The vaccination of rHN1201gE−/gI−/11k−/28k−TK−/ could elicit an earlier and higher level of gB antibody, and the neutralizing antibodies elicited by rHN1201gE−/gI−/11k−/28k−TK−/ were effective against both PRV classical and variant strains. Clinically, the body temperature of the pigs immunized with rHN1201gE−/gI−/11k−/28k−TK−/ was significantly lower than that of the classical PRV vaccine immunized pigs, and the recombinant PRV could provide effective protection against the challenge with the PRV variant. These results imply that the rHN1201gE−/gI−/11k−/28k−TK−/ could be a promising vaccine candidate for the prevention of the current epidemic of pseudorabies in China.In China, the re-emerging pseudorabies virus (PRV) variant has caused large-scale outbreaks of pseudorabies in swine herds with classical PRV vaccine immunization since late 2011. Here, a recombinant PRV with TK/gI/gE/11k/28k deletion was constructed based on variant HN1201 strain isolated in 2012, by the bacterial artificial chromosome infectious clones. Compared with the parental virus, the recombinant PRV rHN1201 Pseudorabies (PR), also called Aujeszky's disease, is caused by the infection of an alpha-herpesvirus Pseudorabies virus (PRV) . The douThe herpesvirus PRV has a broad host range, which is known to cause acute fatal disease in a variety of mammals –5. The Pvia multiple passages on pig kidney cells and chicken embryos, and their companion differential serological tests have been widely used to control PR during the past several decades  infectious clone is widely used for the studies of viral genome manipulation, and then be used for evaluating the efficacy of vaccine candidates –20. In hin vitro .TK−  used in this study were tested free of PRV, porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and porcine circovirus 2 (PCV2). All the animal samples were collected according to the protocol approved by the Animal Care and Ethics Committee of National Research Center for Veterinary Medicine (Permit 20170625005).KP722022.1) isolated in 2012 has been described previously  competent cells DY380 that harbor the plasmid pBAC-HN1201TK− were obtained as described previously  gene with homologous arm sequence of Cat-OriS from pBeloBAC11 plus the inverted repeat fragment of the homologous arm of PRV TK gene] was transformed into the DY380 cells containing the plasmid pBAC-HN1201gE−/gI−/11k−/28k−TK−/ was assessed, and then was compared with that of the parental virus HN1201. After infection by the parental and rescued virus (MOI of 1.0), the supernatants of Vero cells were harvested at 0, 4, 8, 12, 16, 20, 24, 28, 32, and 36 h post-infection (hpi) and stored at −80°C. The virus titers were determined by the 50% tissue culture infectious dose (TCID50). Growth kinetics for each virus were tested in triplets and the resulting titers were averaged.One-step growth curve of the rescued rHN12013 TCID50 of the virus was aspirated at 1 h after incubation, and then the cells were overlaid with 1% low-melting-point agarose in DMEM supplied with 2% FBS for plaque formation. For each virus, 100 plaques were randomly selected, and the plaque size was determined by ImageJ software .Plaque sizes were determined at 48 hpi in Vero cells. Briefly, in the 6-well plates with monolayer cells, the culture medium (DMEM supplied with 2% FBS) containing 1.0 × 10n = 5). The piglets in groups 1 and 2 were vaccinated intramuscularly with 1.0 × 105.0 TCID50 rHN1201gE−/gI−/11k−/28k−TK−/ and one dose of Bartha-K61, respectively. DMEM medium was used as the placebo in group 3 (unvaccinated). After vaccination, rectal temperature and clinical signs were recorded daily. The pig serum samples in days post-vaccination (dpv) of 0, 8, 10, 12, 14, and 21 were collected to monitor gB and neutralizing antibodies (NAbs). All pigs were challenged with HN1201 (1.0 × 106.0 TCID50 per pig) intranasally at 21 dpv. At 14 days post-challenge (dpc), all pigs were euthanized and necropsied, and organ samples were collected for immunohistochemistry (IHC) assay.A total of 15 pigs (28-day-old), free of PRV, PRRSV, CSFV, and PCV2, were randomly divided into 3 groups (2 TCID50) in 96-well plates and incubated at 37°C for 1 h in an atmosphere with 5% CO2. After incubation, 100 μl of PK-15 cell suspension containing 2.0 × 104.0 cells was added to each well. The inoculated cells then were incubated at 37°C in an atmosphere with 5% CO2 for 5 days, and the titers of PRV-specific NAbs were determined based on cytopathic effect (CPE), and the titers were expressed as the reciprocal of the highest dilution at which infection of the PK-15 cells was inhibited in 50% of the culture wells.For antibody testing, gB antibodies of the serum samples were evaluated by the Aujeszky gB (Pseudorabies Virus) Antibody Test Kit  according to the instructions. The PRV-specific NAbs titers were tested by serum-neutralization test (SNT). Briefly, serum samples were inactivated at 56°C for 30 min prior to the SNT. Two-fold serially diluted serum (50 μl) was mixed with an equal volume of the HN1201 or Bartha-K61 , gB-r (5′-GTCYGTGAAGCGGTTCGTGAT-3′), and Probe-gB (FAM-ACGTCATCGTCACGACC-TARAM), on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The copy number for each sample was expressed as log10 copies per gram of samples.Brain, cerebellum, tonsils, and lung samples were collected from the pigs from 3 groups for IHC assay. The samples were fixed with 10% formaldehyde, processed into paraffin blocks, and cut into sections. The sections were stained with hematoxylin and eosin. PRV antigen in the infected pigs' tissues was detected using an IHC with a PRV monoclonal antibody 3B5 (a gB-specific IgG antibody), as described previously , 29, andt-test. Differences were considered statistically significant when p < 0.05.Data were presented as mean ± SD. The survival rates were analyzed by the Kaplan–Meier test. The differences in plaque areas of viruses, body temperature, and antibody titers of piglets between groups were determined by using Student's TK− by homologous recombination in E.coli DY380 /I-SceI/Amp/SE(28k) was applied to replace the fragment SE(gI)/gI/gE/11k/28k/SE(28k) in pBAC-HN1201li DY380 . The 5-gt 72 hpi . PlaquesgE−/gI−/11k−/28k−TK−/ were virtually identical to that of parental virus HN1201 in PK-15 cells. However, the plaque areas of the rescued virus were smaller than those formed by HN1201 in PK-15 cells  exhibited high fever , which were earlier and higher than that of the Bartha-K61 group   , indicatt HN1201 , which igE−/gI−/11k−/28k−TK−/ group than those in the Bartha-K61 group (p < 0.05)  , but notgE−/gI−/11k−/28k−TK−/.In the IHC assay of tissue samples, as shown in gE−/gI−/11k−/28k−TK−/, was generated based on the RPV variant strain HN1201. The full protection in pigs immunized with rHN1201gE−/gI−/11k−/28k−TK−/ indicated that it is a safe and protective vaccine candidate to control the PR caused by new epidemic PRV variants.The widely used PRV vaccines of classical strain, such as the strain Bartha-K61, were effective in PR control during the past several decades in China , 8. HoweThe 3-kb deletion in the unique short (US) region of Bartha genome, partial loss of 28k, most of US7 (gI), and complete deletion of US8 (gE) and 11k  indicateAs reported, PRV gE and gI are required for efficient cell-to-cell spreading, gE/gI participates in the envelopment of nucleocapsids into cytoplasmic membrane vesicles , and delIn herpes simplex virus, gI and gE null mutants lead to the formation of small plaques , and in The rapid improvement of biotechnology promoted the research of genetically modified PRV in recent years, such as the application of BAC system in gene deletion , 37–39, gE−/gI−/11k−/28k−TK−/ was constructed by the BAC system, which elicited an earlier and higher level of gB antibody, and the NAbs elicited by rHN1201gE−/gI−/11k−/28k−TK−/ were effective against both PRV classical and variant strains. The rHN1201gE−/gI−/11k−/28k−TK−/ vaccination could provide effective protection against the challenge with the PRV variant. Therefore, it is a promising vaccine candidate for the prevention of the current epidemic of PR in China.In summary, here, a recombinant PRV rHN1201The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The animal study was reviewed and approved by Animal Care and Ethics Committee of National Research Center for Veterinary Medicine.FT, BH, and KT conceived and designed the research. SY, XB, YZ, LG, TW, and YW conducted the experiments. FT, YZ, and BH analyzed the data. SY, FT, BH, and KT conceived the study, carried out additional analyses, and finalized the manuscript. All authors contributed to the article and approved the submitted version.Mycoplasma hyopneumoniae (201200211200).This study was supported by the project of R&D and industrialization of genetically engineered vaccines for swine pseudorabies, swine ring, and 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This n = 20 per group) including low translucent (LT) , high translucent (HT) , and multilayered system (ML) . Each monolithic zirconia group was equally subdivided according to be either air-abraded with 110 µm aluminium oxide particles or left untreated (control). After thermocycling, the flexural strength was measured by using a universal testing machine. Two-way ANOVA followed by Tukey's post hoc and independent samples t-test were used for the statistical analyses (P < 0.05).  Sixty bar-shaped specimens were fabricated from three different types of presintered monolithic zirconia (P = 0.010). Having controlled the effect of surface treatment, the flexural strength of HT and LT zirconia was found to be significantly higher than the ML zirconia system (P ≤ 0.001). Airborne particle abrasion could significantly decrease the flexural strength of monolithic zirconia only in ML zirconia (P = 0.002).  Surface treatment and types of zirconia were found to have a significant interaction ( Multilayered zirconia system had the lowest flexural strength among all groups. Moreover, the flexural strength of this system was attenuated by surface treatment with airborne particles abrasion. Its Bilayered core-ceramic systems were designed to cover the opaque face of zirconia under a tooth-like appearance while having the strength of yttria-stabilized tetragonal zirconia polycrystalline (Y-TZP). A common complication of bilayered zirconia is porcelain veneer chipping due to the dense and unreactive surface of zirconia and its lower adhesion to ceramic veneers , 4, 5, aThe inherent semitranslucency of monolithic zirconia impedes a natural tooth-like appearance ; howeverAttaining information regarding the flexural strength is of importance, particularly the mechanical strength of monolithic zirconia after surface treatments , 13. Lon2O3) on the flexural strength of aged monolithic zirconia. The null hypothesis was that different translucencies and surface treatment would not affect the flexural strength of thermocycled monolithic zirconia.Not enough studies have addressed the effect of different translucencies and types of monolithic zirconia especially multilayered system and the combined effect of translucency and surface treatment on the flexural strength of monolithic zirconia. This in vitro study was designed to evaluate the effect of three different translucencies and air abrasion with aluminum oxide (Aln = 20 per group) including low-translucent (LT) monolithic zirconia , high-translucent (HT) zirconia , and multilayered (ML) system of monolithic zirconia . The specimens were manufactured according to ISO 6872, with ± 0.02 mm accuracy [In this experimental in vitro study, a bar-shaped specimen (25 × 5 × 2 mm) was designed , based on which 60 specimens were milled  out of three different types of presintered monolithic zirconia (accuracy  and sint2O3 particles (n = 10 per subgroup). Both sides of the specimens were air-abraded with 110 µm laboratory Al2O3 particles  for 10 seconds at 400 kPa air pressure from a 10 mm distance. The particle size was adopted with respect to the previous studies claiming that larger particles enhance the surface abrasion, wettability, and cement bond strength [Each group was subdivided to be either left untreated as the control or air-abraded with Alstrength , 23 ; images were taken at ×500 and ×1500 magnifications to check the surface properties.M = 3Wl/2bd2, where W is the applied load (N), l is the test span (mm), b is the specimen width (mm), and d is the specimen thickness (mm) [The specimens were, then, subjected to 1000 thermal cycles  between 5 °C and 55 °C, ultrasonically rinsed with distilled water for 10 minutes, and air-dried for 20 seconds. To measure 3-point flexural strength, a force  was applied on a universal testing machine in the middle of the specimen on a 20 mm fixture at a speed of 1 mm/min . The foress (mm) .t-test for pairwise comparisons (α = 0.05).Statistical analyses were done by IBM SPSS for Windows . Kolmogorov–Smirnov and Shapiro–Wilk tests were used to confirm normal distribution and the homogeneity of variances. Two-way ANOVA was used for the descriptive data (mean and standard deviation (SD)), and one-way ANOVA was used to evaluate the effect of each factor, followed by Tukey's post hoc test and independent samples P ≤ 0.001), while the effect of surface treatment with Al2O3 was insignificant (P = 0.249). Moreover, a significant interaction existed between the surface treatment and zirconia type (P = 0.01). Having controlled the effect of surface treatment, one-way ANOVA showed that zirconia type significantly affected the flexural strength (P ≤ 0.001). Tukey's post hoc test showed that, in both the control and APA subgroups, the flexural strength of both HT and LT zirconia specimens was significantly higher than that in the ML system (P ≤ 0.001) (≤ 0.001) .P = 0.978) and HT (P = 0.182) groups in neither the control nor the APA subgroups. Independent t-test showed that the surface treatment had no significant effect on the flexural strength in the HT (P = 0.176) or LT zirconia group (P = 0.110). But, in the ML zirconia system, air-abrasion significantly reduced the flexural strength compared with the control group (P = 0.002)  .2O3 created porous and irregular surfaces in all types of zirconia. In the control group without surface treatment, ML system and HT zirconia had the most and least irregular surfaces, respectively. However, after airborne abrasion with Al2O3, LT and HT zirconia showed rougher surfaces , which included three grades as ultratranslucent multilayered zirconia, supertranslucent multilayered zirconia, and multilayered zirconia . Flinn eμm are not usable in smaller dimensions due to the impossibility of phase transition [Pereira et al.  and Parkansition . Spyropoansition  evaluateThe current findings showed that the flexural strength was not significantly different between the LT and HT zirconia. Likewise, Matsuzaki et al.  found thIn line with the present findings about ML zirconia compared with HT and LT zirconia, Mao et al.  found thμm Al2O3 particles on the ﬂexural strength of monolithic zirconia [Another finding of the present study was the significant interaction between the surface treatment and zirconia type. APA creates the clean and rough surface required for higher adhesion to dental cement or veneering porcelains , 33, 34.zirconia , 23, 34.2O3 particles (0.28–0.40 MPa) [Contrary to what the present study found about LT and HT zirconia, some studies showed concerns about the superficial damage after airborne particle abrasion with 50–120 µm Al.40 MPa) –38. Howe.40 MPa) , 39. Dis.40 MPa) , 39, 40.Mao et al.  examinedA systematic review detected that the translucency and flexural strength were significantly influenced by the composition, microstructure, and surface treatment . ReviewiKurtulmus et al.  investigAmong the limitations of the present study, was the nonclinical in vitro nature, which restricts free interpretation of the results for the clinical conditions. Further studies are recommended to consider other types of zirconia with different surface treatments.2O3.Within the limitations of this study and considering the in vitro conditions, it can be concluded that, between the three different translucencies of monolithic zirconia, the multilayer zirconia system had the lowest flexural strength than the two other types of monolithic zirconia. Besides, the flexural strength of this system decreased by surface treatment with airborne particle abrasion with Al


The homogeneous diffusion of selenium particles in the produced spheres is credited with an improved electrochemical performance.Carbon–selenium composite positive electrode (CSs@Se) is engineered in this project using a melt diffusion approach with glucose as a precursor, and it demonstrates good electrochemical performance for lithium–selenium batteries. X-ray diffraction (XRD) and scanning electron microscopy (SEM) with EDS analysis are used to characterize the newly designed CSs@Se electrode. To complete the evaluation, electrochemical characterization such as charge–discharge (rate performance and cycle stability), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) tests are done. The findings show that selenium particles are distributed uniformly in mono-sized carbon spheres with enormous surface areas. Furthermore, the charge–discharge test demonstrates that the CSs@Se cathode has a rate performance of 104 mA h g Due to the increasing environmental damage caused by the massive burning of coal, gasoline, and natural gas, there is a growing demand for the effective use of clean energy. Storage of energy is critical for a clean energy system. The ever-increasing need for energy storage can be met by rechargeable batteries with high specific capacity and energy density ,2,3,4,5.−1, which is lower than sulfur (S), but its higher density compensates for this shortcoming. Furthermore, selenium has a higher electrical conductivity (1 × 10−3 S m−1) than sulfur (5 × 10−28 S m−1) [−1 at 100 mA g−1 after 10 cycles. Abouimrane et al. [−1 at 50 mA g−1 current density.Selenium, which has electrochemical characteristics comparable to sulfur, can produce selenides when it reacts with lithium. Selenium has a theoretical gravimetric capacity of roughly 670 mA h g8 S m−1) ,10 resul8 S m−1) . However8 S m−1) ,13. Thes8 S m−1) ,23,24,258 S m−1) , and oth8 S m−1) . Wu et a8 S m−1)  developee et al.  created Despite the fact that the performance of selenium composite cathodes has increased over time, there is still insufficient contact between the active material and the electronic conductors. Many attempts have been made to improve the electrochemical performance of lithium–selenium batteries; for example, a porous carbon structure has proven to be an effective positive electrode. Wang et al.  injected−1 up to 400 °C, remaining for 3 h in an Ar/H2 protection gas. Following that, a sample of CSs@Se was obtained through natural cooling. The synthesized materials were characterized using different microstructural techniques and electrochemical tests for Li–Se batteries.Synthesis of CSs@Se: All the chemical reagents used in this experiment were purchased from Sigma Aldrich and were not purified further. Carbon spheres (CSs) were created using a straightforward one-step hydrothermal procedure. A known amount of glucose (11.2 g) was introduced to 130 mL of de-ionized water and swirled for half an hour with a constant magnetic stirrer to achieve uniform dispersion. The solution was then poured to two Teflon-lined autoclaves (100 mL each), and the reaction was carried out in a vacuum oven at 180 °C for 6 h to obtain a regular and uniform shape. The autoclaves were then cooled naturally in air to room temperature, and the black solid precipitates were recovered by centrifuge after three washes with ethanol and water (three times each) and desiccated at 80 °C for 12 h (named CSs) in a vacuum oven. To produce CSs@Se, known quantities of carbon spheres and selenium powder (1:2 mass ratio) were mashed together. The mixture was then transferred to a tubular furnace (OTF-1200X) and heated at a rate of 5 °C minUsing a field-emission scanning electron microscope , we determined the structure and morphology of CSs and CSs@Se composite. The EDS instrument installed in the SEM was used to determine the elemental mapping of the elements. The phase composition of the material was determined using an X’Pert PRO diffractometer , with the measuring angle ranging between 5–70° and a scanning rate of 0.8°/s. The thermogravimetry (TG) examination was carried out using a vertical zero friction dilatometer L75VS Linseis  at temperatures ranging from 30 to 800 °C in an argon environment in order to determine the selenium weight percent contained in the composite material, and the surface area of the material was calculated using the Brunauer–Emmett–Teller (BET) (ASAP2020) technique.6 solution in an electrolyte mixture of Ethyl carbonate (EC) and Diethyl carbonate (DEC)  and a porous Celgard 2400 separator. Half-cells were produced in a glove box with argon flowing , then left for 8 h before being tested for electrochemical performance. The LAND battery testing station was used to undertake rate performance and cyclic stability tests in the voltage range of 0.005–3 V (vs. Li/Li+) at various current densities. For the first five cycles, a cyclic voltammogram (CV) test was performed on workstation CH1611E with a voltage window of 0.005–3 V at 0.5 mV/s. The coin cell impedance was measured at 10 mV over a frequency range of 0.001–100,000 Hz.Using a half-cell, the electrochemical performance of bare carbon spheres (CSs) and CSs@Se composite was investigated. The cathodes were made by combining 70 weight percent active material (CSs/CSs@Se), 10 weight percent PVDF binder, and 20 weight percent carbon black (Super-P). A small amount of N-methyl pyrrolidone (NMP) was also added to the mixture, which was then agitated for 12 h with a continual magnetic stirrer to achieve a homogenous mixture. Finally, this homogeneous mixture (slurry) was applied on the foil (copper). The coated foil was then dried overnight at 60 °C. The foil was punched into disc shapes to make electrodes. The anode was a lithium foil disc with a 1M LiPF2/g, as shown in Low and high magnification SEM pictures of bare carbon spheres are shown in TGA of CSs@Se composite was conducted in l radius . At the  spheres c. The ba−1 for five cycles from 0.005–3 V (vs. Li+/Li) are shown in 2Se corresponds to one cathodic peak centered at 1.03 V for the first curve (black one). The occurrence of only one peak indicates that no polyselenides phase exists during the lithiation process and hence no shuttle effect. The presence of two anodic peaks at 1.07 and 2.3 V (for the first curve) suggests that the reduced Li2Se was oxidized back during the de-lithiation process. After the first cycle, the anodic and cathodic peaks at 1.07 and 2.6 V have little displacement around 0.96 V for the second cycle, and for the other cycle, the curves practically coincide, indicating that CSs@Se composites are reversible. The charge–discharge pattern at various current values is shown in 2Se. After the first discharge curve, the slope voltage for the others shifts somewhat. The slope voltage plateaus on the charge curves correlate to the two peaks on the cyclic voltammetry curve.The electrochemical testing of a CSs@Se composite positive electrode for lithium–selenium (Li–Se) batteries is performed with a 2032 type coin cell and a lithium foil negative electrode. The cyclic voltammetry (CV) curves of CSs@Se composite at 0.5 mV s−1 to 2500 mA g−1). The composite cathode has an average specific capacity (at current densities) of 294 (50 mA g−1), 279 (100 mA g−1), 244 (250 mA g−1), 209 (500 mA g−1), 156 (1000 mA g−1), and 104 mA h g−1 (2500 mA g−1). Furthermore, when the current returns to its initial amount, a specific capacity of 290 mA h g−1 is measured, demonstrating that the CSs@Se composite, as a positive electrode, is highly reversible. At the same current densities as above, the capacities of the bare CSs are 47.8, 43.06, 32.6, 26.6, 18.4, and 14.9 mA h g−1. −1. Due to the limited electrochemical kinetics in the CSs@Se electrode, the Li+ are not fully intercalated into the CSs@Se composite at higher densities. −1 was preserved at 50 mA g−1 for 100 cycles, but the specific capacity of 270 mA h g−1 was preserved at 25 mA g−1.Meanwhile, In addition, the rate performance of the current CSs@Se composite is compared to other materials, such as S/C composite , G-Se@CN+ in the composite. The Rct for CSs@Se composite before and after 70 cycles is shown in + can be calculated from the slopes of ω−1/2 and Z’  and diffusion coefficient (D) of Li2 and Z’ b, where This equation is utilized to determine the diffusion coefficient for the composite before and after cycling ,38,39,40−1 at 50 mA g−1 and 104 mA h g−1 even at 2500 mA g−1) and cyclic performance (270 mA h g−1 even after 100 cycles). The improved electrochemical performance owes to the rapid intercalation/de-intercalation of Li+ into the CSs@Se composite and the decreased generation of undesirable soluble-polyselenides.A simple hydrothermal procedure was used to successfully create CSs@Se composite, which is used as the positive electrode in lithium–selenium batteries. According to the synthesized substance, it is extremely homogenous and favors easy selenium loading. This composite’s electrochemical performance was evaluated, and it demonstrated good rate performance (294 mA h g


Mary Rose, which sank tragically in the Solent in 1545 AD, presents a rare archaeological opportunity to research individuals for whom the precise timing and nature of death are known. A long-standing question surrounds the composition of the Tudor navy and whether the crew were largely British or had more diverse origins. This study takes a multi-isotope approach, combining strontium (87Sr/86Sr), oxygen (δ18O), sulfur (δ34S), carbon (δ13C) and nitrogen (δ15N) isotope analysis of dental samples to reconstruct the childhood diet and origins of eight of the Mary Rose crew. Forensic ancestry estimation was also employed on a subsample. Provenancing isotope data tentatively suggests as many as three of the crew may have originated from warmer, more southerly climates than Britain. Five have isotope values indicative of childhoods spent in western Britain, one of which had cranial morphology suggestive of African ancestry. The general trend of relatively high δ15N and low δ13C values suggests a broadly comparable diet to contemporaneous British and European communities. This multi-isotope approach and the nature of the archaeological context has allowed the reconstruction of the biographies of eight Tudor individuals to a higher resolution than is usually possible.The great Tudor warship, the  The overarching aim is to extend the biographies of these men to provide wider insights into Tudor life under the reign of King Henry VIII (r. 1509–1547), at the individual level. The known timing and circumstances of death, the excellent preservation of their skeletal remains and the fact that there is evidence for their professions  present a very rare opportunity to apply a multi-isotope and ancestry estimation study to provide higher resolution information on individual osteobiographies than most archaeological studies allow.This study presents new multiple isotope analyses (strontium (. 1.1Mary Rose was a successful warship for Henry VIII (r. 1509–1547) for 34 years, from 1511 until 1545  marks the end of the medieval period and the beginning of the early modern era in England. This period is characterized by substantial social and economic change, notably the break from Catholicism. However, this is a largely arbitrary historical division rather than a major transition in terms of the lives of the populace. Many medieval customs continued; diet remained mostly unchanged and established trade links with continental Europe and the Mediterranean region continued . ConsequMary Rose soon after she sank was attempted by a West African diver, Jacques Francis [In the Tudor period, England had trade links with The Netherlands, France, Italy, Germany and the Mediterranean . Althoug Francis .13C and δ15N values at sites from this period [Historical research into the diet of British medieval populations has largely produced generalizations of nation-wide diet and have mainly focused on the English perspective . Cerealss period ,29, but s period ,31. Howes period .. 1.3Mary Rose [Elizabeth of Newcastle was reinforced with a contingent of Devon men in 1513 [Mary Rose may have been manned by a semi-permanent, professional crew as those sampled were young but suffered many age-related vertebral pathologies potentially caused by continuous work aboard a warship [Henry VIII was an ambitious king, keen to wage war and demonstrate his power in Europe, and the early Tudor period was a time of great development for the royal navy . Althougary Rose –35. In 1ary Rose . Substan in 1513 . Foreign in 1513 . Sources in 1513 . A palae warship . However warship . Numbers warship .Mary Rose crew in the form of various ceramic types, including English, French, Low Countries', Rhenish and Iberian wares [There are indications of foreign connections among the an wares . These man wares . In addian wares .Mary Rose crew, using δ13C, δ15N and δ18O isotope analysis on 18 individuals [13C and δ15N analysis on bone collagen provided comparable results to those at other British medieval sites [et al. [18O results demonstrate that at least one-third, and up to almost two-thirds, of the individuals in their sample originated from a more southerly, or warmer, locale than Britain and make the claim that miscommunication between the multi-national crew contributed to the ship's sinking. This paper was heavily criticized by Millard & Schroeder [et al.'s [18O values in British humans [There has been one isotope study into diet and provenance of the ividuals  . Theal sites ,29,41. Bchroeder , who argh humans ,43 suppoh humans  conclusi. 1.4Since the 1970s, isotope analysis has been employed by archaeologists to investigate the diet, origins and mobility of past humans and animals (see §§2.3 and 2.4 for detail on isotope notation). Childhood diet and origins are investigated through isotope analysis of dental samples. Dental tissues do not substantially remodel; therefore, the isotope composition of primary dentine and enamel represents the formation period during childhood .87Sr/86Sr isotope ratios are incorporated into skeletal tissues from diet and principally relate to the geology of the area where food was produced [87Sr/86Sr analysis is useful for exploring origins. Time-integrated radioactive decay of the rubidium isotope 87Rb to 87Sr, compared with the stable 86Sr isotope, is responsible for the variable 87Sr/86Sr. Very old and rubidium-rich bedrocks have higher 87Sr/86Sr, such as granites (up to 0.720 in Britain), whereas younger and low-rubidium bedrocks exhibit lower 87Sr/86Sr values, for example, basalts (approx. 0.705) [The produced . As past. 0.705) .18O/16O isotope ratios (δ18O) are primarily derived from ingested fluids, reflecting local drinking water values and are therefore also useful for investigating origins. Values of δ18O vary according to temperature, altitude, latitude, coastal proximity and precipitation and thus relate to particular climatic zones [18O values within archaeological populations by Lightfoot & O'Connell [18O values [18O values of the water in liquids and food [18O values, and therefore, it is a complex isotope proxy to interpret, a point returned to in the discussion. However, in this study, comparisons will be made with δ18O data from other medieval sites, where the impact of culinary practices is likely to have been similar.The ic zones . A globa'Connell  found thO values . Studiesand food ,50,51. T87Sr/86Sr and δ18O analyses have long been used in parallel to investigate origins (e.g. [34S (34S/32S isotope ratio) analysis of bone and dentine collagen has also become useful in both dietary and mobility studies in humans and animals [34S has a wide range; relatively low and negative values are common in inland areas of waterlogged ground and/or impervious lithologies [34S values, and food grown and grazed on coastal soils can be influenced by a ‘sea-spray effect’ which results in δ34S values which approach marine sulfate composition (+20‰) [34S values than those sourcing their diets from further inland, although estuarial regions also tend to have high biosphere δ34S [Combined ns (e.g. ) but in  animals –57. Bioshologies . By contn (+20‰) , althougn (+20‰) . Therefoere δ34S . Values ere δ34S , with exere δ34S .13C (13C/12C isotope ratio) and δ15N (15N/14N isotope ratio) values is based on the premise that the isotope values in skeletal tissues reflect the average δ13C and δ15N of consumed food (primarily dietary proteins) [13C and δ15N values, as diet is not a major focus. The premises behind these methods have been described in detail elsewhere [13C principally vary in relation to the consumption of marine resources and different types of plants (C3 and C4 photosynthesizing plants). Values of δ15N are closely related to the trophic level of the consumer, with humans consuming more animal protein having higher values. The consumption of fish also tends to result in higher δ15N values due to elongated aquatic food chains. As an example, humans that consume mostly marine protein are expected to have δ13C values up to −12‰ and high δ15N values up to 15‰ [15N values [13C and δ15N variation. Origins also have an impact, as varied landscape baseline values mean that the same foodstuffs will have different values depending on where they are produced [Palaeodietary reconstruction using δroteins) . It is blsewhere , so are p to 15‰ . HoweverN values . Dietaryproduced .13C and δ15N [87Sr/86Sr and δ18O [More recently, multiple isotope proxies have been employed in combination to investigate archaeological diet and mobility to increase interpretative potential. However, studies that integrate five isotope systems, such as this one, remain rare ,56,66–69and δ15N ,29,72–74and δ18O .. 2. 2.1The Mary Rose Trust has attributed professions to the skeletal remains of seven individuals who are investigated in this project: Cook (FCS-12), Royal Archer (FCS-70), Archer (FCS-75), Carpenter (FCS-81), Officer (FCS-84), Gentleman (FCS-85) and Purser (FCS-88)  root for δ13C, δ15N and δ34S analysis. The same sampling method was followed for all individuals. A ca 4 mm strip of enamel was extracted from the lower half  of the lingual surface of the M2. The strip was divided into mesial and distal sections, with the larger being analysed for 87Sr/86Sr and the other for δ18O. A whole M1 root was sampled for each individual. Due to the exceptional preservation of the teeth and the fact that maxillary and mandibular molars should produce similar isotope ratios [Enamel samples were extracted from the second molar (M2) for e ratios , teeth/r18O values and limiting issues surrounding intra-individual variation [Mary Rose were from a 10 year old [Each tooth provides information from different periods of childhood . M1 rootariation . The youyear old ,6, so thyear old ,80.13C and δ15N isotope analysis to investigate diet in the years before death, prompted by the only instance of severe, bilateral buccal cemento-enamel junction caries among the individuals recovered from the Mary Rose.In addition to the dental samples, a collagen sample from a rib bone  was tak. 2.387Sr/86Sr (approx. 20–40 mg) and δ18O (approx. 2.5–5 mg) analysis were then divided.Enamel samples (20–50 mg) were cut from teeth and cleaned using a diamond saw and burr to remove all adhering dentine and greater than 10 μm of the enamel surface. Samples for 87Sr/86Sr analysis, samples were ultrasonically cleaned in deionized water and transferred to a clean working area  for further preparation and 87Sr/86Sr analysis at Cardiff Earth Laboratory for Trace element and Isotope Chemistry (CELTIC). Samples were digested in 8 M HNO3 and heated overnight at 120°C. Strontium extraction from enamel samples used Sr Spec™ resin using a revised version of the protocol of Font et al. [3. Matrix elements (including Ca and traces of Rb) were then removed in several washes of 8 M HNO3 before Sr was eluted and collected in 0.05 M HNO3. Samples were placed on a hotplate (120°C) to dry overnight before this process was repeated for a second pass for the effective removal of any remaining traces of Ca. Once dry, purified samples were re-dissolved in 0.3 M HNO3. The 87Sr/86Sr ratios were measured using a Nu Plasma II multi-collector inductively coupled plasma mass spectrometer (MC-ICP-MS) at Cardiff University. Samples were introduced using an Aridus II desolvator introduction system. All data were first corrected for on-peak blank intensities, then mass bias corrected using the exponential law and a normalization ratio of 8.375209 for 88Sr/86Sr [87Rb) interferences were monitored and corrected for using 82Kr and 83Kr  and 85Rb (85Rb/87Rb = 2.5926), respectively. Analysis of NIST SRM 987 during the analytical session gave a 87Sr/86Sr value of 0.710292 ± 0.000007 , and all data are corrected to NIST SRM 987 values of 0.710248 [87Sr/86Sr ratio in NIST SRM 1400 , giving an average 87Sr/86Sr ratio of 0.713111 ± 0.000014 , which is consistent with the published value . At the Cardiff University Stable Isotope Facility, samples were acidified for 5 min with greater than 100% ortho-phosphoric acid at 70°C [18O/16O) normalized to the VPDB scale using an in-house carbonate reference material (BCT63) calibrated against NBS19 certified reference material. The δ18Ocarbonate values are then converted into the VSMOW scale (VSMOW = 1.0309 x δ18O VPDB + 30.91 [18O BCT63 is ±0.03‰ (1σ). The standard deviation of replicate δ18O measurements is 0.049‰. The δ18Ocarbonate values were converted to δ18Ophosphate (δ18Op) values . The loollowing ) to alloequation .. 2.413C, δ15N and δ34S analysis following a modified Longin method [−1) and gelatinized in a pH3 solution (acidified with HCl) at 75°C for 48 h. The supernatant was then filtered , frozen and freeze dried. Samples were weighed and analysed in duplicate. Ratios of δ34S, δ13C and δ15N are reported in per mil (‰) relative to VCDT, VPDB and AIR standards, respectively. The δ34S analysis was undertaken at the National Environmental Isotope Facility, Keyworth, Nottinghamshire. The instrumentation comprises a ThermoFinnigan EA IsoLink coupled to a Delta V Plus isotope ratio mass spectrometer via a Conflo IV interface. Values of δ34S were calibrated using IAEA S-1 and S-2 with secondary checks comprising in-house standard M1360P (powdered gelatine) with laboratory-accepted delta value of 2.99‰ and modern cattle bone collagen . The average 1σ reproducibility across the standard material was ±0.28‰. M1360P was used to calculate %S . The 1σ standard reproducibility was ±0.25‰. Both δ13C and δ15N ratios were measured at the Cardiff University Stable Isotope Facility using a Flash 1112 elemental analyser coupled to a Thermo Delta V Advantage. Ratios of δ13C and δ15N were calibrated against caffeine  and an in-house supermarket gelatine standard, which is calibrated against IAEA-600 (δ13C and δ15N), IAEA-CH-6 (δ13C) and IAEA-N-2 (δ15N). The 1σ (n = 54) reproducibility was ±0.06 for δ13C and ±0.07 for δ15N. Different weights of caffeine were analysed to establish a calibration equation for the abundance of C and N against signal intensity in the mass spectrometer, which was used to calculate %N and %C in actual samples. The content of caffeine  was calculated from its chemical formula (C8H10N4O2). All samples adhered to published quality control indicators [Collagen was prepared for δn method . Approxidicators ,91.. 2.518O values and good cranial preservation. Further information on these methods is provided in the electronic supplementary material.During a photogrammetry study of FCS-09 , it was Craniometrics  were ana. 3. 3.187Sr/86Sr, δ18O and δ34S analyses are displayed in 18O and 87Sr/86Sr data, including quality control indices). All three proxies produced relatively wide-ranging results. The 87Sr/86Sr values range from 0.70872 to 0.71070 (σ). Overall, the δ18Op values are high for a British dataset, ranging between 18.4‰ and 21.2‰ (σ). The mean is skewed by one clear outlier, FCS-70 (21.2‰), who has one of the highest values measured in a human from a British archaeological context, and therefore, the median of 18.9‰ provides a more balanced average. Three individuals  have δ18Op values that fall more than two standard deviations outside of the British mean . Half of the individuals  have high (greater than 14‰) δ34S values (15.7 to 17.4‰). One individual (FCS-88) has an intermediate value (12.6‰), and the others  have low δ34S values (−3.3 to 6.9‰).The isotope data from  0.71070 , with a nd 21.2‰ , with a n = 615, ). One inn = 615, . The δ34. 3.213C values range between −20.1‰ and −18.3‰, with a mean of −19.4‰  and δ15N values range between 9.4‰ and 12.8‰ (σ). Two outliers were observed in terms of δ15N. FCS-88 has the highest δ15N value (12.8‰) and FCS-75 has a markedly lower value than the rest of the dataset (9.4‰). FCS-12 has the highest δ13C (−18.3‰) and δ34S value (17.4‰) as well as a high δ15N value (11.3‰). When comparing the δ34S and δ13C data, the three individuals with the lowest δ34S values also have the lowest δ13C values  and the three individuals with the highest δ34S values also have the highest δ13C values . Although sample sizes are deemed too small for statistical analysis, δ34S and δ15N isotope data show no clear relationship.The isotope results are presented in tables nd 12.8‰ , with a 15N was observed between the early developing dentine and later developing rib samples. The dietary signals were well within the range of the other crew members, and the buccal caries are likely to result from a cultural practice that had a negligible dietary impact.FCS-09 was the only individual for which different elements were analysed to investigate temporal changes in diet. Only a minor elevation in δ. 415N and low δ13C values for all eight of the crew are characteristic of a largely terrestrial protein-rich childhood diet in a C3 ecosystem and consistent with isotope data from other late medieval British sites .Mary Rose individuals have 87Sr/86Sr values (0.70872 to 0.71070) compatible with British origins, which Evans et al. [87Sr/86Sr values are not particularly diagnostic, with values in this range potentially deriving from various areas across Britain [87Sr/86Sr values such as these have been recorded at various locations across Europe, the Mediterranean, Africa and the Middle East and are by no means unique to Britain [The eight s et al.  estimate Britain . Further Britain ,110–118.18O analysis was designed to address challenges and ambiguities in the interpretation of provenance, but it is not possible to discount all issues of equifinality. Recommended methods were employed [Mary Rose provides a particular challenge, as it cannot be treated as a settled community from which outliers can be identified statistically, as advocated by Lightfoot & O'Connell [The approach to δemployed , samplinemployed ,79, drinemployed , and intemployed ,43. The 'Connell . The sam18O values suggest that as many as three individuals may be from warmer climates than Britain. FCS-70, FCS-81 and FCS-85 have high δ18Op values  that are more than two standard deviations from the British mean of 17.7‰ ±1.4‰  is markedly lower than in this study , although there is considerable overlap in the datasets. This difference may derive from chance sampling, as both studies relate to only a small number of individuals or may relate to varied sampling and pre-treatment methods.The δn = 615; ,43). The boiling . Neverthes lower . Once coer [18Op , the mea34S values (greater than 14‰) that suggest coastal origins [34S value whereas FCS-70 and FCS-81 have very low δ34S values suggesting ‘inland’ residence, potentially in areas of wetland and/or impervious lithology [34S values  also had the highest δ13C values, supporting a coastal residence [Three of the five ‘British’ individuals  have high δ origins ; FCS-88 ithology . The thresidence .The two potential sub-sets of the crew are discussed below, with more detailed biographical reconstruction for individuals with isotope values outside of the British range.. 4.118Op value of 18.4‰ and a 87Sr/86Sr value of 0.71070, FCS-09 sits well within British isotope ranges and is discussed in more detail in a later section, as ancestry analyses were also undertaken.Five of the individuals  have isotope values consistent with a childhood spent in Britain. With an δ18Op values (group mean = 18.6‰) closer to the mean value for western Britain  than for eastern Britain  [18Op values [All five have δ n = 83) . These cp values , althoug18Op = 18.4‰, 87Sr/86Sr = 0.71070, δ34S = 16.7‰; FCS-75: δ18Op = 18.6‰, 87Sr/86Sr = 0.71055, δ34S = 15.7‰) and may have originated from the same region. Their δ18Op values suggest a western or southern British origin, and their δ34S values indicate a childhood spent in close proximity (less than 30 km) to the sea [87Sr/86Sr close to 0.7110 are commonly found on Palaeozoic rocks  that are common on the western side of southern Britain, such as in South Wales, Devon and Cornwall, as well as Carboniferous grits in North Devon [FCS-09 and FCS-75 have very similar isotope values , FCS-84 (0.70915) and FCS-88 (0.70979) relate to rocks of various ages across vast swathes of Britain [87Sr/86Sr values may relate to limestone geologies although there are various other possibilities within Britain (and beyond). Values between 0.709 and 0.710 are very common in the British biosphere, leading Montgomery [34S values of these three individuals, FCS-12 has a very high δ34S value (17.4‰) indicative of coastal origins, most likely from more exposed western areas. FCS-88 (12.6‰) has a more intermediate value, whereas FCS-84 (6.9‰) had a more inland childhood abode [87Sr/86Sr values, it is clear that all three came from different areas. A range of western coastal zones provides potential areas of origin for FCS-12. FCS-88 could derive from the Thames estuary or other southern/western areas, though probably not from the most exposed coastal zones of the west. FCS-84 could come from the southern end of the English midlands or inland areas of Wessex that are not on chalk lithology (e.g. north Wiltshire). However, pinpointing origins is not possible, and these assignments must be considered as suggestions based on the current mapping.Suggesting origins for the remaining three British individuals is more difficult. The δ Britain . All are Britain ,111. Mos Britain . The 87Sntgomery  to descrod abode . DespiteMary Rose and the threat of French invasion, she never ventured further than the seas around Britain and coastal areas of northern France. Apart from refitting in the River Thames prior to the Third French War, she is believed to have mostly kept to the Channel [Lizard comprised contingents from the coastal towns of Beaumaris, Plymouth, Hull and Tynemouth [Due to the size of the  Channel . Recruitynemouth . The his. 4.218O values beyond the expected British range [18Op values (greater than 19‰) in humans are common in warmer climates than Britain, such as southern European coasts, southwest Iberia and North Africa [18Op values relative to the other individuals  have δsh range ,43. Highh Africa ,124. It  source; ). Howeve source; ,125,126,18Op value of 19.3‰, a ‘coastal’ δ34S value of 17.4‰ and a 87Sr/86Sr value (0.70983) consistent with various Mesozoic and late Palaeozoic sediments found extensively around the Mediterranean coastline, including southern Spain, Italy and Greece [FCS-85 may well have originated from a southern European coastal site as he has a high δd Greece ,124,127.87Sr/86Sr values consistent with various Mesozoic lithologies around the Mediterranean [18Op values than would generally be expected of residents of southern Europe , especially FCS-70 [34S values , which excludes origins within coastal zones and raises the possibility of areas of wetland/impervious geology [18Op value of 19.8‰, an upbringing in inland southern Europe is a possibility for FCS-81 (the ‘Carpenter’). Material evidence from the carpenter's cabin suggests that at least one of the six carpenters on board may have been Spanish. Four adzes with a ‘stirrup’ design, commonly found in Spain during this period were recovered, and there were a few coins identified as Spanish in an embroidered leather pouch found in a personal chest in the cabin [FCS-70 and FCS-81 have erranean ,127. They FCS-70 –130 (buty FCS-70 ). They a geology ,60,61. W18Op value of 21.2‰ is rather high for residence in Europe [18O values due to dietary factors, it is possible that this individual was an Iberian Moor. This is supported by the dietary isotopes of FCS-70 which indicate a C3-based diet, consistent with Spanish origins [4 which is more expected for residents of more arid climates [3 crops were widespread by the medieval period, and so low δ13C values do not rule out more arid climates than Britain, such as North Africa, where populations cultivated European crops such as wheat, similar to other Mediterranean agricultural communities [87Sr/86Sr value of FCS-70, regions such as northeastern Morocco [Although an δn Europe –130 (butn Europe ), warmern Europe . Conside origins , opposedclimates , like Afmunities . Conside Morocco  and loca Morocco  are poss. 4.3To improve the clarity of the isotope data and with the possibility of North African origins for FCS-70 and FCS-81 and potential African ancestry in FCS-09, further craniometric and morphoscopic analyses were conducted . Furthermore, it was only possible to analyse FCS-81 using one ancestry estimation method, so the results are not as strong. The δhis time . Althoug. 5Mary Rose crew. Our findings point to the important contributions that individuals of diverse backgrounds and origins made to the English navy. This adds to the ever-growing body of evidence for diversity in geographic origins, ancestry and lived experiences in Tudor England. Although isotope analysis is a useful tool for investigating provenance, it should be used with caution and suggested interpretations in this research are supported by artefactual evidence. As drivers of isotope variation become better understood and biosphere maps become higher resolution, the confidence with which origins can be reconstructed will improve [Mary Rose will result in a more complete narrative of both Henry VIII's navy and the wider Tudor world.There has been extensive historical research on the Tudor period, yet direct analyses of its population are lacking. The multi-factorial approach taken by this study has combined artefactual and historical evidence with multi-isotope and ancestry estimation analyses to gain a greater insight into eight members of the  improve . As such improve ), ancestClick here for additional data file.


Mycobacterium avium subspecies paratuberculosis (MAP) may be an etiological agent in some or all of IBD. We hypothesized that the clinical efficacy of methotrexate and 6-MP in IBD may be to simply inhibit the growth of MAP.Clinical improvement in inflammatory bowel disease (IBD) treated with methotrexate and 6-mercaptopurine (6-MP) is associated with a decrease in pro-inflammatory cytokines. This has been presumed to indicate the mechanism of action of methotrexate and 6-MP. Although controversial, there are increasingly compelling data that M. avium using a radiometric (14CO2 BACTEC®) detection system that quantifies mycobacterial growth as arbitrary “growth index units” (GI). Efficacy data are presented as “percent decrease in cumulative GI” (% −ΔcGI).The effect on MAP growth kinetics by methotrexate and 6-MP were evaluated in cell culture of two strains each of MAP and The positive control antibiotic (clarithromycin) has ≥85% −ΔcGI at a concentration of 0.5 µg/ml. The negative control (ampicillin) has minimal inhibition at 64 µg/ml. MAP ATCC 19698 shows ≥80% −ΔcGI for both agents by 4 µg/ml. With the other three isolates, although more effective than ampicillin, 6-MP is consistently less effective than methotrexate.in vitro. Each of the four isolates manifests different % −ΔcGI. These data are compatible with the hypothesis that the clinical improvement in patients with IBD treated with methotrexate and 6-MP could be due to treating a MAP infection. The decrease in pro-inflammatory cytokines, thought to be the primary mechanism of action, may simply be a normal, secondary, physiological response. We conclude that henceforth, in clinical studies that evaluate the effect of anti-MAP agents in IBD, the use of methotrexate and 6-MP should be excluded from any control groups.We show that methotrexate and 6-MP inhibit MAP growth  Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease In the therapy of IBD,  both methotrexate and 6-MP are used because of empirical efficacy, even though their precise mechanism of action is unknown. Both methotrexate and 6-MP interfere with DNA replication. Methotrexate acts by inhibiting dihydrofolate reductase, folate generation and the consequent production of adenine.M. avium isolates in culture. The effect of methotrexate has been evaluated on E. coliSalmonella typhimuriumM. tb. We hypothesized that the clinically relevant mechanism of action of “low” dose methotrexate and 6-MP in the therapy of IBD, may, in whole or part, be due to the inhibition of MAP growth. If this hypothesis is correct, the decrease in the pro-inflammatory cytokines, heretofore considered the primary mechanism of action of these two agents, could simply represent a secondary phenomenon. The observed decrease in pro-inflammatory cytokines could be ascribed to the treatment of the instigating MAP infection. To evaluate this hypothesis we have studied the effect of methotrexate and 6-MP on MAP and other This study was approved by the Research & Development Committee at the VAMC Bronx NY (0720-06-038) and was conducted under the Institutional Radioactive Materials Permit (#31-00636-07).M. Avium subspecies avium strains  were ATCC 25291 (veterinary source) and M. avium 101 In this study, we used four well-characterized strains of mycobacteria. Two were MAP, a bovine isolate, ATCC 19698 (ATCC Rockville MD) and “Dominic” a human isolate from an individual with Crohn's disease  renders clinically resistant strains of MAP inappropriately susceptible to antimicrobials in cell culture. M. avium & specific subspecies including silveticum) as described. To confirm the identity of the species studied, DNA was obtained from the isolates (High Pure Template Prep. Kit Roche Diagnostics Indianapolis IN), PCR amplified using primers for IS 900 (MAP) The positive antibiotic control clarithromycin (gift of Abbott Chicago IL) was dissolved in methanol. The negative control antibiotic was ampicillin (Sigma St Louis MO) which was dissolved in water. Methotrexate and 6-mercaptopurine (both Sigma) were dissolved in NaOH at a maximal final concentration of 50 mM (Sigma). Control inocula were performed using the maximum concentration of each diluent. Agents were tested in serial dilutions from a minimum of 0.05 µg/ml to a maximum of 64 µg/ml . Aliquots of chemicals being evaluated were prediluted, stored at −80°C, thawed, used once and discarded.ΔcGI), compared to the control cGI of that isolate in the diluent (e.g. methanol or NaOH) that was used for the specific chemical being evaluated. cGI data for each experiment is presented until the day prior to any GI reading exceeding the assay limit of “999” (Data are presented as cumulative growth index (cGI) units±SD . The effect (or lack thereof) of each agent is presented as the percent decrease in cGI units (% −of “999” . Raw datM. avium isolates are approximately 10 fold higher (∼1×107 CFU's/ml), compared to MAP (∼1×106 CFU's/ml)  . Becausethan MAP .M. avium ATCC 25 291 was positive for IS 902 and M. avium 101 was negative for both (Data not presented).Both of our MAP isolates (ATCC 19698 & Dominic) were Mycobactin J dependant (data not presented), were IS 900 positive and had ≥99% homology with the GenBank accession NC_002944 of MAP (data not presented). ΔcGI at the lowest concentration evaluated (0.5 µg/ml). The negative control antibiotic  has a minimal effect (21% −ΔcGI) at the 32 µg/ml. In contrast, 6-MP has an initial ≥43% −ΔcGI starting at 1 µg/ml increasing to ≥84% −ΔcGI at 4 µg/ml. Methotrexate has 40% −ΔcGI inhibition at 2 µg /ml and ≥75% −ΔcGI at 4 µg/ml.  compared to MAP 19698 (84%−ΔGI at 4 µg/ml). Both M. avium isolates show less susceptibility to 6-MP than to methotrexate  bacterial infections.As is conventional with antibiotic susceptibility studies, we compared agents on an equal weight basis. However, methotrexate (MW 450) is a much larger molecule than 6-MP (MW 170) with a molar ratio of ∼3∶1. Thus, in comparison to 6-MP on a molar basis, methotrexate is an even more potent inhibitor of growth than our data indicate. Additionally, a simple extrapolation of our data to a comparison with conventional “antibiotics” therapy is difficult. The dosages of methotrexate and 6-MP in clinical use have not been titrated according to standard antibiotic conventions. Dosage has been individualized, influenced by such factors as hematological toxicity and patient tolerance.2 by IV infusion over 2–24 hours (for a 70 kg man this could equate to 7500 mg in 24 hours.)There is a remarkable discrepancy between the doses of methotrexate and 6-MP, used to treat different diseases, that merit discussion. Each agent has a “high” dose, (used in to treat reticuloendothelial malignancies Our data are compatible with the thesis, but do not conclusively prove, that MAP may be zoonotic. Corroboration of our data will be necessary and multiple additional studies, both basic and clinical need to be performed. However, we suggest that, as a consequence of our observations antecedent clinical studies that have evaluated anti-MAP agents need to be reevaluated and that henceforth such studies will need to exclude methotrexate and 6-MP from “control” or placebo subjects.We show that both methotrexate, as well as 6-MP, interfere with the growth of MAP, an organism that may be the etiological factor for some, or all of IBD. Some of the implications of these observations are discussed.


As we show [cyt1Δ mutant is not maximized at the level of restriction used by Lin et al. [cyt1Δ cells [As in our prior work showing that life span extension by calorie restriction (CR) occurs independently of Sir2 , we haven et al. . Thus, w1Δ cells . cyt1Δ cells, the claims made by Lin and Guarente [In their correspondence, Lin and Guarente  divert aGuarente  are untecyt1Δ cells, we show that CR at either glucose concentration significantly increases the life span of rho0 cells, which completely lack mitochondrial DNA [In addition to responding to the statements of Lin and Guarente , we wishrial DNA . Furtherrial DNA ,8,9. In


During the 1990–1991 Gulf War, approximately 700,000 U.S. troops were deployed to the Persian Gulf theater of operations. Of that number, approximately 100,000 have presented medical complaints through various registry and examination programs.Widespread symptomatic illness without defining physical features has been reported among veterans of the 1991 Gulf War. We ascertained changes in symptom status between an initial 1995 symptom evaluation and a follow-up in 2000.We assessed mailed symptom survey questionnaires for 390 previously surveyed members of the U.S. Department of Veterans Affairs Gulf War Registry for changes over the 5-year interval in terms of number and severity of symptoms.For the cohort as a whole, we found no significant changes in symptom number or severity. Those initially more symptomatic in 1995 showed some improvement over time, but remained much more highly symptomatic than those who had lesser initial symptomatology.The symptom outbreak following the 1991 Gulf War has not abated over time in registry veterans, suggesting substantial need for better understanding and care for these veterans. During the 1990–1991 Gulf War (GW), approximately 700,000 U.S. troops were deployed to the Persian Gulf theater of operations. Large-scale involvement of U.S. forces in combat in this region largely ended by the winter of 1991. Nevertheless, significant numbers of the 700,000 U.S. service personnel deployed to the region in 1990–1991 presented with medical complaints in the years following operations. Of that number, approximately 100,000 have presented medical complaints through various registry and examination programs . The symOnly one study has made an attempt to longitudinally follow GW veterans to determine how their symptomatic presentations have evolved—whether there has been improvement, stability, or decline in the self-reported health of individuals and groups of GW veterans , 2004. TWe have studied, both clinically and by survey, various groups of GW veterans since 1995 . Our oriParticipants were U.S. military veterans from the Gulf War Health Registry maintained by the VA. In 1995 (time 1), veterans residing in seven states—Delaware, Illinois, New Jersey, New York, North Carolina, Ohio, and Pennsylvania—were selected by VA’s Environmental Epidemiology Service using a simple random sampling procedure. The veterans were originally selected as part of recruitment for clinical studies of GW illness . A randoAt time 1, we mailed introductory letters and questionnaires with postage-paid response envelopes to 2,011 veterans. We followed this with reminder postcards, a second  letter and questionnaire, and a maximum of three follow-up phone calls at intervals of approximately 2 weeks until a response was received, ultimately yielding 1,161 responses (60%).At time 2, The U.S. Department of Defense’s Defense Manpower Data Center (DMDC) provided updated addresses and demographic information on those who had responded at time 1. These veterans were sent a letter describing the project and requesting their consent to be interviewed. Records from the Internal Revenue Service and national directory assistance databases  were used to locate veterans whose letters were returned without forwarding information. These databases did not include cellular telephone numbers.This 5-year follow-up of the registry cohort was included as a part of a larger investigation of a national random sample of deployed GW veterans . As partParticipants were asked to examine a list of 48 symptoms identical to that used at time 1 . For eaca) veterans reporting good health and few moderate/severe symptoms , and b) veterans reporting fair/poor health and endorsing an average of 37 symptoms, 75% as moderate/severe . Thus, there was a total of 390 respondents who completed both the time 1 and time 2 symptomatology assessments  to examine the main effect of time on the severity of 48 symptoms by controlling for age (mean centered), rank, race, marital status, sex, education, branch of service, duty , smoker status, and cluster membership (“mildly” or “highly” symptomatic as classified at time 1). In addition, we performed an ANCOVA  to examine the main effect of cluster membership at time 1 on change in number of symptoms over the 5-year period. The change score was calculated by subtracting time 1 scores from time 2 scores. We used the following variables as controls: age (mean centered), sex, rank, race, marital status, education, branch, duty, and smoking status. For tests of main effects and interactions, we adopted p < 0.05 as the critical value. To adjust for multiple comparisons when comparing changes in the 48 individual symptoms, we employed a Bonferroni correction, adopting as a critical value p < 0.001 (0.05/48).To assess changes in symptoms over time, we conducted paired samples The results for time 1 have been fully described by Logistic regression analyses suggested no significant differences in response rates attributable to sex, date of entry into the registry, branch of service, type of duty , or rank . Multivariate analysis of variance also revealed no significant differences in response rates attributable to either specific symptoms or specific diagnoses as determined by VA examining physicians and independent questionnaires completed at the time of registry enrollment .n = 20), not deployed to the Gulf (n = 39), incarcerated (n = 3), or incapacitated . Of the remaining 1,091 eligible to participate, 640 (60%) were mailed a questionnaire, of which, 453 (71%) were completed and returned. Forty-five (7%) could not be delivered, and no forwarding address could be found. Of those who completed the questionnaire, 426 were contacted by telephone and by mail, and 398 of these completed both time 1 and time 2 assessments. Thus, 62% of those mailed a questionnaire at time 2 completed it and the subsequent telephone interview.Updated DMDC data and data collected by telephone indicated that 70 of the original 1,161 registry veterans were ineligible to participate in the follow-up study. These included veterans who were deceased , and education . In addition, t-tests with Bonferroni correction revealed no significant differences in the number of symptoms or their average severity reported at time 1 by the 390 current respondents and the remainder of the time 1 cohort.These 390 respondents did not differ significantly from the 1,161 respondents who participated at time 1 in terms of sex ; rank ; duty status at mobilization ; or branch . They were significantly different from the 1,161 originally studied in terms of age at time 2 , racial composition  = −1.48; p = 0.14]. Average symptom severity also increased slightly from 0.83 ± 0.62 at time 1 to 0.84 ± 0.62 at time 2. Again, this change was not significant . Results of ANCOVA on change in number of symptoms controlling for age (mean centered), sex, rank, race, marital status, education, branch, duty, and smoking status revealed that the effect of time 1 cluster membership was significant   = 15.11; p < 0.001] and age . Black veterans reported 4.26 more symptoms at time 2, whereas white veterans showed a slight decrease . Increase in the number of symptoms over time was associated with older age .At time 1, the mean number of symptoms (± SD) reported by the sample of 390 was 22.07 ± 12.92. At time 2, the mean number of symptoms increased slightly (0.67 symptoms) to 22.74 ± 12.89; however, this change was not significant [< 0.001] . Those vF = 2.24; p < 0.001], race , and cluster membership   = 1.57; p < 0.05] and a significant time × cluster membership effect .A repeated measures MANCOVA indicated that there was no significant difference over time in the severity of the 48 symptoms. However, we observed multivariate effects on severity for age  . In addip < 0.001 level, with F values ranging from 32.96 to 329.59. Veterans who were highly symptomatic at time 1 had higher severity scores at time 2 than those who reported mild symptomatology.Next, we looked at tests of between-subject effects for the variables that yielded significant multivariate tests to examine the severity changes in individual symptoms. The main effect of age was significant for “persistent or recurring problems” with “arms, hands, and shoulders,” “back problems,” “frequent or painful urination,” “sexual or genital problems,” “pain in arms or legs,” and “pain in more than one joint,” with the severity of all these symptoms increasing more in older individuals. The main effect of race was significant for “persistent or recurring problems” with “constipation,” “hair,” and “sweating not due to exercise,” with all three symptoms increasing more in severity among African-American veterans . The mait-tests showed that while the changes in this symptom were not significant for those who never smoked and for former smokers, the increase in symptom severity for the current smokers was significant . The time × cluster membership interaction term was significant for “persistent or recurring problems,” with 18 symptoms listed in t-tests for 18 symptoms for the cluster originally reporting mild symptomatology showed that after Bonferroni correction (0.05/18 = 0.002), the increases over time in the severity of “abdominal gas,” “muscle aches or cramps,” “numbness or tingling sensations,” “pain in arms or legs,” and “difficulty concentrating” were significant. For the cluster that reported high symptomatology at time 1, the decreases in the severity of “headaches,” “difficulty breathing,” “unrefreshing sleep,” “sleeping more than usual,” “sudden mood changes,” and “feeling depressed or blue” were significant after Bonferroni correction  are relevant in the absence of substantial improvement in the severity and number of symptoms experienced by Gulf War veterans over time.


The model is analyzed using nonlinear dynamical system theory. For small Na+ window current, the neuron exhibits high-frequency tonic firing. At current threshold, the spike response is almost instantaneous for small d-current conductance, gd, and it is delayed for larger gd. As gd further increases, the neuron stutters. Noise substantially reduces the delay duration and induces subthreshold oscillations. In contrast, when the Na+ window current is large, the neuron always fires tonically. Near threshold, the firing rates are low, and the delay to firing is only weakly sensitive to noise; subthreshold oscillations are not observed. We propose that the variability in the response of cortical FS neurons is a consequence of heterogeneities in their gd and in the strength of their Na+ window current. We predict the existence of two types of firing patterns in FS neurons, differing in the sensitivity of the delay duration to noise, in the minimal firing rate of the tonic discharge, and in the existence of subthreshold oscillations. We report experimental results from intracellular recordings supporting this prediction.Cortical fast-spiking (FS) interneurons display highly variable electrophysiological properties. Their spike responses to step currents occur almost immediately following the step onset or after a substantial delay, during which subthreshold oscillations are frequently observed. Their firing patterns include high-frequency tonic firing and rhythmic or irregular bursting (stuttering). What is the origin of this variability? In the present paper, we hypothesize that it emerges naturally if one assumes a continuous distribution of properties in a small set of active channels. To test this hypothesis, we construct a minimal, single-compartment conductance-based model of FS cells that includes transient Na About 25% of the neurons in the mammalian neocortex are inhibitory, namely reduce the activity of neurons they contact. These inhibitory neurons exhibit diversity of morphological, chemical, and biophysical properties, and their classification has recently been the focus of much debate. Even neurons belonging to a single class of “fast-spiking” (FS) display a large variety of firing patterns in response to standard square current pulses. Previous works proposed that this class is in fact a discrete set of neuronal subtypes with biophysical properties differing in a discontinuous way. In this work, we propose an alternative theory, according to which the biophysical properties of FS neurons are continuously distributed, but distinct firing patterns emerge due to highly nonlinear dynamics of these neurons. We ascertain this theory by exploring with mathematical techniques a biophysically based model of FS neurons. We demonstrate that variable firing responses of cortical FS neurons can be accounted for if one assumes heterogeneity in the strength of some of the ionic conductances underlying neuronal activity. Our theory predicts the existence of two main firing patterns of FS neurons. This prediction is verified by direct recordings in cortical slices. Among inhibitory neurons in the neocortex, the “fast-spiking” (FS) compose the most prominent type. These neurons are characterized by brief action potentials with a width smaller than 0.5 ms followed by a deep monophasic afterhyperpolarization (AHP) ,2. Delay+ current )+ window current contribute strongly to variability in the minimal firing rate.A large variability is observed between FS neurons in their minimal firing rates in response to steady current. Whereas many FS cells have high steady-state minimal firing frequencies on the order of tens of Hz or more ,23, the + current, this bifurcation can be of Hopf or SN types. As a consequence, the properties of the neuron during the delay period depend also on the Na+ window current. In particular, subthreshold oscillations are found during that period . This is consistent with the observation of subthreshold oscillations at frequencies in the gamma-range (20–100 Hz) in FS neurons in cortex [INa window current is small . In addMarder and colleagues proposed that for a specific pattern of activity, one can find parameter subspaces within which the model displays qualitatively, and even quantitatively, similar behavior ,42. Theytdelay, decreases with the amplitude of the current step, Iapp, and disappears at a non-zero value as Iapp is elevated . As shown by [θh to more hyperpolarized potentials in pyramidal neurons, decreasing their Na+ window current. It also facilitates the persistent Na+ current INaP by shifting its activation curve leftward [θh more negative in pyramidal cells, and also reduces the maximal conductance of INaP [+ currents in FS cells, our results suggest that they may modify qualitatively the firing patterns of these neurons.The modeling results presented in this paper can be applied to understand the effect of some neuromodulators on the firing patterns of FS cells. Dopamine attenuates the d-type K neurons . Consist neurons  . Na+ cushown by , it shifleftward . Similarof INaP . If one V is the membrane potential of the neuron, C = 1μF/cm2 is the membrane capacitance, and the parameters of the leak current are gL = 0.25 mS/cm2 and VL = −70 mV. The external current injected into the neuron is denoted by Iapp.Our model of FS cells is based on that of ,28, with+ current INa is given by:h and m, follow:gNa = 112.5 mS/cm2, VNa = 50 mV, mσ = 11.5 mV, hθ = −58.3 mV, hσ = −6.7 mV, thθ = −60 mV, thσ = −12 mV [+ window current, controlled by the parameter θm, on the dynamics of the neuron.The Na= −12 mV . In this+ current IKdr is of the Kv3.1–Kv3.2 type. It is responsible for the brief duration of the spike, about 0.5 ms [gKdr = 225 mS/cm2, VK = −90 mV, nθ = −12.4 mV, nσ = 6.8 mV, tnθ = −27 mV, thσ = −15 mV [The delayed rectifier Kt 0.5 ms , and fort 0.5 ms . It is g= −15 mV .+ current Id incorporated in the model [gd are fixed: aθ = −50 mV, aσ = 20 mV, aτ = 2 ms, bθ = −70 mV, bσ = −6 mV, bτ = 150 ms [gd is varied to study the effect of the strength of this current.The Khe model ,11 has f= 150 ms ,52. The Inoise, of the form:ξ(t) is a Gaussian white noise with an average 0 and a unit variance, and D has the units of μA2 × ms/cm4 .Finally, to study the effect of noise in the external input on the firing pattern of the neuron, we add an additional external input, Numerical methods. Simulations were performed using the fourth-order Runge-Kutta method with a time step of 0.01 ms implemented as a C program or within the software package XPPAUT [e XPPAUT , which wDelay. The delay duration tdelay is defined to be the time from the onset of current injection, or, if the neuron fires transient 1–3 spikes, from the last transient spike to the first spike of the sustained firing. We define that the neuron shows a delay if tdelay is at least twice as large as the inter-spike interval during steady-state spiking tISI, or if it is larger than both 100 ms and 1.2 tISI.Fourier spectrum. Discrete Fourier transforms of subthreshold oscillations were calculated numerically over a time window of TFT ending TBS = 5 ms before the first spike of the steady-state firing. The absolute values of the Fourier components were averaged over nR repetitions of the same stimulus. Parameters for TFT = 120 ms, nR = 20. Parameters for TFT = 90 ms, nR = 5 (A), TFT = 120 ms, nR = 13 (B), TFT = 120 ms, nR = 11 (C).b varies depend on the shape of the function VFP(b), where VFP is the value of the membrane potential of the neuron at the fixed point of the dynamics for fixed b. Equivalently, one can relate the bifurcations to the shape of the curve b = b(VFP), in the b-VFP plane (the “b-VFP curve”), which is defined by (see VFP. The numerator therefore is positive in the relevant range of VFP for which b > 0. The functions IL(VFP) and IKdr(VFP) are increasing with VFP. Only the function INa(VFP) may decrease with VFP. Therefore, for a small Na+ window current, b decreases monotonously with VFP. This happens for instance for mθ = −24 mV (+ current is sufficiently large [INa(VFP) in b(VFP) be non-monotonous. This happens for instance for mθ = −28 mV . For large bτ, V follows the curve VFP(b) during the delay period (bx̃ the solution of the equation Vx̃ ≡VFP( bx̃) Note that the fixed point of the fast subsystem for b = bx̃ is unstable if the neuron fires following the delay. When Iapp is near Ith, VFP(b) is approximated by Vx̃ [b is approximated bytdelay is justified because tdelay is determined mainly by the slow dynamics of b when it is near bx̃. Before the current step is applied at time t = 0, the system is at rest with b = brest. The subsystem converges immediately to its fixed point on the slow time scale, and the solution to tdelay is determined by the equationbHopf is the value of b at the Hopf bifurcation and α is a constant. Substituting Ith, tdelay >> τb, and Iapp−Ith is small, bx̃ depends only weakly on Iapp and bHopf − bx̃ depends linearly on Iapp−Ith. Therefore, tdelay scales as (Iapp−Ith) −1.y period A. We dend by Vx̃ , and the13a from , tdelaySaddle-node bifurcation of the fast subsystem. The dynamics are very slow near the SN bifurcation, occurring at . Neglecting the changes in VFP during the evolution, bISN = b∞(VSN). Namely,Iapp−Ith is small, bSN−bISN depends linearly on Iapp−Ith. Therefore, tdelay scales as −log(Iapp−Ith).tdelay on the noise variance, D, for weak noise and weak window INa. In this case, the delay ends because the fast subsystem is destabilized via a Hopf bifurcation. According to Theorem 4.1 in [D, is neither too large nor too small, tdelay is determined by the equationAx̃ and Bx̃ are constants. As above, tdelay is determined mainly by the slow evolution near bx̃, and therefore one can use the approximation VFP(b) ≈ Vx̃. Substituting tdelay, in the limit tdelay >> τb, we obtainWe calculate the dependence of m 4.1 in , assumingd, gd = gd1, and a step current with an amplitude Iapp, there is a solution to the equation F(b) = b. We prove here that for a large enough gd, a solution to this equation does not exist for this value of Iapp. The function F(b) is defined only when the limit cycle exists, namely only for b ≤ bSNP. Since the current Id depends on gd and b only through the product gdb  is very small for large gd.We consider the case that, for a certain value of ct gdb , bSNP fF(b) is the time-average of the function b∞(V(t)) over LC(b) (b∞(V) is a positive, decreasing function of V  ≥ F(b∞(VNa)). From the fact that bSNP(gd) decreases with gd ) > bSNP(gd). Since F(b) is defined only for b ≤ bSNP(gd), we obtain that F(b) > b, and there is no solution to the equation F(b) = b if gd is large enough. Therefore, the full system cannot exhibit a tonic firing state. If the rest state of the neuron is unstable, the neuron stutters. In practice, F(b) is much larger than F(b∞(VNa)) because the membrane potential spends a large fraction of its period in subthreshold values, and therefore gd should not be extremely large to prevent a solution of the equation F(b) = b.We continue by noticing that: (1) er LC(b) ; (2) b∞ion of V ; and (3)with gd , one finhttp://www.campdeninstruments.com) and then transferred to a holding chamber where they were kept at room temperature for at least 1 h before recording, continuously bubbled with 95% O2, 5% CO2. Recording was done in a chamber mounted on an upright microscope equipped with IR/DIC optics (Nikon physiostation EC-600), where they were held at 32–34 °C and constantly perfused. The normal bathing solution contained (in mM): 124 NaCl, 3.5 KCl, 2 MgSO4, 1.25 NaHPO4, 2 CaCl2, 26 NaHCO3 and 10 dextrose, and was saturated with 95% O2, 5% CO2 (pH 7.4). Whole-cell recordings were made from neurons in the barrel field. Patch recording micropipettes (4–6 MΩ) were filled with a solution containing (in mM) 125 K gluconate, 5 NaCl, 2 MgCl2, 10 EGTA, 10 HEPES, and 2 Na2-ATP, pH 7.2, 280 mOsm). Voltages were recorded with a patch clamp amplifier , and digitally sampled at 10 kHz. Data acquisition and analysis were performed with Labview . Series resistance was typically <15 MΩ. During all recordings, 50 μM DL-2-amino-5-phosphopentanoic acid  and 6,7-dinitroquinoxaline-2,3-dione  were present in the bath to block excitatory transmission.Mice  were deeply anaesthetized with pentobarbital, decapitated, and their brains quickly removed into cold (5 °C) physiological solution. Coronal cortical slices (400 μm thick) were cut with a vibratome  it fired brief spikes with fast, deep, monophasic AHPs [dV/dt to falling phase dV/dt was smaller than 2. Previous studies revealed that the morphological correlate of FS neurons can be either “basket” [“basket”  or “neur“basket” ,54,55. M“basket” , but oth“basket” . Thus, tFigure S1θm = −24 mV, gd = 0.39 mS/cm2, Iapp = 0, Iapp of the hyperpolarizing current step is −8 μA/cm2, VL = −60 mV.(A) The neuron does not fire rebound spikes in response to release from hyperpolarizing current steps. Parameters: INa and depolarized VL, that is raised to −60 mV from the reference parameter set of −70 mV. Parameters: θm = −24 mV, gd = 1.8 mS/cm2, Iapp = 1.7 μA/cm2.(B) The neuron exhibits stuttering without delay for weak window (68 KB PDF)Click here for additional data file.Figure S2θm = −24 mV, θh = −54.3 mV, gd = 0.39 mS/cm2. Left: voltage trace for Iapp = 2.38 μA/cm2. Right: the steady-state f–Iapp curve of the neuron. The average firing frequency goes to zero at firing threshold.(A) Parameters: θm = −28 mV, θh = −62.3 mV, gd = 0.39 mS/cm2. Left: voltage trace for Iapp = 1.95 μA/cm2. Right: the steady-state f–Iapp curve is discontinuous at the current threshold. The minimal frequency is 23.3 Hz.(B) Parameters: (87 KB PDF)Click here for additional data file.


Idiopathic pulmonary fibrosis (IPF) is characterized by the insidious onset of dyspnea or cough. However, a subset of patients has a short duration of symptoms with rapid progression to end-stage disease. In this study, we evaluated clinical and molecular features of “rapid” and “slow” progressors with IPF.26 patients with <6 months of symptoms before first presentation [rapid progressors] and 88 patients with >24 months of symptoms [slow progressors] were studied. Survival was analyzed by the Kaplan-Meyer method and proportional hazard's model. Lung microarrays and tissue proteins were measured in a subset of patients. No differences were found in age, physiologic impairment and bronchoalveolar lavage (BAL) cellular profile. There were more males  and smokers  in the rapid progressors group. Survival from the beginning of symptoms was significantly reduced in rapid progressors  and there was a tendency for decreased survival from the time of diagnosis . We identified 437 differentially expressed genes. Lungs of rapid progressors overexpressed genes involved in morphogenesis, oxidative stress, migration/proliferation, and genes from fibroblasts/smooth muscle cells. Upregulation of two of these genes, adenosine-2B receptor and prominin-1/CD133, was validated by immunohistochemistry and were expressed by alveolar epithelial cells. BAL from rapid progressors showed a >2-fold increase of active matrix metalloproteinase-9, and induced a higher fibroblast migration compared with slow progressors and controls [238±98% versus 123±29% (p<0.05) and 30±17% (p<0.01)].A subgroup of IPF patients, predominantly smoking males, display an accelerated clinical course and have a gene expression pattern that is different from those with slower progression and longer survival. These findings highlight the variability in the progression of IPF, and may explain, in part, the difficulty in obtaining significant and reproducible results in studies of therapeutic interventions in patients with IPF. Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing interstitial lung disease of unknown etiology characterized by progressive dyspnea, reduced lung volumes, impaired gas exchange, and the histopathologic signature of usual interstitial pneumonia (UIP). This disease, which is the most common of the idiopathic interstitial pneumonias, is unresponsive to current therapy and most patients die within 5 years after diagnosis The purpose of this study was to determine whether “rapid” and “slow” progressor IPF patients could be distinguished by clinical, biological or molecular features. Better identification and understanding of these differences may provide insights into the pathogenesis and assist in the development of therapeutic interventions.This study included 114 individuals from a cohort of 167 consecutive patients with IPF evaluated at the National Institute of Respiratory Diseases, Mexico, between 1995 and 2004. The study was approved by the Ethics Committee of the National Institute of Respiratory Diseases, México. Diagnosis of IPF was made based on established criteria and confirmed by lung biopsy in 31% of the subjects Smoking status was characterized as “never”, “former” (patients who stopped smoking at least 12 month before presentation), or “current” (patients who were either still smoking or stopped smoking less than a year before presentation) Patients were treated with several regimens: prednisone, or prednisone plus azathioprine, or inhaled beclomethasone. Most patients also received colchicine. There were no differences between the type of initial treatment among both groups (data not shown).Seven healthy volunteers (43.4±11.9 years) were selected as controls for the gelatin zymography study, and five of them were assayed for fibroblast migration. Lungs from patients who died from non-respiratory causes (53.7±6.7 years) and from patients with chronic hypersensitivity pneumonitis (56.1±8.9 years) were used as controls for immunohistochemistry.2) levels were performed at the baseline and after 6 months follow-up using Wilcoxon rank-sum tests. A longitudinal test for group differences at 6 months controlling for baseline oxygen saturation, was performed using analysis of covariance (ANCOVA) to adjust for regression toward the mean.Pulmonary function tests, including spirometry, plethysmography, and arterial blood gases were performed as described elsewhere High resolution computed tomography (HRCT) was performed with 1.0- or 1.5-mm-thick axial sections taken at 1-cm intervals throughout the entire thorax and were reconstructed using a high spatial frequency algorithm. Between 20 and 25 CT images were acquired in each patient. HRCT scans were scored on a scale of 0-5 for ground glass attenuation, extent of septal thickening and honeycombing as described As part of the diagnosis process, bronchoalveolar lavage (BAL) was performed in 85 out of the 114 patients as described 2.Tissue samples were obtained by open lung biopsy in 8 from 26 “rapid” and 27 from 88 “slow” progressors. None of the patients had been treated with corticosteroids or immunosuppressive drugs at the time of biopsy. There was no mortality related to the surgical procedure and all patients were discharge from the hospital. One “rapid” progressor patient and two “slow” progressors showed surgical morbidity which included prolonged air leakage  and hemothorax in 1 “slow” progressor patient. Lung samples were fixed with 10% formaldehyde and handled routinely for light microscopy. A pathologist, blinded to the clinical data, scored the lesions from 0–2 : 1) extent of honeycomb; 2) hyperplasia of smooth muscle cells; 3) hypertensive changes; 4) extent of fibrosis 5) extent of interstitial inflammation; 6) hyperplasia of type 2 cells. The assessment was done as previously described 2BAR) (5 µg/ml)  and mouse anti-human prominin-1/CD133 (10 µg/ml) , were applied and samples were incubated at 4°C overnight. A secondary biotinylated anti-immunoglobulin followed by horseradish peroxidase-conjugated streptavidin  was used according to manufacturer's instructions. 3-amino-9-ethyl-carbazole  in acetate buffer containing 0.05% H2O2 was used as substrate. The sections were counterstained with hematoxylin. The primary antibody was replaced by non-immune serum for negative control slides.Lung tissue sections from 7 “rapid” progressors 8 “slow” progressors, 5 hypersensitivity pneumonitis, and three control lungs were treated as previously described 7 controls (1.5 µg of protein) were analyzed in 8.5% SDS-PAGE gels containing gelatin (1 mg/ml) and a final concentration of 0.3 mg/ml heparin as previously described To identify gelatinolytic activity, BAL fluid samples from 8 “rapid” progressors, 8 “slow” progressors and BAL fluid samples were 50× concentrated by lyophilization and solubilized in water. Aliquots containing 35 µg of protein were mixed with 2× Laemmli buffer (V/V) and separated on 10% SDS-polyacrylamide gels. Proteins were electroblotted onto nitrocellulose membranes . After blocking with 5% (w/v) non-fat dried milk in PBS, the membranes were incubated overnight at 4°C with polyclonal antibody against human adenosine A2B receptor , and monoclonal antibody against CD-133 . Membranes were subsequently washed, incubated with the corresponding secondary antibody conjugated to horseradish peroxidase  for 1 h at room temperature, and visualized with Enhanced Chemiluminescence (ECL) detection system  using radiograph film  according to the instructions of the manufacturer. Films were digitalized and quantified using image analysis software .http://www.dom.pitt.edu/paccm/genomics/ACC/index.htm.Lung samples from 4 “rapid” and 4 “slow” progressor patients were among the samples previously described by us http://compbio.cs.huji.ac.il/scoregenes/). For Data Mining and visualization, we used Genomica (http://genomica.weizmann.ac.il/) and Spotfire Decision Site 8.0 . Correction for only transcripts that had an Entrez Gene annotation was included in the analysis.Statistical analyses were performed as described To identify genes that best distinguish between “rapid” and “slow” progressors, we used the Threshold Number of Misclassification (TNoM) score 5 cells) were added to the upper chamber. The lower chamber contained 0.3 ml of medium with 5% BSA alone or with 50% BAL fluid. After incubation for 8 h at 37°C the non-migrating cells on the top of the chamber were scraped and washed. The migrating cells were determined according to manufacturer's instructions. Briefly, the cells were stained and the color was eluted with 300 µl of extraction buffer and aliquots of 150 µl were measured in an ELISA plate reader at 545 nm. The number of cells that migrated in absence of BAL was used as control (0% migration). All assays were performed in duplicate.Migration of normal human lung fibroblasts was performed using 24-well collagen-coated Boyden chambers  as described Data were analyzed with STATA software. In August 2005, we obtained the vital status of each patient by reviewing the clinical charts and using telephone or telegrams in all cases that had been lost to follow up. We obtained the vital status on 85% of the “rapid” progressors and of 80% of the “slow” progressors. For the remaining patients, their vital status was considered alive. Data are summarized using mean±SD, median, and range for continuous variables, and frequency and percentage for categorical variables.2 test for categorical variables. The time elapsed between the beginning of symptoms and the first consult either at our Institute or another Hospital was recorded. For survival analysis, “time zero” was defined in two ways: (1) from the beginning of symptoms as reported by the patient; (2) from the “index visit”, which was defined as the date the patient was first seen at the National Institute of Respiratory Diseases during the study period (1995 to 2004). Cumulative survival probabilities were estimated using the Kaplan-Meier method. In addition, a Cox proportional hazards regression model was used to compare survival as a function of several variables including age, gender, clubbing, forced vital capacity (FVC) as percentage of predicted, PaO2, SpO2 at rest and after exercise, smoking (current and cumulative as pack-years) and duration of symptoms before diagnosis. In all cases, two-tailed p values <0.05 were considered statistically significant.Patient demographics were compared between “rapid” progressors and “slow” progressors, using the two-sample, rank-sum test for continuous variables, Fisher's exact test and a chiIn univariate analysis of the whole cohort (n = 167), time elapsed between the beginning of symptoms, smoking, masculine gender, FVC%, PaO2, SpO2 at rest and during exercise, were significant predictors of mortality. In the multivariate Cox model, time elapsed between the beginning of symptoms and the first consult, smoking, masculine gender, and FVC%, remained significant.Analysis of survival of the whole cohort (n = 167) showed that patients consulting shortly after the beginning of symptoms  displayed a worse survival compared with those with ≥24 months of symptoms (n = 88) or intermediate   and the median survival was 27 months (CI: 13–41). For the “slow” progressors, the median follow-up time from beginning of symptoms was 60.5 months (CI: 52–69.4), and the median survival was 93 months (CI: 68–105). When examined from the time of diagnosis, the “rapid” progressors had a median follow-up time of 10 months (CI: 7.4–21), while the median survival was 25 months (CI: 10–38). For the “slow progressors”, the median follow-up time from diagnosis was 17 months (CI: 13–22) and the median survival was 32 months (CI: 25–42).No differences in age, lung functional alterations, oxygen saturation, extent of HRCT changes and BAL cell profile were found between the groups Table 1.2 versus 4.9±3.8 FF/cm2 from “slow” progressors (p = 0.2).Thirty one percent of the patients were biopsied (8/26 “rapid” and 27/88 “slow” progressors). None of the patients had been treated with corticosteroids or immunosuppressive drugs at the time of biopsy. Among those biopsied, no differences were found in the morphologic parameters explored: interstitial inflammation, pulmonary hypertension changes, smooth muscle hyperplasia, type 2 cell hyperplasia, and extent of fibrosis or honeycombing. Hyaline membranes were not observed. “Rapid” progressor patients showed 6.9±4.1 FF/cmWe obtained the vital status on 85% of the “rapid” progressors and of 80% of the “slow” progressors. For the remaining patients, their vital status was considered alive. As illustrated in 2 at rest. As shown in 2 between rapid and slow progressors at baseline (p = 0.90) but at 6 months follow-up, 75% of rapid progressors had oxygen saturation values below the median of slow progressors. ANCOVA model with 6-month oxygen saturation as response, and adjusting for baseline oxygen saturation revealed a significant difference between rapid and slow progressors (p = 0.027).At 6 months follow-up, no differences were found in FVC . However, rapid progressors showed a significant reduction of SpOGene expression profiles were obtained in 8 patients who fulfilled the criteria for “rapid” (n = 4) or “slow” (n = 4) progressors. Rapid progressors (59±6.5 years) were all male, one of them current and two former smokers. Slow progressors (61±7.0) were also male, and two of them were former smokers. These patients showed no differences in pulmonary function tests and BAL cell profile (data not shown). These subjects were included in a previous analysis of gene expression; however, the data presented here were not reported in the earlier study We could not find genes with a false discovery rate 2BAR and prominin-1, two highly upregulated genes in “rapid” progressor patients, was determined by immunohistochemistry in “rapid” and “slow” progressor IPF lungs as well as in hypersensitivity pneumonitis and normal control lungs. In IPF lungs from “rapid” progressors, the immunoreactive A2BAR protein was strongly expressed in reactive alveolar epithelial cells and fibroblasts (2BAR.The cellular source of AProminin-1/CD133 was detected mostly in the lungs of “rapid” progressor patients (5 from 7). The immunoreactive protein was observed in some areas of hyperplastic type 2 pneumocytes and attenuated alveolar epithelial cells covering fibroblastic foci as well in some areas of bronchiolar metaplasia  may play an important role in determining the eventual clinical phenotype, and that some modifiers genes are inducing a more aggressive IPF phenotype. We are aware that our study has limitations: the retrospective data collection, the dependence on patient reporting of the duration of symptoms, and the small number of tissues available for microarray analysis. However, the relatively large number of studied patients, our ability to confirm the results of gene expression by immunohistochemistry and the convincing BAL data, support the validity of these observations. Taken together with reports of the impact of acute exacerbations of IPF on morbidity and mortality, our results further highlight the variability in the progression and outcome of IPF. These findings may explain, in part, the difficulty in obtaining significant and reproducible results in studies of therapeutic interventions in patients with IPF Table S1Upregulated Genes in Rapid Progressors(0.44 MB DOC)Click here for additional data file.Table S2Downregulated Genes in Rapid Progressors(0.16 MB DOC)Click here for additional data file.