applied for thin lesions and long-term responses can be improved. Different strategies have been investigated to improve the results such as ALA penetration enhancers, the use of ALA ester derivatives, certain physical methods such as tape stripping and iontophoresis and the development of carriers such as liposomes or nanoparticles –11. A alAn approach we have been investigating is the used of light fractionation with a single long dark interval. We have shown that light fractionation significantly increases the effectiveness of ALA-PDT both in preclinical and clinical studies –19. The et al. showed that ALA penetrates more easily into the circulation whereas MAL remains at the site of application [The mechanism responsible for the increased effectiveness of light fractionated ALA-PDT is not simply the result of the utilization of re-accumulation of PpIX during the dark interval between the two light fractions . Furtherlication . We havelication . Based oBF-200 ALA, a recently approved nanoemulsion-based gel formulation containing 7.8% ALA (10% ALA hydrochloride), is under investigation in several phase III trials for the treatment of actinic keratosis (AK) –29. In pThe current study is the first to compare BF-200 ALA with ALA in preclinical models. We investigated the fluorescence kinetics of BF-200 ALA in normal hairless mice and compared that with ALA and MAL. We also investigated the fluorescence distribution both in-vivo and ex-vivo in pig and mouse skin. We investigated the PDT induced damage after BF-200 ALA and ALA using light fractionation and compared the visual skin damage and the vascular responses of mouse skin. If light fractionation can be shown to enhance the efficacy of PDT using BF-200 ALA there is the potential to adopt this approach in the clinic.The animal ethics committee of the Erasmus MC approved the experimental protocols of the study. We investigated the pharmacokinetics of PpIX distribution using  fluorescence imaging and assessed PDT efficacy after the administration of different porphyrin precursors in a number of cutaneous murine and porcine models. An overview of these protocols and the number of animals in individual groups is shown in Four different animal models were used: the normal skin of female outbred hairless mice  aged between 8 and 10 weeks, the mouse skin-fold window chamber, ex-vivo porcine skin and ex-vivo murine skin –32. The 4 (Sigma-Aldrich) in distilled water.Ex-vivo porcine and murine skin was prepared as described previously . Ex-vivoBF-200 ALA (marketed as Ameluz) a 10% aminolevulinic acid HCl nano-formulation,  and MAL  were supplied by the manufacturer ready-for-use. ALA was used in two formulations. To compare the pharmacokinetics of PpIX distribution after ALA and BF-200 ALA application  with 514 nm used in previous studies were translated to 532 nm based on the in-vivo absorption spectrum of PpIX in mouse skin and the photon energy [2 at a irradiance of 89 mW/cm2. PDT was performed in a single illumination delivered at 4 hrs or according to a light fractionation scheme where the illumination is performed 4 and 6 hours after the administration of porphyrin precursor. In the light fractionated scheme the light dose of the first fraction was based on the delivered PDT-dose i.e. the light dose that led to 48% PpIX photobleaching, which is considered to be the optimal light dose for the first fraction [PDT efficacy was determined in SKH1-hr mice by visually scoring the response of skin to PDT group 5, . PDT andn energy . This refraction . The PDTfraction .The vascular response to PDT was investigated intra-vitally using the skin-fold chamber model and ex-vivo in skin samples harvested 24 hours after PDT.™ Compound (Sakura Holland B.V.) and 5 and 50 μm cross-sections were cut with a cryostat, mounted on glass slides  and air-dried. The 50 μm sections were used for fluorescence labelled IHC staining of CD31 and CD144 on the same day and the staining procedure was performed as described previously [The PDT induced vascular damage was histologically investigated in SKH1-hr mice by collecting the skin in the illuminated area and of the contralateral side at day 1 after PDT group 6, . The PDTeviously .In a last group of SKH1-hr mice the vascular response to ALA-PDT and BF-200 PDT was investigated at depth in skin using the chamber model and intra-vital confocal microscopy group 7, . Confoca2 at 50 mw/cm2) used in previous experiments with 514 nm were translated to 630 nm based on the in-vivo absorption spectrum of PpIX in mouse skin and the photon energy as described above. This resulted in a light dose of 130.6 Jcm-2 at 65.3 mWcm-2 either delivered in a single illumination or according to a light fractionation scheme. In this skin-fold window experiment photobleaching during PDT could not be monitored and the illumination of the first light fraction was standardized to be 6.53 Jcm-2, which is consistent with 5 Jcm-2 at 514 nm. The transmission images were also used to determine the vascular area pre and post PDT. The area of a vessel was measured by drawing regions of interest in the LSM aim software. While the animal is repositioned between measurements care was taken to measure the same length of vessel for each series of images pre and post PDT.PDT was performed using a 630 nm laser  and a microlens . The total light dose and irradiance  and distribution  and PDT induced damage  was determined using Student’s t test, ANOVA or SNK test and considered significant when The PpIX fluorescence increase was similar up to 4 hours after topical application of ALA, MAL and BF-200 ALA . Betweenr after ALA application was not significantly different from BF-200 ALA. The correlation after MAL and vehicle showed both statistical significant differences with ALA and BF-200 and with each other .Representative examples of confocal PpIX fluorescence images obtained are displayed in the left column of r after ALA application was not significantly different from BF-200 ALA . After photobleaching during the first illumination, PpIX fluorescence was accumulated again to the starting value . The photobleaching curves are shown in The surface fluorescence intensity at the start of treatment was not significantly different between the single and light fractionation group treated with ALA or BF-200 ALA or between the ALA and BF-200 ALA groups . The mean light dose delivered in the first light fraction that lead to 48% photobleaching was 2.80±1.12 J/cmThe change in vascular area during PDT determined in the intra-vital microscopy images collected low in dermis of the skin-fold chamber model is shown in The area of the venules did not change significantly as determined with the paired t-test . There wThe CD31 fluorescence content in the upper dermis showed no difference between ALA and BF-200 ALA independent of the illumination scheme used. This is the first study to compare ALA and BF-200 ALA. We have previously shown that light fractionation can significantly increase the PDT response in both preclinical and in clinical studies –19. BF-2The concentrations of ALA used in the present study are different and were chosen to correspond to those used in the clinic; ALA-hydrochloride at 20% and BF-200 ALA contains an ALA concentration comparable to 10% hydrochloride. Several researchers have shown that different percentages of ALA or different volumes of a standard percentage of ALA does not lead to different fluorescence kinetics in normal mouse or human skin –36.et al despite the use of the same ex-vivo model. They reported PpIX fluorescence only in the upper part of the epidermis after 3 hours and even 8 hours of topical application of MAL and BF-200 ALA, where BF-200 ALA lead to more fluorescence at depth than MAL [All three porphyrin precursors  showed PpIX fluorescence extending over the whole epidermis of mouse skin and ex-vivo pig skin after 4 hours of application as we have shown before for MAL and ALA in mouse skin . Our resthan MAL . This mathan MAL ,37.Using the mouse skin-fold chamber model and intra-vital confocal microscopy we observed differences in the fluorescence intensities at depth in the dermis. We detected significantly more fluorescence after BF-200 ALA application suggesting a deeper penetration compared to ALA. This can be explained by the stabilising effect of the nanoemulsion in BF-200 ALA which is a soybean lecithin-based emulsion gel –40.We note that we were unable to maintain viable mouse skin ex-vivo and that the topical application of ALA, MAL or BF-200 ALA did not lead to PpIX accumulation.We have demonstrated that light fractionated BF-200 ALA-PDT results in a significant increased efficacy just as we have previously shown for ALA . SpecifiIt is important to consider the wider applicability of the present results obtained in normal mouse skin. While tumours and their vasculature might show different results, the optimization of treatment schemes in this model has previously served as a good predictor of clinical response –19. The We have hypothesized that the mechanism behind the increased response to light fractionation is based on a cellular mechanism in addition to the treatment parameters, also the spatial distribution, in particular endothelial accumulation, is important . Cells aBoth ALA and BF-200 ALA but not MAL-PDT show significant increased response to light fractionation. The significant lower PpIX accumulation in endothelial cells after MAL application, shown in the present and previous studies, suggests the involvement of the vasculature . In the As mentioned previously, an optimized response to light fractionated PDT is dependent on the damage induced by the first light fraction, which is influenced by the choice of irradiance, light dose and the PpIX concentration . This meIn summary, we compared the temporal and spatial fluorescence distribution and PDT response to ALA-PDT with BF-200 ALA-PDT. Both formulations show similar fluorescence kinetics in normal mouse skin and similar PpIX fluorescence intensities within endothelial cells. Both formulations show significantly increased response to light fractionation compared to a single illumination as shown by the visual skin damage response in mouse skin. There are however small differences. BF-200 ALA showed more PpIX fluorescence at depth in the dermis of living mouse skin compared to ALA suggesting a deeper penetration. Also more loss of VE-cadherin was found one day after BF-200 ALA-PDT compared to ALA-PDT. The current results show the potential of light fractionated BF-200 ALA for clinical translation where ALA is currently used and in more difficult to treat conditions such as vulval or cervical intraepithelial neoplasia or lesions in the oral cavity.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S3 Text(DOCX)Click here for additional data file.S4 Text(DOCX)Click here for additional data file.


Macroporous gelatin microcarriers, CultiSpher-S, were applied to in vitro wounds and cultured for 21 days. Tissue sections showed incorporation of wound edge keratinocytes into the microcarriers and thicker neoepidermis in wounds treated with microcarriers. Thickness of the neoepidermis was measured digitally, using immunohistochemical staining of keratins as epithelial demarcation. Air-lifting of wounds enhanced stratification in control wounds as well as wounds with CultiSpher-S. Immunohistochemical staining revealed expression of keratin 5, keratin 10, and laminin 5 in the neoepidermal component. We conclude that the CultiSpher-S microcarriers can function as tissue guiding scaffold for re-epithelialization of cutaneous wounds.The possibility to use a suspended tridimensional matrix as scaffolding for re-epithelialization of  The use of scaffolds for dermal regeneration in combination with cultivated keratinocytes is well accepted ,2. SeverGelatin is a well-tolerated substance with low antigenicity  and has in vitro and in vivo studies have revealed that a variety of cell lines exhibit survival and proliferation when cultured on CultiSpher-S microcarriers [Several carriers . In the carriers .vitro model for wound healing. Tissue culture wound models are of value for investigating the wound healing process in human skin and a large number of identical wounds in vitro enables a more rapid discovery process and supports planning of subsequent in vivo studies [in vitro wounds inflicted in excised human skin and the wounds were cultured for 21 days. The effect of the CultiSpher-S microcarriers was evaluated by studying the ingrowth of wound edge keratinocytes into the microcarriers and the formation of a neoepidermis.Based on the previous reports supporting the use of CultiSpher-S in wound healing and work performed by the group on injectable microcarriers as dermal scaffolding we have investigated the use of these microcarriers on re-epithelialization without additional cell transplantation. There is a need to evaluate the efficacy of these microcarriers for use in guided tissue regeneration without cellular pre-seeding . The pre studies . MicrocaHuman skin samples were rescued from tissue discarded during routine abdomenoplasty and de-identified to comply with Swedish ethical regulations governing the use of human materials. Skin was processed within 12 hours. Wounds extending over the dermo-epidermal junction were prepared as previously described . In brieTo investigate the effect of gelatin in solution in the wounds, six wounds were cultured with addition of 2% gelatin . Gelatin solution was deposited directly in the wound area and wounds were left free of maintenance medium for 48 hours as described above, and then cultured submerged in medium for the remaining culture time.Additionally, experimental and control wounds were treated as mentioned above but cultured submerged in maintenance medium for the first 14 days of culture after which the wounds were air-lifted using cell culture inserts  for seven days to observe stratification of the neoepidermis.All wounds were collected after 21 days of culture and snap-frozen in liquid nitrogen. Specimens were mounted to sample holder with OCT  and sectioned in 10 μm and 50 μm sections with a Leica 3050CM cryostat .Sections were air dried for 30 minutes before washing in PBS and subsequently stained with routine Mayer’s hematoxylin and eosin (HistoLab). Sections were mounted for microscopy with Mountex mounting medium (HistoLab).For immunohistological staining procedures sections were air dried for 60 minutes and fixated with 4% paraformaldehyde or acetone. Sections were washed in PBS and blocked with bovine serum albumin (BSA) at 2.5% for 30 minutes. For staining of 10 μm sections incubation with primary antibody took place in a humidified chamber for 60 minutes, for 50 μm sections at 4° C overnight. Primary antibodies were used at the following dilutions; pancytokeratin AE1/AE3 1:200 , keratin 5 1:200 , keratin 10 1:400 , laminin 5 1:100  and Ki-67 1:200 . Secondary anti-mouse or anti-rabbit antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 546  were applied for 60 minutes in a humidified chamber. After washing and draining, sections were mounted with ProLong Gold mounting medium  containing 4, 6-diamidino-2-phenylindole (DAPI).Stained sections were visualized with an Olympus BX41 light/fluorescence microscope and images captured using an Olympus DP70 CCD camera . Thickness of the neoepidermis in experimental and control wounds was measured digitally. The wound bed area of interest was delimited by tracing the base of the wound. Monochrome thresholding was applied to pancytokeratin staining (Alexa Fluor 546 red) and an average height of neoepidermis across the wound base was calculated. Three tissue sections per wound were included in the measurements. Measurements were performed in Fiji  and resuin vitro wounds. Wounds were cultured in DMEM supplemented with 10% FCS for 21 days. Five experimental wounds out of twelve showed complete re-epithelialization and incorporation of microcarriers, and where microcarriers were not completely retained almost complete or partial ingrowth into the remaining carriers could be observed. The partially re-epithelialized experimental wounds were included in the thickness measurements.To investigate the effects of CultiSpher-S as a gelatin scaffold on the re-epithelialization of cutaneous wounds the microcarriers were applied to Hematoxylin-eosin staining of cryosectioned tissue showed microcarriers incorporated in the re-epithelialized wound bed . ControlWounds treated with microcarriers showed a significantly thicker neoepidermis (128.0 μm±17.4) on average compared to control wounds (47.6 μm±3.09) . SectionWounds cultured with 2% gelatin solution did not re-epithelialize fully over the culture time of 21 days but the neoepithelial tongue closest to the wound edge was observed to be thicker than in normal controls. Additionally, single cells and clusters of cells could be observed dispersed over the length of the wound bed .Immunohistological staining revealed expression of keratin 5 and keratin 10 in the neoepidermis of wounds with microcarriers  in compain vitro wounds a porous scaffold in the form of CultiSpher-S gelatin microcarriers. We utilized an in vitro wound healing model based on viable human skin in culture. The tissue culture model provides a tool for investigating healing of standardized wounds over a time span of several weeks with cells present in the biopsy in their native environment within the tissue biopsy. Avoiding animal studies is always desirable and using a model based on the target tissue enables investigations of the complex process of wound healing in a more contextual manner than by utilizing cell assays. Previous work by Seland et al. [in vitro wound healing model is the lack of a systemic response of the vascular and immune systems. However, the process of foremost interest for the study was re-epithelialization. This implies activation, proliferation and migration of wound edge keratinocytes and these responses to wounding are present in the model, visualized by re-epithelialization of standardized wounds within a delimited time. We measured re-epithelialization and observed epidermal regeneration. In our experimental setup the wounds are cultured for three weeks to allow re-epithelialization under normal conditions. Wounds with microcarriers presented re-epithelialization with visible microcarriers present in the wound bed. This indicates that the microcarriers are stable enough to provide scaffolding for the keratinocytes over a time-span required for successful wound healing.We investigated the effects of providing resident cells in cutaneous d et al.  reportinThe porous structure of the microcarriers enables cell migration into the carriers, creating an open scaffold for wound edge keratinocytes to cover during the re-epithelialization process. Wound edge keratinocytes populated the microcarriers and re-epithelialized the wound with a thicker epidermal component compared to control wounds. When staining with antibody against Ki-67, a proliferation marker, no increase in cells expressing the antigen could be found at the wound edges indicating that the main event leading to microcarrier incorporation is migration rather than proliferation. The epithelial origin of the populating cells was confirmed by immunohistochemical staining using a mixture of antibodies that detect both acidic and basic keratins (pancytokeratin) . Confocain vitro wounds with antibodies against keratin 5 and 10 was performed. Keratin expression by keratinocytes is compartmentalized and dependent on state of differentiation. Basal keratinocytes express keratins 5 and 14 [The incorporation of microcarriers resulted in a disorganized appearance of the neoepidermis. To establish the organization we investigated the hallmarks of a regenerated epidermis, which are the terminal differentiation of keratinocytes  and the 5 and 14 ,19. UponThe wound bed with incorporated CultiSpher-S microcarriers showed positive staining for the major basal membrane component laminin 5. The dermo-epidermal junction is a complex network of proteins, and laminins function as key components in the anchoring complexes that ensure communication between the two separate tissue compartments . A stablAir-lifting of wounds was undertaken to investigate the process of stratification in wounds with microcarriers. Retaining sheets of epidermal cells in the air-liquid interphase, combined with inducing agents, leads to stratification and cornification of the epidermal layer within seven to ten days . After aWe investigated whether a gelatin coating of the experimental wound bed would exert similar effects as the Cultispher-S gelatin microcarriers on re-epithelialization by adding 2% gelatin in solution to the wounds. The gelatin was left to settle in the wound area for 48 hours before the wound was submerged in culture medium. The results showed only partial re-epithelialization of all experimental wounds with a thick neoepidermal outgrowth. Keratinocytes suspended in semi-solid medium undergo terminal differentiation due to loss of contact to ECM ,24 whichDisruption of the skin barrier by external wounding leads to vast cellular and biochemical actions to restore barrier integrity. A main event of the proliferative phase of wound healing is the activation of keratinocytes at the wound edge, aimed to migrate and re-epithelialize the wounded area. In the case of extensive damage, or stagnation into a chronic wound, the possibility for successful migration of wound edge keratinocytes is limited due to an overall imbalance of the wound healing microenvironment attributed to ongoing inflammation, lack of substrate and inappropriate signaling cues . Chronicin vitro cutaneous wounds without additional cell transplantation. The in vitro model is based on viable human skin with cells present relevant for re-epithelialization giving strong indications for the in vivo situation. The microcarriers facilitated the formation of a thick epidermis while supporting maturation of the neoepidermis. Previous work shows the microcarriers to be suitable as a guiding scaffold for dermal regeneration and together with our findings we conclude that the CultiSpher-S microcarriers can be beneficial as provisional matrix for cutaneous wound healing. A microcarrier suspension is easy to store and handle, administration is direct and the microcarriers in suspension can fill in wounds of various shapes and depths which infers that the CultiSpher-S microcarriers show an advantage over static sheet shaped scaffolding.The present study shows that Cultispher-S microcarriers can function as tissue guiding scaffold for keratinocytes for re-epithelialization of S1 DatatableDescription of the measurement procedure in Fiji and the obtained measurement values for wound sections analyzed in the study.(PDF)Click here for additional data file.S1 FigHematoxylin-eosin staining of wound cultured with microcarriers for 21 days. The organization of collagen fibers is longitudinal adjacent to the microcarriers and no adverse effects can be observed. Asterisk (*) indicates blood vessels in the dermal wound bed. Scale bar = 200 μm.(TIF)Click here for additional data file.S2 Fig(A and B) Hematoxylin-eosin staining of section of wound cultured with 2% gelatin. Note single cells and cell clusters indicated by asterisk (*) in the wound bed in magnification (A). (C) Immunohistochemical staining against pancytokeratin (red) and nuclear staining with 4, 6-diamidino-2-phenylindole (DAPI). Arrow indicates neoepidermis, arrowheads indicate wound edges. Scale bars = 200 μm for (A), 1000 μm for (B) and (C).(TIF)Click here for additional data file.S1 Imageset(ZIP)Click here for additional data file.


Altered brain anatomy in specific gray-matter regions has been shown in patients with posttraumatic stress disorder (PTSD). Recently, white-matter tracts have become a focus of research in PTSD. The corpus callosum (CC) is the principal white-matter fiber bundle, crucial in relaying sensory, motor and cognitive information between hemispheres. Alterations in CC fibers have been reported in PTSD and might be assumed to underlie substantial behavioral and cognitive sequelae; however most diffusion tensor imaging (DTI) studies in adult-onset PTSD failed to address the clinical correlates between imaging and PTSD symptoms severity, behavioral manifestation and cognitive functions. In the current study we examined (a) to what extent microstructural integrity of the CC is associated with memory performance and (b) whether imaging and cognitive parameters are associated with PTSD symptom severity. DTI data were obtained and fractional anisotropy (FA) values were computed for 16 patients and 14 controls. PTSD symptom severity was assessed by employing the clinician administered PTSD scale (CAPS) and memory was tested using a task probing item and associative memory for words and pictures. Significant correlations were found between PTSD symptoms severity, memory accuracy and reaction-time to CC FA values in the PTSD group. This study demonstrates meaningful clinical and cognitive correlates of microstructural connectivity. These results have implications for diagnostic tools and future studies aimed at identifying individuals at risk for PTSD. Post-traumatic stress disorder (PTSD) is a debilitating condition that can develop in response to an acute traumatic event and is characterized by re-experiencing, avoidance, negative alterations in cognition and mood and hyper-arousal . NeuroimThe majority of studies testing the exact structural and functional topography of white-matter in PTSD used brain-magnetic resonance imaging (bMRI) that allowed volumetric/area measurements. Several studies in PTSD have shown a reduction in the volume and/or area of the corpus callosum (CC), with or without generalized white-matter atrophy –11. WhilThe corpus callosum (CC) is the principal white-matter fiber bundle connecting neocortical areas and plays an integral role in relaying sensory, motor and cognitive information . The CC To date, DTI studies in adult PTSD patients were limited to childhood trauma  or to chIn the current study we aimed to examine (a) to what extent predefined imaging (FA of the CC) and cognitive measurements (memory accuracy and response time) are associated in PTSD, and (b) whether imaging and cognitive parameters are associated with PTSD symptom severity, in a group of adult-onset PTSD patients without TBI. Specifically and based on previous literature, we hypothesized that high symptom severity is associated with lower CC micro-structural integrity as well as with impaired memory performance.Participants in the current study were 16 PTSD patients  recruited from the trauma-center at Soroka University Medical Center (demographic and characteristics of PTSD subjects are presented in All PTSD patients had experienced a traumatic event 6–36 months preceding the study. PTSD was diagnosed during a structured psychiatric interview using DSM-IV criteria performed by a board-certified psychiatrist (current co-morbid disorders were exclusionary). PTSD symptoms severity was assessed using the Clinician-Administered PTSD Scale  which iTo assess for anxiety levels in both groups we used the Spielberger's State-Trait Anxiety Inventory (STAI); a self-report questionnaire that measures state and trait anxiety . The totTo probe for memory performance, we employed a previously used item-association memory paradigm see , 37–38) –38 37–38Participants performed the items recognition task, followed by the associative recognition task. In the items task, participants viewed 12 stimuli, one at a time. Of these, 6 were targets , and 6 were distracters , mixed randomly. Participants were informed that the list included targets and distracters, and were instructed to respond to each stimulus with a designated “yes” key (“1”) for targets and a “no” response key (“0”) for distracters. In the associative recognition task, participants viewed 12 stimuli-pairs; of those 6 were intact pairs from the learning phase  and the other 6 pairs were distracters . Participants were informed that the list included intact and recombined pairs, and were again instructed to respond using the same keys. Stimuli that were used in the items test were not used in the associative test and vice-versa. Response-time  was recorded.To assess performance, for each experimental block  we computed two outcome measures: for items performance and for associative performance, both calculated by the difference between the proportions of hits (responding "yes" to a target that had appeared in learning list of that specific block) minus the proportion of false-alarms (responding "yes" to a distracter that had not appeared in learning list of that specific block). This resultant in two items and two association performance measurements for each task (words and pictures). For analysis purposes, we averaged each measure (items and associative performance) across each task's blocks (words and pictures), thus remained with one items and one associative averaged performance measurements, for the words and pictures tasks separately. Subsequently, we created an associative deficit index (ADI) reflecting the difference between the averaged item recognition and the averaged associative recognition performance separately for each task . The ADI was calculated as the averaged items recognition proportion minus the averaged associative recognition proportion; thus, higher ADI score reflect greater associative deficit.Structural MRI scans were acquired on a 1.5-Tesla scanner , using a 6-channel SENSE head-coil. The pulse sequence used was a TIw-FFE (spoiled gradient echo). A T1-weighted whole brain anatomical scan was collected for each subject using the following parameters: repetition-time = 15ms, echo-time = 4.6ms, flip-angle = 30°, matrix-size 256x256, field of view 25.6 cm, 150 sagittal slices (1×1×1 mm resolution).2. A TR/TE = 5711/95 ms was used without cardiac gating for a total acquisition time of 3:08 min for each dataset.DTI data were acquired on the same scanner using a single-shot echo-planar imaging (EPI) sequence with SENSE parallel imaging (reduction factor = 2.5). Axial slices of 3.0 mm thickness were acquired parallel to the anterior-posterior commissure line (AC-PC). The in-plane acquisition resolution was 2.88 × 3.58 mm. 42 slices were acquired with zero gap to cover the entire hemisphere and brainstem. The DTI data were acquired along 16 directions with b = 1000 s/mmb-value = 1000) by applying affine linear registration [All fiber tracking was performed using DtiStudio . At the first stage, we performed automatic image registration, i.e. registered the diffusion-weighted images to the b0-image  and state-trait anxiety parameters were compared, and found significantly different with higher scores for the PTSD patients group  separately. Correlations are reported for the patients group only since no significant correlations were found in controls. To ensure that our results in the PTSD group are not influenced by participant's age or education we computed the correlation between these variables and cognitive and imaging parameters. Since the age of patients was found highly correlated to RT in the associative pictorial memory task  and the FA values of the anterior  and the posterior  CC portions, correlations reported below involving these variables were controlled for age.To answer the question whether imaging and cognitive parameters are associated with PTSD symptom severity we computed the correlation between the total CAPS score and FA of the CC as well as ADI and RT in the PTSD group. As stated before, correlations involving RT in the associative pictorial memory task and the FA values of the anterior CC portions were controlled for age. A significant linear correlation between CAPS symptoms severity  and the total CC FA value was found . To test whether this effect is specific to distinctive CC portions, we computed the correlation between the CAPS total score and each CC portion, and found a consistent pattern for most portions . To test whether this effect is specific to distinctive PTSD symptom clusters  we computed the correlation between the CAPS sub-clusters and CC FA values using semi-part correlations  to white-matter microstructural integrity (FA). The significant correlation between CC FA values and PTSD severity, as measured by the CAPS questionnaire, support the hypothesis that alterations in CC white-matter architecture are associated with core PTSD pathophysiology, rather than a specific symptom cluster. In addition, the findings of the current study significantly expand the results reported in our previous work  showing According to the scheme proposed by Witelson  for CC sOverall, it seems that while the volume of the CC is important for accurate associative recognition of words as well as pictures , the aniNotwithstanding the origin of CC alterations in PTSD is unclear: The question whether anatomical connectivity architecture abnormalities in PTSD patients are a preexisting predisposition due to developmental/genetic factors, or are a consequence of the traumatic exposure itself, remains open. The preexisting predisposition hypothesis relates to premorbid vulnerability, i.e., abnormal white-matter integrity as a risk-factor predisposing individuals to PTSD and predicting its severity. Although the cross-sectional nature of this study prohibits confirmation of this possibility, it is supported by earlier studies of psychiatrically healthy youths with variable family histories of depression and major depressive disorder  that demSome general limitations of DTI analysis must be acknowledged. Lateral projections of the CC are difficult to reveal and quantify due to mixture of other fiber bundles with different orientations, especially association fiber bundles that are located in a region lateral to the callosal fibers. This limitation of tractography raises a concern about the validity of the DTI-based fiber reconstruction and resultant parcellation maps. Partial volume effects due to the use of voxel sizes larger than the typical diameter of a specific fiber bundle (or tract) result in apparently reduced diffusion anisotropy and correspondingly lower FA values by averaging white-matter tracts with more isotropic tissues such as gray matter or even CSF. Moreover, there are limitations of the simple diffusion tensor model if fibers merge, branch, or cross. Although the aforementioned challenges emerge as a general concern for fiber tractography, they did not arise as a major obstacle for studying the topology of fiber connections in the human CC. Another limitation to be considered is the small sample size in the current study; Based on the correlation analysis and the sub-group comparisons we concluded that the difference in FA values between extremely-severe and mild-severe PTSD patients is categorical in its nature, yet it is possible that a larger sample size would have revealed a continuous difference rather than a categorical one. Future studies with larger sample size are warranted to further explore the nature of the difference between these groups.In summary, the current study reveals novel findings regarding the functional relationship between microstructural integrity of specific CC portions and altered cognitive, and specifically memory, performance in PTSD. The correlations observed in the current study have consequences for diagnostic tools and future studies aimed at identifying individuals at risk for PTSD, and in turn, develop individual-based treatments that assist in preventing the chronicity of this disorder.S1 File(ZIP)Click here for additional data file.


Guided by preclinical data we ultimately hope to discern whether a particular pattern of transduced T cell migration within patients reflects early development of graft vs. host disease. Current difficulties in terms of choice of suicide gene, biodistribution of radiolabeled tracers in humans vs. animal models, and threshold levels of genetically modified cells needed for detection by PET/CT are discussed. As alternative suicide genes are developed, additional radiolabel probes suitable for imaging in patients should be considered.Clinical trials increasingly incorporate suicide genes either as direct lytic agents for tumors or as safety switches in therapies based on genetically modified cells. Suicide genes can also be used as non-invasive reporters to monitor the biological consequences of administering genetically modified cells to patients and gather information relevant to patient safety. These genes can monitor therapeutic outcomes addressable by early clinical intervention. As an example, our recent clinical trial used  Suicide genes convert innocuous prodrugs into cytotoxic products. Injecting viruses carrying suicide genes directly into solid tumors destroys the virus-infected cells and nearby neighbors through diffusion of cytotoxic products. Inserting suicide genes into cells that migrate to tumors  can accomplish the same task.Leukemia patients are often administered allogeneic T cells to attack their cancerous cells. Unfortunately T cell replete transplants with the most robust anti-tumor activity tend to have the greatest potential to cause graft vs. host disease (GvHD), a frequent and sometimes fatal side effect of allogeneic stem cell transplantation. GvHD manifests itself early after transplantation and after the overt elimination of leukemia. T cells genetically modified to express the herpes simplex virus thymidine kinase (TK) suicide gene can be killed by administering the prodrug ganciclovir (GCV) to induce apoptosis at the first sign of GvHD . Althougex vivo to act as in-patient factories to produce biological molecules, such as insulin, to alleviate the need for repeated injections (Safety switches (suicide genes) are of particular value in therapies dependent upon long-lived and/or proliferating cells. Genetically modified cells carry an inherent and potentially life-long hazard of cancerous transformations. Stem cells administered to regenerate tissues damaged by disease or treatment, correct congenital malformations, or rejuvenate aging tissues may have unknown risks . Likewisjections .ex vivo manipulations of transduced cells affect their therapeutic potential and biodistribution? The limited penetration of visible light through human and large animal bodies makes answering these questions by tagging the cells with bioluminescent reporter probes (luciferase) untenable. Also, there are no Food and Drug Administration (FDA) approved reagents for generation and measurement of bioluminescent imaging in humans. In these situations higher energy reporters are needed, such as those suitable for imaging via positron emission tomography (PET) scans of patients.Monitoring the location of genetically modified cells in patients could have important ramifications in terms of either safety or the general biology of these therapeutic interventions. Do the cells reach their target or migrate to an unexpected location, cause damage to a normal tissue, or contribute to tumor progression? If a cancer relapses, do modified immune cells reactivate, proliferate, and target the new tumor or ignore it? Is an organ regenerated primarily from genetically modified stem cells recruited to enhance tissue repair? What is the relative amount of modified cells at various sites? Do All enzyme-based suicide genes should amplify tracer signals, but to be a successful reporter the product of the reaction must also be retained in the cells. Conversion of 5-fluorocytosine by cytosine deaminase to a molecule that diffuses out of the cell, 5-fluorouracil, makes it a poor reporter. In contrast TK phosphorylates cellular substrates  into membrane impermeable phosphorylated forms and entraps them within cells. This makes TK a better choice for imaging studies.124I-labeled 2′-fluoro-2′-deoxy-1β-D-arabino-furanosyl-5-iodo-uracil butyl]guanine (18F-FHBG) as a substrate for TK . To achieve both greater sensitivity to GCV and more accumulation of the phosphorylated 18F-FHBG for imaging, we used, TK75, a hyperactive mutated version of the enzyme , is the only additional clinical study of which we are aware that uses e enzyme . TK75 wa18F-FHBG-PET/CT imaging. The remaining six individuals were scanned at three time points. Because in a DLI setting acute GvHD is usually not evident before 30 days post administration of T cells, we chose to do one baseline scan before the DLI, and additional scans on ∼days +14 and +30 after the infusion of the CD34-TK75 genetically modified DLI.Our preclinical bioluminescence studies suggested that allogeneic T cells migrate in a distinctive pattern when they cause GvHD in mice . Finding18F-FHBG in this or any other patient at baseline and later time points . We used a portion of the same CD34-TK75 transduced donor T cells and 18F-FHBG prepared for the patients under GMP conditions in a parallel transplant of 2 NOD-SCIDg-/-(NSG) mice for each patient. As expected the xenogeneic CD34-TK75 transduced T cells induced GvHD in NSG mice, and the 18F-FHBG-microPET/CT scans revealed transduced T cells in their thymic region . We previously demonstrated by BLI that this site is one of the preferred locations for migration and expansion of human T cells during xenogeneic GvHD (18F-FHBG.Only one of our six imaged patients developed GvHD (day 64) potentially attributable to the genetically modified T cells. Unfortunately no discernable difference was observed between the biodistribution of eic GvHD . Our curex vivo manipulations prior to infusion.Data from other trials suggested that the therapeutic potential of T cells is affected by the means in which T cells are activated pre-transduction and the method of expansion thereafter review . Our cel18F-FHBG accumulation was negligible throughout the body in all human DLI recipients. In contrast, it was difficult or impossible to track CD34-TK75 expressing human T cells trafficking to the abdomen of mice due to the high background accumulation of 18F-FHBG in the abdomen. As discussed earlier, a threshold level of radiotracer may be required before it can be detected by imaging. Thus it is likely that due to the very limited number of transduced T cells available for infusion (0.1–1.3 × 106 CD3/kg), we failed to reach that threshold in any patient. Alternatively a diffuse distribution of the transduced cells, an inappropriate choice of time points, or a combination of these factors may have limited our ability to detect an alteration in the pattern of uptake of 18F-FHBG in the tissues of human recipients after DLI. In collaboration with MolMed we have initiated a second trial of 18F-FHBG-PET/CT imaging  in which patients that have poor immune recovery after haploidentical transplant will be infused with TK transduced T cells from the same haploidentical donor between 40 and 60 days post transplant. In this trial ≥50 times as many TK transduced cells/kg will be administered.We noted that background A concern for long-term cell-based gene therapies is that genetically modified cells might be immunologically recognized and eliminated. Although patients in our trial exhibited neither a cellular nor serum immune response to CD34-TK75, an immune response against unmodified TK has been noted in other trials reviews . To addr18F-FHBG and 2′-deoxy-2′-[18F]fluoro-5-methyl-1-β-D-arabinofuranosyluracil (18F-FMAU), has been compared in healthy volunteers (18F-FMAU than 18F-FHBG accumulated in the liver, bladder, and myocardium while more 18F-FHBG than 18F-FMAU was concentrated in the gall bladder and gastrointestinal tract. This observation indicates that one TK substrate for PET imaging may be preferable over another depending upon the anticipated location of the genetically modified cells. This study also demonstrated that preclinical models may not accurately reflect the distribution of the substrates in humans since overall there was more background in mice when using 18FHBG than 18F-FMAU, particularly in the gastrointestinal tract, bladder, and spleen. In comparison, there was less overall accumulation of 18F-FHBG in patients than in mice, just as in the study we report here. The reverse was true for 18F-FMAU.The biodistribution of 2 radioactive substrates used with TK, lunteers . More 18There are currently a number of suicide genes under preclinical investigation. Developing additional highly retained radiolabeled probes for imaging that have high signal to noise ratios could be of significant value to patients and the general biological understanding in this field.Suicide genes such as TK should be considered an adjunct to any clinical gene therapy trial in order to exploit their dual safety and monitoring functions. Many factors govern which suicide gene system is optimal. Among these are the anticipated urgency to rid a patient of the cells, whether it is better to be able to leave non-proliferating genetically modified cells intact or to kill all transduced cells, the overall potency of a particular system, the importance of bystander-cell killing, and immunogenicity. The potential to gather safety or general biological information by directly imaging the genetically modified cells in the patient should be added to these considerations.18F-FHBG, each of which required specialized facilities, highly trained individuals, and separate clinical protocols. Nonetheless, because preclinical models do not always reflect the biology of either healthy humans or patients, there is a marked benefit to including a tool to gather additional biologic information in a non-invasive manner. As transplant biology and regenerative stem cell therapy advance, there is an increasing need to monitor and understand the behavior of various exogenously added cells in patients.There are hurdles for performing any gene therapy trial. Adding a component that requires synthesis of a radioactive probe admittedly introduces additional complexities, but these are not insurmountable. FDA and institutional oversight was required for the cGMP quality manufacturing of both our genetically modified cells and All authors contributed to the conception, design, acquisition, analysis, or interpretation of the work. All authors also drafted the work or critically evaluated it for intellectual content, approved the final version, and agree to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The immediate-early response mediates cell fate in response to a variety of extracellular stimuli and is dysregulated in many cancers. However, the specificity of the response across stimuli and cell types, and the roles of non-coding RNAs are not well understood. Using a large collection of densely-sampled time series expression data we have examined the induction of the immediate-early response in unparalleled detail, across cell types and stimuli. We exploit cap analysis of gene expression (CAGE) time series datasets to directly measure promoter activities over time. Using a novel analysis method for time series data we identify transcripts with expression patterns that closely resemble the dynamics of known immediate-early genes (IEGs) and this enables a comprehensive comparative study of these genes and their chromatin state. Surprisingly, these data suggest that the earliest transcriptional responses often involve promoters generating non-coding RNAs, many of which are produced in advance of canonical protein-coding IEGs. IEGs are known to be capable of induction without de novo protein synthesis. Consistent with this, we find that the response of both protein-coding and non-coding RNA IEGs can be explained by their transcriptionally poised, permissive chromatin state prior to stimulation. We also explore the function of non-coding RNAs in the attenuation of the immediate early response in a small RNA sequencing dataset matched to the CAGE data: We identify a novel set of microRNAs responsible for the attenuation of the IEG response in an estrogen receptor positive cancer cell line. Our computational statistical method is well suited to meta-analyses as there is no requirement for transcripts to pass thresholds for significant differential expression between time points, and it is agnostic to the number of time points per dataset. Cells respond to stimuli through a set of genes that are primed for rapid activation. These genes, known as immediate-early genes (IEGs), are regulated at the level of transcription of the messenger RNA, and at subsequent RNA processing levels. These rapid responders are then rapidly switched off in normal cells. Immediate-early genes are involved in many cellular processes, including differentiation and proliferation, that are often dysregulated in cancer where they become continuously active. We characterise IEGs in a genome-wide sequencing dataset that captures their transcriptional response over time. Using a novel analysis technique, we identify both protein-coding and non-coding genes that are activated comparably to IEGs and investigate their properties. We examine how IEGs are switched off, including through microRNAs, small non-coding RNAs that act to control the level of key IEGs. We identify a novel set of microRNAs responsible for the attenuation of the IEG response in an estrogen receptor positive cancer cell line. Immediate-early (or primary response) genes are induced in response to a stimulus without the requirement of de novo protein synthesis . The souκB and CREB generally found in IEGs [Many immediate-early genes (IEGs) encode transcription factors which regulate secondary response genes (SRGs) . Necessa in IEGs . Chromat in IEGs . The pre in IEGs , 6. Howe in IEGs .A more comprehensive understanding of the initiation of the immediate-early response, and its less well studied attenuation are required. Studies of IEG induction show distinct differences between the kinetics of pre-mRNA and mature mRNA which are particularly evident for delayed IEGs where the mature mRNA may peak up to 3 hours later than the precursor . A transIEG expression is tightly regulated at multiple levels, including control of transcription initiation and elongation as well as subsequent co-transcriptional and post-transcriptional events . Recent There has been intense interest in the roles of long non-coding RNAs (lncRNAs) in cellular differentiation. This class of transcripts is currently under-studied, but lncRNAs are differentially expressed during differentiation, are preferentially localised in chromatin and have been proposed to ‘fine-tune’ cell fate via their roles in transcriptional regulation –16. Genohttp://fantom.gsc.riken.jp/5/.The FANTOM5 project has recently produced the most comprehensive expression atlas for human and mouse cells, based upon cap analysis of gene expression (CAGE) data . In partβ , as well as human MCF7 breast cancer cells treated with EGF and HRG . Aortic smooth muscle cells are primary cells which are components of blood vessels. They are normally growth-quiescent in the normal adult vessels, but are activated by injury, or exposure to growth factors (including FGF2) and pro-inflammatory cytokines (including IL1b). These cues are sensed by these cells through changes in immediate-early gene expression, and can lead to increased proliferation and migration.Four kinetic signature functions were defined as illustrated in et al. (2015) [All transcripts for protein-coding genes were represented by conservatively thresholded FANTOM5 CAGE clusters . More t. (2015) , and ref. (2015)  describe1p), maximal change in expression (2p) and time of the change (st). The peak and dip signature functions require a rate constant δ which is not an explicit parameter of the model. Instead, the rate is calculated from the switch time st , and by 240 minutes-end time  of the experiment respectively.CAGE clusters assigned to approximately 200 known IEGs showed significantly elevated expression at the start of the time course, hence the importance of including the ures see . The ratThe fit between kinetic signatures and the time series CAGE data was assessed using the nested sampling algorithm to calculate the log of Bayesian evidence , log Z, using existing algorithms . The likThe percentage of CAGE time series that could be reliably annotated with a kinetic signature varied from 21%–39% across the cell types and protein coding or RNA biotypes. The assignment of clusters to signatures showed a slight preference for the early peak category . In contAssignments of time series to models allowed commonalities and differences between cell types and stimuli to be identified, even in the presence of varying numbers of time points sampled for each cell type. Eleven genes exhibited the early peak response in all four data sets, including eight known IEGs , indicating that early peak genes have a major impact on the regulation of transcription and splicing, consistent with previous studies of IEGs [Gene ontology terms associated with early peak clusters that were significantly over-represented in HRG treated MCF7 cells are listed in  of IEGs . When geκB) signalling pathways also provide a route for signals to pass from the extracellular environment to the nucleus [κB pathways.Pathways in the Panther database  that wer nucleus , 27, 28.ts in the range 1-100 min were more prevalent for early peak clusters in comparison with clusters annotated with the dip signature  [ts values are binned in 30 min intervals . This pattern was less apparent in the MCF7-HRG cell line where the proportion of known IEGs found in an interval exceeded the overall average towards the end of the time course. Further, in AoSMC-FGF2, AoSMC-IL1b and HRG treated MCF7 cells there was a peak in IEGs around 3 hours after stimulus (180-210min) suggesting genes currently described as IEGs may also have a role later in the immediate-early response than would be expected. Surprisingly, the proportion of nucleotide binding genes was maximal in the 60-90 min interval for the AoSMC-IL1b and MCF7-EGF data, and in all cases many nucleotide binding genes were activated concurrently with IEGs in contrast with previous reports [It has been proposed that nucleotide binding proteins are among the feedback regulators responsible for the attenuation of the immediate-early response to EGF . This se. (2007) , we conslus 180-2min sugge reports .Thus, cellular responses to FGF2, EGF, IL1b and HRG may be distinguished by the variable timing of factors (whether they are known IEGs or nucleotide binding proteins) that constitute and repress each response.Further exploration of model parameters yielded other insights. The timing of IEG induction and that of known transcription factors (TFs) is contrasted in Targets of the MAPK cascade in Immediate early genes are typically shorter in length than the genome-wide average . The meats could be shown for early peak genes by Pearson correlation . Fig ts for known IEGs assigned to the early peak signature with the densities of early peak nucleotide binding genes. Known IEGs were typically shorter in length and had lower ts than nucleotide binding genes . Surprisingly, ts. Thus the typically short length of IEGs will decrease the time required for their transcription, but IEGs are not necessarily induced with equally rapid kinetics. The time at which short IEGs reach their transcriptional peak was up to three hours after the stimulus suggesting their activation rates coordinate their expression with diverse processes and pathways: late-acting IEGs are not delayed due to gene length. Further, many early peak genes not known to be IEGs fell within the range of characteristics of known IEGs: length from 1.2Kb-240Kb, ts less than 210 min.However, a weak positive correlation between gene length and ts for the completion of transcription given the estimate for the 5’ kinetics and the length of the gene. For example, the contour plotted at 120 min curves leftwards to identify those longer genes which were transcribed with lower ts whose complete transcription would peak at 120 min . The density for known IEGs was predominantly within the 120 min contour. In contrast, the density for nucleotide binding proteins was beyond this contour. Comparing the counts of early peak IEGs, nucleotide binding genes and TFs within the 90 min contour with those outside this contour shows 28% of early peak IEGs, 17% of nucleotide binding genes and 22% of TFs lie within this contour in comparison with the 18% average for early peak genes. IEGs were significantly enriched within the 90 min contour (p = 5.1e-5 by hypergeometric test). Both early peak IEGs and TFs were enriched within the 120 min contour (p = 1.5e-4 and p = 0.04 respectively by hypergeometric test). While currently identified IEGs have distinct characteristics, our analysis also identifies a number of TFs induced on a similarly rapid timescale that may be part of the initial phase of the immediate-early response, or its attenuation.The contour lines plotted in Fig et al.[The assignment of genes to kinetic signatures could also be used to explore their association with promoter-proximal pausing through calculation of the travelling ratio. The travelling ratios for the union of all genes with CAGE clusters annotated as early peak in the two MCF7 data sets and for the set of known IEGs are presented in et al., we founCAGE clusters for RNA genes were assigned to kinetic signatures using the procedure described above. A lower threshold was used for the initial data selection: A minimum sum of 3 TPM normalised by relative log expression (RLE) over the time course was used as a threshold to increase the number of time courses from the more conservative 10 TPM criteria used for protein coding genes. GAL genes for rapid activation [ts for lncRNA, snoRNA, snRNA and miRNA assigned to the early peak category are shown in ts of less than 30 min.The role of ncRNA in the immediate-early response is not well understood. A small number of mature miRNA that respond to EGF signalling have been identified , 35, andtivation . Clusterts and length is shown in The association between ncRNA Studies of known IEGs have suggested that they are transcribed from loci with constitutively permissive chromatin structure . To deteConsidering the accessibility of CAGE clusters for non-coding genes, we found no significant difference between early peak clusters and the remaining set of non-coding clusters. However, early peak lncRNA had significantly greater DNaseI counts than the remaining non-coding clusters . Genome-wide analysis of enhancer activity was then performed. Multiple enhancer expression values arising from the many-to-many assignment of distal enhancers to genes , 39 wereThese results indicate that the promoters of IEGs and early peak genes are in accessible chromatin (but not more so than average in the case of IEGs), and are poised for transcription as shown by the high travelling ratios, but prior to stimulus they are not driven by enhancer expression. It is likely that this state is determined by specific transcription factors for IEGs and early peak genes.It has been previously demonstrated that in response to EGF stimulation a set of 23 mature miRNA show a rapid reduction in expression that upregulates a large number of target mRNAs in non-tumorigenic MCF-10A breast epithelial cells . These mObserving that the transcription of the host gene for hsa-mir-155 (an ID-miR) showed clear peaks in the time courses of AoSMC-FGF2, AoSMC-IL1b and MCF7-HRG cells , and thaUtilising a small RNA sequencing dataset for MCF7 cells stimulated with HRG , we applied the kinetic signature assignment procedure once again to identify regulated species. Of the 716 mirbase miRNA that passed a minimum expression criterion , 11.6% were assigned to a kinetic signature  a somewhat lower proportion than for CAGE data reflecting a greater variation between replicates. http://www.targetscan.org) we found all targets for dip miRNAs. For 12 of the 15 dip miRNA present in TargetScan we observed a greater representation of miRNA targets in early peak genes than in a reference set of unregulated genes . The targets of seven of these miRNA were significantly overrepresented (by hypergeometric test): hsa-mir-139 (p = 2.6e-2); hsa-mir-224 (p = 1.1e-2); hsa-mir-522 (p = 1.5e-6); hsa-mir-548n (p = 9.8e-7), hsa-mir-676 (p = 3.2e-2), hsa-mir-3163 (p = 2.1e-7) and hsa-mir-3662 (p = 3.5e-3) and are candidate ID-miRs for MCF7 cells. The data for mature hsa-mir-3163 and two of its targets FOSB and EGR3 are shown in Reasoning that miRNA-mediated repression will be reflected in the CAGE signals, either by direct action on mRNA or indirectly through transcriptional inactivation processes, we sought to establish a connection between the targets of mature miRNA that are assigned to the dip signature, and protein-coding genes with CAGE clusters assigned to the early peak signature in MCF7 cells stimulated with HRG. Making use of the TargetScan database of miRNA targets  satisfied the expression criterion in the small RNA sequencing data. CAGE and miRNA expression data for these transcripts are presented together in It has long been known that miRNA host genes may be protein coding or lncRNAs . As noteWe have identified seven immediately and transiently downregulated mature miRNA that preferentially target early peak genes. While these regulatory miRNA differ from those previously associated with the immediate-early response, the regulatory role appears to be the same. The fine-tuning of signalling pathways by miRNA has been previously described , 42–44 aWe have defined kinetic signatures, including a signature representing the classical IEG response, as the basis for a novel approach to time series data analysis. We have shown that large numbers of transcripts can be categorised according to the kinetic signature their expression profiles fit best, if any, and the categories can then be explored using standard enrichment statistics. These methods have successfully identified known IEGs as well as many other transcripts displaying the known characteristics of IEGs. A Bayesian approach utilising the nested sampling algorithm , 45 is uregulation of transcription from RNA polymerase II promoter and the TGF-beta signalling pathway. Nucleotide binding genes have been proposed as components of the negative feedback architecture of the immediate-early response. We found these genes to be activated concurrently with IEGs in many cases, but, considering the time for completion of transcription where gene length is accounted for, the translation of these genes would peak later.Genes assigned to the early peak kinetic signature were over-represented in gene annotations and pathways that are biologically relevant to the immediate-early response, including In common with known immediate-early genes, early peak genes showed promoter-proximal pausing in MCF7 cells, an RNAPII regulatory pathway previously demonstrated for selected IEGs in macrophages [Intriguingly, certain lncRNAs had properties analogous to known IEGs. Such similarities and differences between the epigenetic regulation of lncRNA and mRNA have been reported previously in genome-wide data . Many ofMature miRNAs previously found to be immediately downregulated in response to EGF  were fouet al[We identify hsa-mir-139 as an immediately downregulated miRNA: miR-139 is a known regulator of several canonical pathways in the metastatic cascade in MCF7 cells , and is et al are respet al.LncRNA are typically expressed at very low levels (NEAT1 and MALAT1 being notable exceptions), as are precursor miRNA, making their analysis problematic for methods that require more strictly thresholded expression data. The model definition and selection methodology we present is not limited by expression level, for example by tests for differential expression, nor do we rely upon the arbitrary thresholding that is common in clustering analyses. Models are specified in advance, and selection is based on the integration of model parameters rather than from a point estimate of best values, an approach which can be sensitive to the optimisation procedure used. Gene sets assigned to the best fitting model can be tested for over-representation of established gene and pathway annotations, and can be integrated with genome-wide data sets to test additional hypotheses.1p), maximal change in expression (2p) and time of the change (st). The decay signature is parameterised by the basal expression (1p), the maximal change in expression  and the half-life (ht). The linear model is parameterised by the expression at time 0 (1p) and the change in expression (2p) from which the rate of increase or decrease can be calculated. See Peak and dip signatures are piece-wise exponential functions parameterised by the basal expression .In the dip model, expression drops by 90% of the change in expression 1p+2p and drops exponentially at rate δ towards 1p. In this case δ is calculated from the half-life ht which is an explicit parameter of the decay model  and the value predicted by the kinetic model (μt). The product of the probabilities of the median observation at time t , log Z  from the samples . MaximisBayesian evidence values and model parameter estimates (and their standard deviations) are computed using nested sampling for all signatures for each time series. CAGE clusters are assigned to one of the exponential kinetic signatures if log Z for that signature is greater than 10 times log Z for the linear model and log Z minus its standard deviation (sd) is greater than log Z plus the estimated sd for any other exponential signature (nested sampling computes log Z for parameters mapped to 0..1 and we used the resulting log Z for the unit cube for model comparison). Clusters are assigned to the linear signature if log Z is 10 times greater than log Z for all exponential signatures. This decision making procedure is designed to minimise the incorrect assignment of exponential signatures to essentially linear data, and has been validated using synthetic data. The theoretical basis of nested sampling is summarised in S1 Text(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 FigtS (or th in the case of the decay signature) and one standard deviation above and below.CAGE TPM values are plotted as circles , predictions of the kinetic signature models using parameter means are shown in blue and the vertical green lines indicate the mean (PDF)Click here for additional data file.S4 Fig(A) early peak; (B) late peak; (C) dip; (D) decay.(PDF)Click here for additional data file.S5 FigtS and one standard deviation above and below.CAGE TPM values are plotted as circles , predictions of the kinetic signature models using parameter means are shown in blue and the vertical green lines indicate the mean (PDF)Click here for additional data file.S6 FigThis analysis was performed with GOrilla  and REVI(PDF)Click here for additional data file.S7 Figts for early peak, late peak, dip signatures and the half-life of the decay signature are shown in blue, the distributions of the subsets of known IEGs in each category are shown in red. The apparent bimodal distibution of ts for peak models is an artefact of the different choices of prior range. When a peak model is run without the early or late restriction the distribution of switch times is not bimodal, however, fewer time courses are assigned to the peak category.The distributions of (PDF)Click here for additional data file.S8 Figts (x axis) and are labelled with the gene name associated with the CAGE cluster.(A) The timing of known IEGs and transcription factors is shown for IEGs (red) and TFs (blue) assigned to the early peak signature in each AoSMC data set. (B) The timing of non-coding genes assigned to the early peak category is shown for lncRNA (red) and all other ncRNA (blue) in MCF7 data and (C) in AoSMC data. Symbols indicate the (PDF)Click here for additional data file.S9 Fig(A) early peak; (B) late peak; (C) dip; (D) decay.(PDF)Click here for additional data file.S10 FigtS and one standard deviation above and below.CAGE TPM values are plotted as circles , predictions of the kinetic signature models using parameter means are shown in blue and the vertical green lines indicate the mean (PDF)Click here for additional data file.S11 Fig(Top) Distributions of early peak DNaseI counts (blue) and non-early peak counts (black) for protein-coding CAGE clusters, and QQ plot. Early peak clusters have significantly higher counts. (Middle) Distributions of early peak DNaseI counts (blue) and non-early peak counts (black) for non-coding CAGE clusters, and QQ plot. There is no significant difference between the distributions. (Bottom) Distributions DNaseI counts for early peak lncRNA (blue) and all other counts (black) for non-coding CAGE clusters, and QQ plot. Early peak lncRNA clusters have significantly higher counts.(PDF)Click here for additional data file.S12 FigtS and one standard deviation above and below.Data is presented for the host lncRNA of hsa-mir-155 (MIR155HG ENSG00000234883) in AoSMC-FGF2, AoSMC-Il1b and MCF7-HRG data sets . CAGE TPM values are plotted as circles , predictions of the kinetic signature models using parameter means are shown in blue and the vertical green lines indicate the mean (PDF)Click here for additional data file.S13 Figth (or tS) and one standard deviation above and below.The data and kinetic signatures are presented for two mature miRNA assigned to the decay signature and for two miRNA assigned to the dip signature. Expression values are plotted as circles , predictions of the model using parameter means are shown in blue and the vertical green lines indicate the mean (PDF)Click here for additional data file.S14 FigEleven ID-miRs were present in the small RNA sequencing data with expression above the minimum threshold. Expression values are plotted as circles , and the dashed purple lines indicate the best-fitting kinetic signature. None of the ID-miR assignments passed the standard statistical criteria, hence these assignments are not significant (NS) and plotted for information only. It is apparent that hsa-mir-155 increases linearly, and that hsa-mir-191 peaks early in the time course. A number of ID-miRs show the expected immediate downregulation including hsa-mir-212 and hsa-mir-320a.(PDF)Click here for additional data file.S15 FigMedian CAGE expression (black circles) of precursor miRNA and median mature miRNA expression (red triangles). Lines are are a spline fitted to the data.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 File(GZ)Click here for additional data file.


Histomorphometrical analyses were made on cortical and trabecular bone of lumbar vertebral body as well as of mid-diaphysis and distal metaphysis of femur. In all rats fed calcium-deprived diet, despite the reduction of trabecular number , an intense activity of bone deposition occurs on the surface of the few remaining trabeculae . Different responses were detected in different sites of cortical bone, depending on their main function in answering mineral or skeletal homeostasis. This study represents the starting point for work-in-progress researches, with the aim of defining in detail timing and manners of evolution and recovery of biochemical osteoporosis.Rats fed calcium-deprived diet develop osteoporosis due to enhanced bone resorption, secondary to parathyroid overactivity resulting from nutritional hypocalcemia. Therefore, rats provide a good experimental animal model for studying bone modelling alterations during biochemical osteoporosis. Three-month-old Sprague-Dawley male rats were divided into 4 groups: (1) baseline, (2) normal diet for 4 weeks, (3) calcium-deprived diet for 4 weeks, and (4) calcium-deprived diet for 4 weeks and concomitant administration of PTH (1-34) 40  Osteoporosis is a systemic skeletal disease that represents a significant public health problem in an increasingly aging society. It is characterized by net bone loss and microarchitectural deterioration of bone tissue, due to an imbalance between the resorption and formation phases in the bone remodelling cycle (increased bone resorption and reduced bone formation), with a consequent increase in bone fragility and susceptibility to fracture –10. OsteIt is well known that rats fed calcium-deprived diet develop osteoporosis, as a result of exaggerated bone resorption, induced by parathyroid overactivity, secondary to nutritional hypocalcemia –25. TherThe aim of the present investigation was to study how the mineral and skeletal homeostasis influence the bone loss in metabolic osteoporosis due to calcium-deprived diet in different sites of the two bony architectures  in both axial and appendicular skeleton. This model seems to be a good starting point for successive studies on bone alterations during unbalanced calcium metabolism, frequently occurring in aging, with the final goal of defining timing and manners of bone mass recovery when calcium is restored in the diet and verifying the eventual differences of recovery between compact and spongy bones . n = 3): sacrificed after 7 days of acclimation;Group 1 : fed normal diet for 4 weeks;Group 2 : fed calcium-deprived diet and distilled water ad libitum for 4 weeks;Group 3 ( n = 5): fed calcium-deprived diet and distilled water ad libitum, plus concomitant administration of PTH (1-34) 40 μg/kg/day, for 4 weeks.Group 4 . All rats were housed individually in single cages, to better check food intake of each rat, and maintained under laboratory controlled conditions . After 7 days of acclimation to housing conditions, the rats were randomized into four groups, indicated as follows:μg/mL), and subcutaneously injected in a volume of 100 μL/100 gr body weight per rat.The calcium-deprived diet is a casein based synthetic diet containing a very low amount of calcium (0.04% Ca). PTH (1-34) was supplied by Eli Lilly and Company , solubilised in saline  15 mg/kg, two days before the sacrifice. Groups 2, 3, and 4 animals received a subcutaneous injection of calcein 15 mg/kg on the first day of the experimental period, a subcutaneous injection of oxytetracycline hydrochloride  30 mg/kg after 20 days, and a subcutaneous injection of Alizarin-Red S  30 mg/kg 2 days before sacrifice, in order to evaluate newly formed bone during animal treatment.The body weight of each animal was recorded at the time of arrival in the housing facility and before euthanasia. At the end of the treatment, all rats were anesthetized with ether and blood samples were collected by cardiac puncture; then, rats were euthanized by exsanguination under ether anesthesia.All experiments were carried out according to the Bioethical Committee of the Italian National Institute of Health. Animal care, maintenance, and surgery were conducted in accordance with Italian law (D.L. number 116/1992) and European legislation (EEC number 86/609).μm thick sections. The sections, taken from the central level of the lumbar vertebral body and from the femur mid-diaphysis and distal metaphysis , were glued to a glass slide and ground to a final thickness of about 40 μm. These sections were superficially stained with Alizarin-Red and scanned with Epson 3200 perfection scanner at 3200 dpi resolution to perform histomorphometry by means of the software Image J . The following static histomorphometric parameters were calculated: trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) of the vertebral bodies and the femoral distal metaphyses; the cortical bone thickness (Ct.Th) of the anterolateral and posterior sides of vertebral bodies; the total cross section area, the cortical bone area (Ct-B-Ar), and the medullary canal area of the femoral middiaphyses; the cortical bone area (Ct-B-Ar) of the femoral metaphyses.Soon after euthanasia, the fifth lumbar vertebra (L5) and the right femur of each animal were removed, deprived of soft tissues, fixed in sodium phosphate-buffered (PBS) 4% paraformaldehyde pH 7.4, dehydrated in graded ethanol, and embedded in methyl-methacrylate resin . The vertebrae and the femurs were transversally cut with a Leica SP 1600 diamond saw microtome cutting system  to obtain serial 200  dynamic histomorphometric parameters were evaluated by means of the image analysis system software Image J . The newly formed bone area (Nf B Ar) and the mineral apposition rate (MAR) were measured between the first two labels  on the anterior and posterior sides of the vertebral cortical bone as well as on the femoral mid-diaphysis. Moreover, the mineralizing surface (MS), marked with Alizarin, was measured on the femoral mid-diaphyses as well as on trabecular bone of vertebral bodies and femur metaphyses. MAR at the femur metaphyseal level was measured taking into consideration the second and third label . All measurements were performed according to the ASBMR histomorphometry nomenclature [The same sections were also further polished to remove the Alizarin-Red staining and used for evaluation of dynamic histomorphometry (by means of bone labelling technique), as a measure of newly formed bone during animal treatment, that is, to distinguish preexistent bone with respect to the bone formed during the period of administration of the calcium-deprived diet. The sections were photographed using a Nikon Eclipse 90i microscope  equipped with a DS-Fi1 Nikon digital camera and driven by the Nikon ACT-2U software;nclature .μm thick) adjacent to those used for histomorphometry was glued to a metacrylate support, cut to obtain 5 μm thick sections (Reichert-Jung 1150/Autocut), and stained with Gomori trichrome. The osteoid surface (OS/BS) was measured on trabecular bone.In order to evaluate the presence of osteoid seams in trabecular bone of both femur metaphyses and vertebral bodies, one section  at 1,500 g for 15 min. Sera were then aliquoted into small volumes and stored at −20°C for successive analyses. The levels of total calcium (Ca) and inorganic phosphorus (P) in serum were determined using the high performance Beckman Coulter analyzer AU680 Chemistry System. The immune-metric assays for the determination of levels of osteoprotegerin (OPG), specific bone alkaline phosphatase (BALP), CTX (Beta CrossLaps), and bioactive-intact-PTH (1-84) in rat serum were provided by Pantec s.r.l. ; all kits are intended for research use only. In particular, rat-OPG and rat-BALP are two ELISA kits produced by SunRed Hotecnology Company ; RatLaps is an EIA kit produced by Immunodiagnostic Systems Ltd. ; rat bioactive-intact-PTH is an ELISA method produced by Immunotopics Inc. . The small amounts of reagents supplied in the kits prevented the possibility to perform automated procedures on laboratory analytical platform. To minimize the variables influencing the test, all good laboratory practice principles were applied: immediate storage of samples after serum separation at −20°C; manual execution of tests in close agreement to the manufacturer's instructions; execution of all tests over two consecutive days in order to avoid repeated freeze/thaw cycles of samples.P < 0.05 indicate significant differences between groups.One-way analysis of variance (ANOVA) with Bonferroni test between treatment groups and controls was performed using the Software STATA 11.0 . Values of The observations reported in the present paper refer to morphological and histomorphometric evaluations performed on transverse sections of the 5th lumbar vertebra and right femur at middiaphyseal and distal metaphyseal levels of all rats.Concerning the vertebral sections, in rats fed calcium-deprived diet, as expected, bone trabeculae are less abundant and cortical bone appears less thick with respect to rats fed normal diet .The results of cortical bone thickness (Ct.Th) in anterolateral as well as in posterior sides of the vertebral body are shown in The results of dynamic histomorphometry show that both in the anterior and posterior cortical bone the mean values of Nf B Ar and of MAR are always similar in all groups . The traGomori trichrome stain performed on vertebral trabecular bone does not reveal the presence of osteoid seams (OS/BS) in all rats of the control group, while the value of OS/BS in all rat fed calcium-deprived diet ranges from 0 to 24%. round-shaped and in sharp-edge-shaped sections. The medullary canal areas are instead always larger in the rats fed calcium-deprived diets (Group 3) with respect to baseline and control ones in both round-shaped and sharp-edge-shaped sections; this difference is sometimes significant.Static histomorphometric parameters are reported in  sharp-edge-shaped sections in all groups; moreover, again in particular in sharp-edge-shaped sections, the endosteal surface shows an irregular outline (index of bone resorption) in all animals fed a calcium-deprived diet. round-shaped and sharp-edge-shaped sections at the periosteal and endosteal level. It must be underlined that in four animals out of five in the two groups fed a calcium-deprived diet and in two out of five animals in the control group we never observed new bone formation at the endosteal level in sharp-edge-shaped sections; this implies high values of standard deviation of Nf B Ar and MAR average.The results of dynamic histomorphometry concerning Nf B Ar, MAR, and MS are shown in Gomori trichrome stain does not reveal the presence of osteoid seams (OS/BS) in all rats of the control group, while the value of OS/BS in all calcium-deprived diet animals ranges between 0 and 22%.In The present investigation analyses how the mineral and skeletal homeostasis influence the bone loss in metabolic osteoporosis due to calcium-deprived diet in different sites of the two bony architectures  in both axial and appendicular skeleton.The first point to discuss concerns the different amount of bone mass recorded between the control group and the two groups of rats fed calcium-deprived diet; in fact, trabecular bone of vertebral bodies and femoral metaphyses Tables  and 7 asThe second point to discuss is that the anterolateral part of vertebral cortical bone shows intense modelling processes in all animal groups. In fact, independently of diet type, new bone deposition always occurs at the periosteal level, as shown by the presence of the 3 labels of osteogenesis in all groups ; thus, t sharp-edge-shaped sections , the excessive individual variability among the animals inside the same group did not allow understanding and discussing the data obtained. recovering bone fragility [ preventive effects, since no significant differences were found between the two groups of animals fed calcium-deprived diet with/without the drug administration.In conclusion, the relevance of this paper performed on the rat model lies in the detailed documentation of the fact that the lack of calcium in the diet does not lead to a unique bone answer. It is to be underlined in fact that the various answers recorded in the different sites of bony architectures pertaining to specific skeletal segments are due to the different main involvement of each skeletal region in maintaining mineral or skeletal homeostasis. As a consequence, the bone modeling processes are differently affected in answering induced biochemical osteoporosis. Regardless of the main topic of the present paper, data here reported on PTH (1-34) seems to indicate that Teriparatide, used with good results as therapeutic support inragility –42, doesstatic and dynamic osteogenesis) and imply different bone quality by the mechanical viewpoint.The present investigation based on animal models represents a good starting point for successive studies on bone alterations during unbalanced calcium metabolism, as frequently occurring in aging, with the aim of studying in detail timing and manners of evolution and recovery in human biochemical osteoporosis with/without administration of PTH (1-34). In particular, the attention will be focused on the type of osteogenesis of the newly formed bone during bone mass recovering that, as previously demonstrated , 44, can