Strongyloides are important parasites of vertebrates including man. Currently, little is known about their germline organization or reproductive biology and how this influences their parasitic life strategies. Here, we analyze the structure of the germline in several Strongyloides and closely related species and uncover striking differences in the development, germline organization, and fluid dynamics compared to the model organism Caenorhabditis elegans. With a focus on Strongyloides ratti, we reveal that the proliferation of germ cells is restricted to early and mid-larval development, thus limiting the number of progeny. In order to understand key germline events , we monitored conserved histone modifications, in particular H3Pser10 and H3K4me3. The evolutionary significance of these events is subsequently highlighted through comparisons with six other nematode species, revealing underlying complexities and variations in the development of the germline among nematodes.Nematodes of the genus The online version of this article (doi:10.1007/s00412-015-0562-5) contains supplementary material, which is available to authorized users. Strongyloides are widespread small intestinal parasites of various vertebrates  to entomopathogenic and even plant parasitic  nematode models Caenorhabditis and Pristionchus spp. (Sommer and Bumbarger Strongyloides ratti (S. ratti) and the sheep parasite Strongyloides papillosus (S. papillosus) have been developed as model representatives of this genus , a facultative parasite of Australian possums  have been shown to undergo an unlimited number of consecutive free-living generations . In addition, all cells in the distal portion of the gonad, which is situated at the dorsal side of the adult worm, open into a common central rachis and form a large syncytium.The gonads in both sexes of the model nematode C. elegans, the appearance and organization of the germ cells differ greatly in S. ratti and other members of the Strongyloididae  to that of C. elegans , with 1C being the DNA content of a haploid set of chromosomes (Hammond and Robinson S. ratti inset 2), proximal to which presumably meiotic nuclei with condensed chromosomes can be observed . Further along the gonad, differentiated oocytes or sperm (depending on the sex) are present, very similar to C. elegans.While the overall morphology of the gonad is very similar to that of C. elegans, many aspects of the control of germline development and gene expression have been shown to act through chromatin modifications, with these being extensively studied and well characterized  and compare this with C. elegans. We confirm that in the adult gonad, the distal arm is occupied by giant nondividing polyploid nuclei, and that there are no mitotically proliferating stem cells here, despite the presence of a DTC-like cell in these species. Additionally, it appears that proliferation of the germ nuclei is restricted to early and mid-larval development. Further, we describe differences in the germline chromatin of Strongyloididae members  and C. elegans, by using conserved histone modifications with a particular emphasis on Histone3 phosphorylated at serine10 (H3Pser10) and Histone3 tri-methylated at lysine4 (H3K4me3). The variations in these histone modifications are further explored through comparisons with three other nematode species , each of which are at an increasing phylogenetic distance to S. ratti, unveiling further complexities in germline development among nematodes. Finally, we discuss the possible implications this has on regulating cell cycles and active transcription for Strongyloididae members.Here, we present the first detailed comparative characterization of the germlines of free-living males and females of Strongyloididae  OP 50 bacteria . Confocal stacks of the entire gonad were taken for manual counting of nuclei and processed in ImageJ (Fiji). For dissected gonad DAPI imaging, the same protocol was followed after gonad dissection.Adult worms (of the desired age) were fixed with ice-cold 100 % methanol (Roth GmbH and Co. KG) and directly mounted (without a rehydration series) on polylysine-coated glass slides in 10 μL of Vectashield  containing 1 μg mLWorms were collected by the Baermann funnel method every 2 h after hatching as described by Basir . Each wo4, 0.5 % UA, 0.5 GA, 2 % H20, warmed up to 0 °C at 4 °C h−1, 5× washed with pre-chilled acetone and infiltrated over a total period of 29 h with increasing concentrations of epoxy resin ; finally, the samples were cured at 60 °C for 48 h in flat embedding molds. Longitudinal semithin sections were stained with osmium tetraoxide and viewed in a FEI Tecnai G2 Spirit transmission electron microscope operating at 120 kV. Images were taken with a Gatan Ultrascan 4000 camera at maximum resolution using the manufacturer’s software.Samples were cryo-fixed with a Baltec HPM-010 high-pressure freezer and were freeze-substituted in a Leica AFS-2 according to following protocol: 56 h at −90 °C in acetone with 5 % gallic acid monohydrate , 3 h warmed up to −60 °C, 5× washed with pre-chilled acetone, 24 h at −60 °C with 2 % OsOStrongyloides spp., males and females of various stages were manually selected and put on plates seeded with labeled E. coli and incubated for 3, 6, 12, or 16 h . At the end of this time period, the larval stage the individuals had reached was scored and the germlines were dissected and used for antibody staining. Young Strongyloides larvae with prolonged exposure to labeled E. coli plates (<16–18-h exposure) were found to be visibly very sick or dead.5-Bromo-2′-deoxyuridine (BrdU) labeling of germlines was done as described by Crittenden et al. , and dis−1 DAPI (Roche). The following primary antibodies were used at the indicated dilutions: Anti-BrdU-FITC labeled , rabbit H3K9/K14ac , rabbit H4K20me1 , mouse H3K27ac , mouse H3K27me3 , rabbit H3K9me1 , rabbit H3Pser10 , mouse H3K4me3 , and rabbit α-tubulin (1:200), rabbit α-Actin (1:200). The following secondary antibodies were used for visualization: Goat anti-rabbit labeled with Alexa Fluor 488 (1:200), goat anti-mouse labeled with Cy3 (1:200), and mouse anti-biotin labeled with Cy3 (1:200).Gonads from adult worms were dissected in egg buffer  containing 0.1 % Tween and immediately fixed in 1 % paraformaldehyde for 5 min. Slides were frozen in liquid nitrogen, freeze-cracked, and then immersed for 1 min in methanol at −20 °C and transferred to PBS-Triton X-100 . Blocking in 0.7 % BSA in PBS-Triton X-100 was then carried out for 1 h. Primary antibodies were incubated overnight at room temperature, slides were then washed three times for 10 min in PBS-Triton X-100, and secondary antibodies were added and incubated for 4–6 h at room temperature. Following three washes for 5 min in PBS-Triton X-100, the slides were counterstained in 10 μL of Vectashield (Vector Laboratories Inc.) containing 1 μg mLAll images were acquired as a stack of optical sections with an interval of 0.65 μm using an Olympus confocal FV1000 microscope and processed in ImageJ (Fiji) and Adobe Photoshop CS.5.w/v dextran sulfate. Fifteen microliters of this mix was added per slide followed by heating the slides at 93 °C for 2 min in a heated block. The slides were then removed and incubated overnight at 37 °C in a humid chamber. For post-hybridization washes, the slides were rinsed in 2× SSCT 50 % formamide at 37 °C for 30 min. This was followed by three washes in 2× SSCT for 5 min each. Then, the slides were blocked in 1 % BSA in 2× SSCT for 30 min. Then, 50 μL of the primary H3Pser10 antibody was added per slide and incubated overnight at room temperature in a humid chamber. Then, slides were washed in 2× SSCT, three times for 10 min each, and incubated with 50 μL of the labeled secondary antibody (1:200) for 4–6 h. Finally, the slides were washed three times in 2× SSCT for 10 min each and were counterstained in 10 μL of Vectashield (Vector Lab. Inc.) containing 1 μg mL−1 DAPI (Roche) and ready for imaging using the Olympus confocal FV1000 microscope.Gonads from adult worms were dissected in egg buffer  containing 0.1 % Tween and immediately fixed in 1 % paraformaldehyde for 5 min. Slides were frozen in liquid nitrogen, freeze-cracked, and then immersed for 1 min in methanol at −20 °C and then transferred to 2× SSCT three times for 5 min each. Then, the slides were dehydrated for 3 min each in 70, 90, and 100 % ethanol after which the slides were left to air-dry. After air-drying, the slides were ready for adding the hybridization mix containing the florescent in situ hybridization (FISH) probes. The amount of probe per slide was between 150 and 200 ng. The final concentration of the hybridization mix after adding the probe was 2× SSCT, 50 % formamide, 10 % Probes (22–25 bp) were ordered from Eurofins MWG Operon (after ensuring preferably a single hit in the genome on the required chromosome). The ready-made probes were labeled internally with biotin along their entire length. They were visualized using a mouse monoclonal anti-biotin Cy3 antibody from Sigma-Aldrich.Strongyloides spp., implying that these organisms do not maintain a population of proliferating stem cells in their adult germlines. In order to test this hypothesis, we first performed a detailed electron and light microscopic characterization of the distal gonad of S. papillosus adult females  found no indication for mitotic activity in any part of the germline, for either S. papillosus males or females. Nevertheless, a DTC was observed, which assumed a somewhat different position than in C. elegans or Pristionchus pacificus  incorporation experiments (BrdU uptake indicates actively cycling cells in S phase) for these species with C. elegans as a control. We first came up with a schematic for how the germline grows in these species, by correlating the development of the gonad at different larval molts under the differential interference contrast (DIC) microscope and with DAPI staining .As the central rachis and syncytium is so prominent in the structure of the lin Fig. , we founC. elegans, there is a constant flow of cytoplasm from the distal tip toward the proximal end of the gonad, indicating active transport. If small lipid droplets are injected at any place in the distal gonad, they are transported proximally via the central rachis in the gonad, where-upon they are incorporated into growing oocytes  been shown to be meiosis specific. We stained dissected gonads of S. papillosus and S. ratti (both sexes) with anti-RAD-51 . For the moment, it must remain open if RAD-51, REC-8, and SMC-3 are indeed not present in Strongyloides gonads or if the heterologous antibodies we used are not suitable to detect these proteins in situ for these species. In order to overcome this problem, we focused on antibodies against conserved histone modifications  and the other marking transcriptionally active chromatin regions , respectively . Nevertheless, it was detected in the band of small compact nuclei, in a diffused and weak pattern . In comparison, a very strong H3K4me3 signal was seen in the giant nuclei along the entire distal arm and in the small compact nuclei in female S. ratti. Additionally, it appears that H3K4me3 assumes a subnuclear localization that was mutually exclusive to the H3Pser10 signal in the small compact nuclei . By contrast, in C. elegans, H3K4me3 was weak but detectable at the distal tip and fairly strong in pachytene nuclei in accordance to previous literature . These species differ in their gonad organization such that S. papillosus, P. trichosuri, and Rhabditophanes KR3021 resemble the gonad organization in S. ratti, while Panagrolaimus PS1159 and Panagrellus PS1163 are similar to C. elegans. Additionally, these species are of varying phylogenetic distance to S. ratti, while being equally distant from the clade V nematode C. elegans , then they did not show any H3Pser10 staining in the distal gonad . Three  of the five clade IV species showed H3Pser10 staining in the gonadal loop region, just like in S. ratti . Even though we used roughly age-matched worms  for these analyses, it must be noted that we did find indications in S. papillosus that staining patterns with H3Pser10 depended at least partially on age . In S. papillosus males, H3Pser10 stained the entire nucleus evenly, with this staining being much stronger in the proximal part of the band of small nuclei , just like our observation in S. papillosus females. In P. trichosuri, condensed spermatogenetic chromosomes proximal to the small nuclei stained strongly for H3Pser10, which was never observed in either of the two species of Strongyloides . Interestingly, in parasitic S. ratti and S. papillosus females, we found H3K4me3 and H3Pser10 staining patterns very similar to the ones in free-living males  showed only weak stainings  are thought to perform this role  yield any indication of active DNA replication in adults. Although we have consistently observed H3Pser10 staining in the small nuclei at the gonadal loop of Strongyloides members, we think that this staining is not indicative of nuclear divisions in these species. This is a major difference compared to other systems including C. elegans, where H3Pser10 staining is directly indicative of cells in M-phase, never staining cells in S-phase  and produce only a few dozen progeny  in these species. The DTC in C. elegans  known to be highly active transcriptionally (Schaner and Kelly Strongyloides and other clade IV nematodes (with the notable exception of P. trichosuri) might hint that the distal tip is a place of high transcriptional activity in these nematodes. The high presence of other transcriptional markers like H3K9/K14ac and H3K27ac at the distal tip  provides further evidence for this. The absence of H3K4me3 in P. trichosuri is surprising and might possibly indicate that this modification is not a mark for active transcription in the giant nuclei of this species.In Panagrellus and Panagrolaimus) and C. elegans, H3Pser10 seems to be specific for dividing nuclei, implying that this role of H3Pser10 in the distal gonad may be conserved. In species without dividing cells in the distal gonad; we consistently did not observe any H3Pser10 staining in this part of the gonad. Surprisingly, in S. ratti males, H3Pser10 specifically marks a portion of the spermatogenic nuclei and based on the limited number of loci that we tested by FISH, this seems to be specific to the X-chromosome. Although some sperm without an X-chromosome (nullo-X sperm) exist in S. ratti , H3Pser10 marks both the condensed meiotic X-chromosomes and the autosomes alike. We never noted this modification on condensed meiotic chromosomes in either S. ratti or S. papillosus males. These striking and varied differences in H3K4me3 and H3Pser10 stainings within the three closely related representatives of the Strongyloididae suggests that this group of nematodes has either recently undergone, or is still in the process of undergoing rapid evolutionary changes. This in turn may be a response to becoming parasitic and/or parthenogenetic (in the parasitic generation). Overall, due to the current limitation in knowledge and tools for Strongyloides species, we must assume that the differences between Strongyloides spp. and C. elegans with regard to the different histone modifications are probably due to a combination of stochastic changes caused either by drift or because of adaptations that have accumulated over the time of their phylogenetic separation . Nevertheless, from our comparative observations of the staining patterns with C. elegans, it appears likely that the small compact nuclei at the gonadal loop in Strongyloides spp. represent nuclei in some stage of meiotic development. If this is true then this small region is equivalent to the extended meiotic zone in C. elegans . This conclusion is supported by two observations: firstly, there is no other region in the gonad of these species which could be meiotic  and secondly, the Strongyloides species we studied are known for their normal diploid genetics  that we have described transform into one another over reproductive life. Due to current technological limitations with working on these species, all our observations so far have been limited to fixed specimens /Parastrongyloides spp. (facultative parasite)/Strongyloides spp. (obligate parasite with a single free-living generation) in comparative studies. Given their multiple advantages, such systems could be used in the future to address questions about the emergence of parasitic lifestyles and the evolutionary consequences of becoming parasitic. Finally, novel insights into germline regulation during development may only be uncovered by looking into atypical species.Our findings presented here enhance our understanding of the biology of a group of fascinating but poorly understood parasites. Additionally, they also illustrate the usefulness of the SupplFig. 1A. DIC image (left) showing a S. ratti larva undergoing the L1 – L2 molt. The gonad is outlined in white in the body of the larva. The Inset (to the right) shows the corresponding DAPI staining (magnified view of the gonad) at L1 – L2 molt showing active proliferation of the germline. B. Dissected gonad from S. ratti L3-stage larva as control showing incorporation of BrdU in both giant and small nuclei . Scale bar 10 μm. Notice that in gonads of worms exposed to BrdU as adults the BrdU signal is seen in the cytoplasm but excluded from the nuclei SupplFig. 2A. Western blots for SMC-3, REC-8 and RAD-51 in P. trichosuri, S. ratti and S. papillosus adult worms (whole worm lysates) showing a single band at expected positions for each, with alpha-Tubulin used as loading control. B. RAD-51 staining (in green) in combination with H3K4me3 (in red) in the S. ratti male germline showing no RAD-51 in the distal gonad, the region with the giant nuclei (top) and no meaningful pattern in the band of small nuclei at the gonad loop (bottom). Much of the RAD-51 signal received here  is probably background noise, given that it was not in the same focal plane as the germ line nuclei . C. A comparison of H3K4me3 and H3Pser10 stainings in the distal gonads (region containing giant nuclei) in the adult males of S. ratti, S. papillosus and P. trichosuri . Note the lack of H3Pser10 staining in S. ratti and S. papillosus in this region, but the presence of H3K4me3. For P. trichosuri, there is a complete lack of both H3K4me3 and H3Pser10 in this region. This pattern is similar to the stainings obtained in the females for each species. Scale bar 10 μm. (GIF 181 kb)High resolution image (TIF 90110 kb)SupplFig. 3A. Western blots for H3Pser10 and H3K4me3 in P. trichosuri, S. ratti and S. papillosus adult worms (whole worm lysates) showing a single band at expected positions, with alpha-Tubulin used as loading control. B. H3Pser10 antibody staining of dissected young (bottom) and old (top) S. papillosus female gonads. The term ‘old’ is used here to indicate a mated female , whereas young is before the L4-adult molt. H3Pser10 is briefly seen on condensed chromosomes at the onset of mating, and from then on in the small nuclei, illustrating possible age related staining patterns in this species. (GIF 56 kb)High resolution image (TIF 42887 kb)SupplFig. 4S. ratti and S. papillosus females. (Top) Distal and gonadal loop panels for S. ratti parasitic female. Inset (bottom) shows a zoom in marked in white in the merge panel. Note the mutually exclusive localization of H3Pser10 and H3K4me3. (Bottom) Distal and gonadal loop panels for S. papillosus parasitic female. Inset (bottom) shows a zoom in marked in white in the merge panel. Note the even localization of H3Pser10 and H3K4me3. (GIF 124 kb)H3Pser10 and H3K4me3 staining in adult parasitic High resolution image (TIF 47892 kb)SupplFig. 5S. ratti germ line. A. Distal gonad panels  from dissected male and female gonads of S. ratti adults stained against transcription activation markers H3K9/K14ac (top 2 panels) and H3K27ac (bottom 2 panels) in combination with H3K4me3 and H3Pser10 respectively. B. Corresponding gonadal loop panels for these males and females stained against the same antibodies. Scale bar 10 μm. (GIF 137 kb)Histone modifications marking active transcription in the High resolution image (TIF 55882 kb)SupplFig. 6S. ratti germ line. A. Distal gonad panels from dissected male and female gonads stained against H3K27me3 (top 2 panels), H3K9me1 (middle 2 panels) and H4K20me1 (bottom 2 panels) in combination with H3Pser10 or H3K4me3 respectively. B. Corresponding gonadal loop panels in these males and females for these same antibodies. Scale bar 10 μm. (GIF 218 kb)Histone modifications marking silencing in the High resolution image (TIF 90110 kb)Below is the link to the electronic supplementary material.


It can be used to assess individuals, journals and universities. Unlike most other citation-based-indicators it equalizes the chances of young scientists (vs. their more experienced colleagues) and of scientists working in less-popular disciplines. The ranking of scientists based on the new indicator is not correlated with the rankings based upon the number of citations or on the Hirsch-index.A new simple bibliometric indicator is based on the number of highly cited papers (as defined by WoSThe online version of this article (doi:10.1007/s11192-017-2526-4) contains supplementary material, which is available to authorized users. Since the seminal work of Garfield  the scieOn top of the total number of citations, many other citation-based indicators have been considered  and of hot papers  from any set of papers. Most hot papers are also highly cited papers, but only a few highly cited papers are also hot papers. The highly cited papers and hot papers have been marked as such also in older versions of the users’ interface of WoS®, but till June 2017 they could only be extracted manually, and this extraction was tedious in large sets of data. Nevertheless, numerous studies were devoted to different aspects of highly cited papers.In June 2017 WoSNoorhidawati et al.  observedDocampo and Cram  studied ®  and of scientists working in less-popular disciplines (their papers need fewer citations to be highly cited). The properties of the new indicator are studied using a set of papers from one city.Let us define a successful paper as a highly cited paper according to the above definition by WoS® core collection was accessed on July 3, and on July 17, 2017. The results were stored, and they were processed afterwards. We are interested in papers with “Lublin” in their address field. Lublin is a city in Poland with 5 large universities, one large research institute, and several medical facilities, which also perform research work. The present author lives in that city and knows many top-scientists from that city personally.The WoSThe scientists who have published at least one highly cited paper or at least one hot paper are characterized by their scientific degree. In principle there are four categories of degrees in Poland, which were assigned the following values: 3 (professor), 2 (Dr.Sci.), 1 (Ph.D.), and 0 (M.Sci.). Foreign citizens who worked in Lublin as post-docs, and authored papers under Polish affiliation are counted as Ph.D.h-index. Those were taken from Scopus. The advantage of Scopus versus WoS® is in easier extraction of certain individual from a set of homonymous authors.The scientists who have published at least one highly cited paper or at least one hot paper are characterized by their total number of citations and their u1, u2, u3, u4 and u5, a research institute marked as i1, and one of the medical facilities, marked as m1 and m2. The scientists who have published at least one highly cited paper or at least one hot paper are characterized by their gender. The association of individuals with institution and with gender (when necessary) was based upon the POLON system, which is a central database of Polish scientists, and on the Web pages of their institutions.The scientists who have published at least one highly cited paper or at least one hot paper are characterized by their institution, which is one of the top universities in Lublin, marked ® in 2007 and about 2400 in 2016, and the number of papers increased linearly over the above period. Thus the number of papers under analysis was about 18,000 in highly cited, and about 4700 in hot papers. Out of these papers, 52 were highly cited, and 4 were hot on July 3, and 54 were highly cited, and 2 were hot on July 17. These numbers are substantially lower than 1% of 18,000, and lower than 1‰ of 4700, which are the expected numbers in a random set of the same size of papers from the database. This result is not surprising, because the scientific institutions of Lublin do not belong to the top universities of the world, and Pareto-type distribution of highly cited papers among the scientific institutions is expected. The number of hot papers from Lublin dropped by a factor of 2 in 2 weeks. This result is also in accordance with the expectations, namely the oldest papers in the set of 4700 have the highest chance of being hot (because they had sufficient time to collect many citations), but they are also sooner removed from the set (of papers which are less than 2 years old).The scientists from Lublin published about 1300 papers indexed in WoSThe results shown here demonstrate an extreme instability in time of any scientific indicator based upon hot papers. However such an indicator can still be used in assessment of top universities in the world, which produce hot papers by dozens  or of top journals . As we focus on papers from Lublin in this paper, from now on we will discuss mainly highly cited papers, which are more stable in time, and will mention hot papers only occasionally. The highly cited papers from Lublin are analyzed one by one, and by author .The highly cited papers from Lublin are sorted by publication year in Table The distribution by year shown in Table The highly cited papers from Lublin are sorted by research area in Table The other research areas received 2 or less of highly cited papers from Lublin each. Table The highly cited papers from Lublin are sorted by journal impact factor (for 2016) in Table Interestingly enough, a substantial fraction of highly cited papers appeared in relatively low-IF-journals. Thus an assessment of publications discussed in this paper is weakly correlated with the assessment of publications by the journal IF.The highly cited papers from Lublin are sorted by the number of authors in Table Most highly-cited papers from Lublin have 10 authors or more. This problem is faced in most citation-based indicators, namely many successful papers are co-authored by numerous individuals, and the actual contribution of certain individual may be very little. The co-authors of certain paper can be from the same scientific institution, but they can very well be from different institution, different city or even from different country. The fraction of papers authored by 10 or more is much higher than the aforementioned analogous fraction of highly cited papers from Malaysia. On the other hand mega-authorship (1000 + authors) which contributed in 6% to highly cited papers from Malaysia was completely absent in highly cited papers from Lublin (the highest number of authors was 60).Let us introduce a fractional authorship from Lublin  as the number of co-authors from Lublin divided by the total number of co-authors. The present definition is different from that by Pislyakov and Shukshina  who perfMost highly-cited papers from Lublin have very low (< 0.2) fractional authorship from Lublin. Many highly-cited papers from Lublin have only authors from Lublin. Relatively few highly-cited papers from Lublin have relatively high (0.2–0.99) contribution of authors from Lublin, but they also have co-authors from other cities/countries. Table Given that most highly cited papers are multi-author papers, the assessment of individuals and or of institutions based on highly cited papers is considered in two versions. In the first version each co-author takes the full credit for the entire paper. In the second version each co-author takes the only a reciprocal number of authors of a paper as a credit for the paper. Equal credit for each co-author is one of possible solutions, and other solutions (for example the first author receives more credit than the other authors) have been also considered in the literature, but detailed discussion of this problem is outside the scope of the present paper.s1, s2, … and they are sorted by the total number of citations.Achievements of authors of highly cited papers from Lublin are summarized in Table S1 in the Online Supporting Material. On top of the authors of highly cited papers, a few top scientists from Lublin (still active), who are not authors of highly cited papers are included for comparison. The scientists are marked h-index, and scientific degree, which are over 0.9 (citations vs. h-index), and about 0.8 , respectively. For example all scientists with > 2000 citations, and all scientist with h > 25 are professors. Table S1 shows a high degree of gender disparity in the number of citations, and in the h-index. For example all scientists but one with h > 25 are male.Table S1 shows that most authors of highly cited papers have rather moderate achievements in terms of their scientific degrees, their total numbers of citations and of their Hirsch index. Holders of M.Sci. and Ph.D. (without habilitation) are commonplace. This should be emphasized that the total number of holders of a professor degree associated with scientific institutions of Lublin exceeds 500, and total number of holders of habilitation in Lublin (excluding professors) exceeds 1200. These numbers are higher by an order of magnitude than the number of highly cited papers from Lublin. Most authors of highly cited papers have fewer than 1000 citations, which is very little in comparison with the highest numbers indicated in the top part of Table S1. Table S1 shows a high degree of rank–rank correlation between the number of citations, u1, 20 scientists represent u2, 3 scientists represent u3, 5 scientists represent u4, 1 scientist represents u5, 3 scientists represent i1, 2 scientists represent m1, and 1 scientist represents m2. These figures are nearly proportional to the overall scientific strengths of particular institutions as expressed by their total numbers of citations, except the achievements of u1 are slightly overrated.The assessment of scientists by the number of highly cited papers as studied on July 3 is presented in Table S2 in the Online Supporting Material. The scientists are ordered by the fractional number of highly cited papers . There are 21 professors, 20 Drs.Sci., 17 Ph.Ds, and 6 Ms.Sci. among the authors of highly cited papers, and 3 Drs.Sci., 3 Ph.Ds, and 1 M.Sci. among the authors of hot papers (no single professor!). 29 scientists represent Most scientists listed in Table S2 authored only one highly cited paper each, with the exception of one  scientist who authored 9, one scientist who authored 3, and 6 scientists who authored 2 highly cited papers each. The achievements in terms of highly cited papers show little correlation with the achievements of the same scientist in terms of their scientific degrees, their total numbers of citations and of their Hirsch index as shown in Table Table u1, 16 scientists represent u2, 4 scientists represent u3, 5 scientists represent u4, 1 scientist represents u5, 3 scientists represent i1, 2 scientists represent m1, and 2 scientists represent m2. The achievements of u1 in terms of highly cited papers are substantially overrated with respect to other institutions as compared with the ranking based on overall scientific strength of this university as expressed by its total number of citations.The assessment of scientists by the number of highly cited papers as studied on July 17 is presented in Table S3 in the Online Supporting Material. The scientists are ordered by the fractional number of highly cited papers. There are 23 professors, 19 Drs. Sci., 18 Ph.Ds, and 5 Ms. Sci. among the authors of highly cited papers, and 2 Ph.Ds among the authors of hot papers (no single professor or Dr. Sci.!). 32 scientists represent Most scientists listed in Table S3 authored only one highly cited paper each, with the exception of one scientist who authored 9 papers, two scientists who authored 3 papers each, and 5 scientists who authored 2 highly cited papers each. The ranks of particular scientists in Tables S2 and S3 are relatively consistent with very few exceptions. S15 advanced from rank 42 on July 3 to rank 5 on July 17 in fractional count of highly cited papers, and s34 who did not have any highly cited paper on July 3 advanced to rank 12 on July 17 in fractional count of highly cited papers. These few exceptions show that the position of particular scientists in the ranking can change substantially just in 2 weeks.The achievements in terms of highly cited papers show little correlation with the achievements of the same scientist in terms of their scientific degrees, their total numbers of citations and of their Hirsch index as shown in Table h scientists.Table ® to another. The rankings of scientists based upon highly cited papers are not correlated with the rankings based upon the academic degrees, numbers of citations or on the Hirsch-indices. The rankings of scientists based upon highly cited papers reduce the inherent advantage of old scientists and of scientists working in more-popular disciplines, which is the case with total number of publications, total number of citations and with the Hirsch-index. The rankings of scientists based upon highly cited papers suffer several shortages, which are common for most citation-based indices, e.g., they can be manipulated by excessive self-citations.The count of highly cited papers  can be used in assessment of scientific performance of individuals. The rankings based upon highly cited papers are extremely unstable in time, and the positions of individuals can substantially vary from one update of the list highly cited papers by WoSSupplementary material 1 (DOC 388 kb)Below is the link to the electronic supplementary material.


Idiopathic membranous nephropathy (MN) is an autoimmune-mediated glomerulonephritis and the most common cause of idiopathic nephrotic syndrome in adult humans. A tumor necrosis factor α (TNF-α)-mediated inflammatory response via TNF receptor 1 (TNFR1) and TNFR2 has been proposed as a pathogenic factor. In this study, we assessed the therapeutic response to blocking TNF signaling in experimental MN. Murine MN was induced experimentally by cationic bovine serum albumin (cBSA); phosphate-buffered saline was used in control mice. In MN mice, TNF was inhibited by etanercept blocking of TNFR1/TNFR2 or the preligand assembly domain fusion protein (PLAD.Fc), a small fusion protein that can preferentially block TNFR1 signaling. Disease severity and possible mechanisms were assessed by analyzing the metabolic and histopathology profiles, lymphocyte subsets, immunoglobulin production, oxidative stress, and apoptosis. cBSA-induced MN mice exhibited typical nephrotic syndrome and renal histopathology. MN mice given etanercept or PLAD.Fc did not exhibit significant reduction of proteinuria, amelioration of glomerular lesions, or attenuation of immune complex deposition. Immune cell subsets, serum immunoglobulin levels, production of reactive oxygen species, and cell apoptosis in the kidney were not altered by TNF inhibition. By contrast, MN mice receiving etanercept or PLAD.Fc exhibited significantly decreased infiltration of immune cells into the kidney. These results show that the therapeutic effects of blocking TNFR1 and/or TNFR2 signaling in experimental MN are not clinically effective. However, TNF signaling inhibition significantly attenuated renal immune cell infiltration in experimental MN. Idiopathic membranous nephropathy (MN) is the most common cause of idiopathic nephrotic syndrome in adult humans and 30–40% of patients with MN will ultimately progress into end-stage renal failure after 10–15 years . During TNF-ɑ is a pleiotropic cytokine produced mainly by immune cells such as macrophages, dendritic cells, and T lymphocytes, and is implicated in immune regulation –12. TNF Previous results of blocking TNF as an MN treatment are conflicting , 18. SimThe cBSA-induced MN mice exhibited typical heavy proteinuria. MN mice from the groups given etanercept or PLAD.Fc did not exhibit significant reduction in proteinuria. No proteinuria was noted in NC mice receiving etanercept or PLAD.Fc Figure .H&E staining showed typical renal histopathology of diffuse thickening of the glomerular basement membrane in the cBSA-induced MN mice. MN-etanercept or PLAD.Fc mice exhibited a similar severity in pathology Figure ; by contMN mice exhibited significant granular immunofluorescence staining for IgG, which showed as a discrete beaded appearance along the glomerular capillary wall. This pattern was similar in the MN- etanercept and MN-PLAD mice, which suggested that inhibition of TNF did not change the deposition of immune complexes Figure . Immunof+ T cells, CD8+ T cells, and CD19+ B cells did not differ significantly between mice in the NC, MN, MN-etanercept, and MN-PLAD groups . MN mice showed an increased number of apoptotic cells in the kidneys compared with NC mice, and inhibition of TNF treatment did not attenuate this effect. This finding suggested that the antiapoptotic effect of TNF inhibition does not have a therapeutic effect against MN.+ T cells , and MN group (D–L) receiving PBS (D-F), etanercept  and PLAD.Fc . Subgroups of immune cells including CD4s Figure , CD8+ T s Figure , and F4/s Figure  were anaMN mice in groups receiving etanercept or PLAD.Fc did not exhibit significant reduction of proteinuria, amelioration of glomerular lesions, or attenuation of immune complex deposition. There were also no effects on the activation of complement, immune cell subsets, serum Ig level, superoxide level, or degree of apoptosis in kidney tissue. However, significantly less infiltration of immune cells was noted in MN mice receiving etanercept or PLAD.Fc. These results show that the therapeutic effects of blocking TNFR1 and/or TNFR2 signaling in experimental MN are not effective. The role of attenuated immune cell infiltration in the kidney has not been determined.In our study, TNF blocking did not improve the clinical outcomes of MN, and the only effect was a decrease in immune cell infiltration in the kidney as shown histologically. The deposited immune complexes subsequently induce complement activation, and oxidative injury, are involved during MN disease progression –6. BecauExperimental models of glomerulonephritis have shown that TNF-ɑ has contrasting proinflammatory and immunosuppressive roles in the kidney. It appears that TNFR1 plays a predominantly proinflammatory role, whereas TNFR2 plays an important role in disease immunoregulation , 22. InhTNF-ɑ is synthesized predominantly by infiltrating T cells and monocytes/macrophages. This cytokine can amplify the renal inflammatory response via increasing the expression of adhesion molecules and inducing the cells to release growth factors, other cytokines, and proinflammatory chemokines in an autocrine and paracrine manner , 21, 22.Although TNF-ɑ has both immunosuppressive and proinflammatory properties, it is not simple to predict the effect of anti-TNF-ɑ treatment in MN , 21, 22.Several factors differentiate MN from other clinical entities and might be involved in the absence of effects in the present trial. It is possible that a longer course of etanercept from an earlier stage of MN would have been clinically effective. The treatment used in our study was initiated very early at the start of MN and lasted the whole process of the disease, but was not effective in preventing the clinical signs of MN. These data do not support the role of TNF in MN and may indicate that the TNF may be a responder rather than a contributor to the MN process. By contrast, pentoxifylline treatment in a similar model significantly decreased proteinuria and caused a high rate of remission, as shown by decreased plasma and urinary TNF-ɑ levels . AlthougA diversity of effects has been reported across various autoimmune diseases, which involve different pathogenic mechanisms and the involvement of organs and/or systems. Anti-TNF-ɑ treatments demonstrate great benefit in autoimmune diseases such as rheumatoid arthritis and Crohn’s disease. There is currently no validated indication for anti-TNF-ɑ treatments in kidney diseases. Some studies have suggested benefits of anti-TNF-ɑ treatments to improve the extent of renal damage in antineutrophil cytoplasmic antibody-associated systemic vasculitis . Some daA previous study showed that 3 months of therapy with etanercept was safe and well tolerated by MN patients . Anti-TNPLAD occurs in the region overlapping the first cysteine-rich domain of P60 and P80 TNFRs and Fas, which are sufficient for mediating specific ligand-independent receptor assembly and subsequent signaling –25. TargUrinary TNF-ɑ excretion correlates with proteinuria in patients with MN and TNF-ɑ gene polymorphism is a risk factor for development of MN. One study demonstrated that the circulating level of the TNFR at the time of initial diagnosis may provide a biomarker for the prediction of renal progression in patients with MN . FurtherThe develop an ideal therapeutic agents effectively and specifically for MN treatment is an important issue to nephrologists. The therapeutic effects of anti-TNF have been demonstrated in many human inflammatory and autoimmune diseases such as rheumatoid arthritis; however, our results did not show therapeutic effects in MN. We assume that anti-TNF does not affect the key pathogenic responses such as the production of Igs and subsequent immune complex deposition, inflammation, complement activation, oxidative stress, and apoptosis. We conclude that anti-TNF may not be a suitable MN treatment.All experiments were conducted according to the National Institutes of Health Guidelines, and all animal experiments were approved by the Animal Care and Use Committee of the National Defense Medical Center, Taipei, Taiwan (IACUC-11-149).n = 5 in each subgroup), each of which received one of three treatments: intravenous injection of TNFR preligand assembly domain fusion protein  twice a week; subcutaneous injection of etanercept  once a week ; or saline  since the time of MN induction. Homogeneous cBSA, construction of the plasmid and purification of the recombinant PLAD.Fc fusion protein were prepared as previously described [The experimental MN model was induced as previously described –37. The escribed , 36–39. Urine samples were obtained for proteinuria screening using Labstix . Samples were group into five grades based on urine protein concentrations: 0 = 0-30 mg/dL, 1+, 30–100 mg/dL; 2+, 100–300 mg/dL; 3+, 300–1000 mg/dL; 4+, ≥ 2000 mg/dL.Renal tissue samples were prepared by snap-freezing, then fixed with acetone for immunofluorescence (IF) or 10% formalin for hematoxylin and eosin (H&E) and immunohistochemistry (IHC). HE staining, IF and IHC were performed as previously described –37. FluoSplenocytes were isolated and stained with marker-specific antibodies: allophycocyanin (APC)-conjugated anti-mouse CD4, phycoerythrin (PE)-conjugated anti-mouse CD8, and FITC-conjugated anti-mouse CD19. All monoclonal antibodies were purchased from BD Biosciences  or eBioscience . The stained cells were analyzed by a FACSCalibur cell sorter and CellQuest software .ROS production was determined with dihydroethidium  labeling, as described previously –37. BrieIn Situ Cell Death Detection Kit  as previously described [Apoptosis was assessed using TUNEL assay and escribed –37. BrieData are expressed as mean ± standard deviation. The data were analyzed using one-way analysis of variance for multiple comparisons, and the Bonferroni test was used to correct for between-group differences. Significance (p ≤ 0.05) was ascertained using SPSS/PC .


The management of comorbidity and multimorbidity poses major challenges to health services around the world. Analysis of scientific research in comorbidity and multimorbidity is limited in the biomedical literature. This study aimed to map global scientific research in comorbidity and multimorbidity to understand the maturity and growth of the area during the past decades.This was a cross-sectional analysis of the Web of Science. Searches were run from inception until November 8, 2016. We included research articles or reviews with no restrictions by language or publication date. Data abstraction was done by one researcher. A process of standardization was conducted by two researchers to unify different terms and grammatical variants and to remove typographical, transcription, and/or indexing errors. All potential discrepancies were resolved via discussion. Descriptive analyses were conducted . Network analyses of collaborations between countries and co-words were presented. During the period 1970–2016, 85994 papers (64.0% in 2010–2016) were published in 3500 journals. There was wide diversity in the specialty of the journals, with psychiatry , surgery , clinical neurology , and general and internal medicine  the most common. PLOS One , the Journal of Affective Disorders , the Journal of Clinical Psychiatry , the Journal of the American Geriatrics Society  and Obesity Surgery  published the largest number of papers. 168 countries were involved in the production of papers. The global productivity ranking was headed by the United States (37624 papers), followed by the United Kingdom (7355 papers), Germany (6899 papers) and Canada (5706 papers). Twenty authors who published 100 or more papers were identified; the most prolific authors were affiliated with Harvard Medical School, State University of New York Upstate Medical University, National Taiwan Normal University and China Medical University. The 50 most cited papers (“citation classics” with at least 1000 citations) were published in 20 journals, led by JAMA Psychiatry (11 papers) and JAMA (10 papers). The most cited papers provided contributions focusing on methodological aspects , but also important studies on chronic diseases .Ours is the first analysis of global scientific research in comorbidity and multimorbidity. Scientific production in the field is increasing worldwide with research leadership of Western countries, most notably, the United States. Over the last three to four decades, substantial progress has been made toward reducing mortality and extending life expectancy worldwide ,2. AlthoAnalysis of scientific research in comorbidity and multimorbidity is limited in the biomedical literature –16. For The scientific landscape has changed considerably in the subsequent years, including the launch of important initiatives for the clinical management of multiple chronic diseases –20, but th 2016. The Web of Science has been considered the world’s leading taxonomic reference for citation analysis and prior to 2004, the only data source on citations available [We conducted a cross-sectional analysis of the Web of Science, Science Citation Index-Expanded (SCI-E) database, from inception to November 8vailable . The seavailable . The seaThe terms of “comorbidity” and “multimorbidity” are often used interchangeably. Many possible definitions and interpretations of the concepts of “comorbidity” and “multimorbidity” have been reported in the biomedical literature –11. For Comorbidity. A widely accepted definition of “comorbidity” is the occurrence or the existence of any distinct additional medical condition to an index disease [ disease . In gene disease ,10.Multimorbidity. Most authors define “multimorbidity” as the co-occurrence of two or more medical conditions in an individual without any reference to an index disease [ disease ,9–11. ThSome authors have introduced other terms to describe the same or closely related concepts. Examples of alternative terms are: “multipathology”, “polymorbidity”, “polipathology”, and “pluripathology” ,13,51.Case example. Consider a 58-year-old woman with coronary artery disease, hypertension, and major depression. Her mental health professional, focusing on the major depression, would consider her coronary artery disease and hypertension as comorbidities. Her primary care physician might describe her as having multimorbidity, giving equal attention to her coronary artery disease, hypertension and major depression.For each included paper, data on the year of publication, the journal title, subject category, keywords, and the authors’ names, institutional affiliation(s), and country was downloaded online through the SCI-E from the Web of Science by one researcher (AA-A) in November 2016. A second researcher (FC-L) verified the data to minimize potential information errors. The SCI-E platform is a database that contains all the above information, including the full addresses of all authors of every paper. We also used the SCI-E to determine the extent to which each paper had been cited in the scientific peer-review literature using the “times cited” number . A process of standardization was conducted by two researchers to bring together the different names of an author or country, and keywords. Specifically, one researcher (AA-A) checked the names by which an individual author appeared in two or more different forms , using coincidence in that author’s place(s) of work as the basic criterion for normalization , and a second researcher (FC-L) verified data. We used both ‘‘author keywords” and ‘‘keyword plus,” which are automatically assigned by the Web of Science from the titles of the references of the articles because this approach has proven to be highly effective in representing the conceptual content of articles. To ensure consistency in the data, one researcher (RA-B) corrected keywords unifying grammatical variants and using only one keyword developed names of the same concept . In addition, the same researcher (RA-B) removed typographical, transcription and/or indexing errors, and a second researcher (FC-L) verified data. All potential discrepancies were resolved via discussion. All these data were entered into a Microsoft Access®  database.http://www.wordle.net/), which is free-software that generates “word clouds” from text that the user provides and places more emphasis on words that appear with greater frequency in the source text. We identified the most frequently used keywords per journal subject category. We also presented the “co-words network” of keywords representing the co-occurrence phenomenon of highly frequent words in the papers. The co-words network reflects the relation among multiple terms, and so is effective in mapping the associations between keywords in textual data [http://www.prisma-statement.org/) guided the reporting of the present analysis , countries, journals and keywords. Data were summarized as frequencies and percentages for categorical items. We have presented in tables the most prolific authors and countries (> 100 papers), and the most cited papers (>1000 citations). We have presented network graphs (or diagrams) to represent data visualization of the structure of the most intense scientific collaboration between countries applying a threshold of 50 papers in collaboration. In order to depict the frequency of the most frequently used keywords, a word cloud was created using Wordle  were identified and included in the analyses . Table 1The number of papers increased exponentially over the study period . ApproxiPLOS One  and the Journal of Affective Disorders  published the largest number of papers, followed by the Journal of Clinical Psychiatry , the Journal of the American Geriatrics Society  and Obesity Surgery . Most papers were classified in one  or two  journal’s subject categories. There was wide diversity in journal’s subject categories, with psychiatry, surgery, clinical neurology, and general and internal medicine the most common  journals published only one paper, 344 (9.8%) journals published two, 220 (6.3%) journals published three, and 2340 (66.8%) published four or more papers. t common .Most papers were written by 4 or more authors  and only 5.5% (n = 4730) of papers were written by one author. The first authors of the papers were based most commonly in North America and Europe; first authors from the United States were responsible for 38.6% (n = 33171) of the papers . We idenOverall, 168 countries worldwide were involved in the sample of papers. The productivity ranking for countries with respect to the number of papers  was headThe most commonly used article/review keywords were “comorbidity” , followed by “depression” , “elderly”  and “mortality”  . The mosArchives of General Psychiatry  with 11 papers and followed by the Journal of the American Medical Association (JAMA) with 8 papers. The list of most cited papers  corresponded to 3596 (4.2%) papers with at least 100 citations. The most cited papers by number of citations (“citation classics” with at least 1000 citations) are listed in d papers  containsd papers  and the d papers ; the widd papers –the concd papers ; and thed papers  to quantIn this cross-sectional analysis, we analyzed the global scientific research in comorbidity and multimorbidity for the period 1970–2016. We have identified the most productive investigators and countries, most common subjects and keywords, most prolific journals and “citation classics” in comorbidity and multimorbidity based on publications in multiple specialties and disciplines. The most striking results are the increasing number of published articles in recent years, with approximately two-thirds of the papers published since 2010. To the best of our knowledge, this is the first comprehensive global mapping analysis of scientific publications in comorbidity and multimorbidity. This analysis complements and expands the perspective of previous studies that analyzed some characteristics of articles in comorbidity , the divIn line with previous research in other areas –57, the Papers on comorbidity and multimorbidity were published most often in journals devoted to neuropsychiatry and neurosciences. Psychiatry has become one of the fastest growing medical disciplines . In factOur analyses suggest that other medical disciplines with a large number of papers on comorbidity and multimorbidity, including surgery ,75, clinThe topic analysis of the most cited papers (“citation classics”) allowed us to determine which topics have attracted the most interest in the research on comorbidity and multimorbidity. These include landmark methodological developments in measuring comorbidity  –34 and dThere are several limitations to our study. We characterized knowledge structures generated by papers included in the Web of Science database, integrating subject categories of journals, keywords of papers and network analyses. However, these methods represent a scoping approach which could be complemented further by more detailed analyses, for example analyzing the content and reporting quality of papers in evidence syntheses (including systematic reviews of the literature ). We onlThe global analysis presented in this study provides compelling evidence of the scientific growth of research on comorbidity and multimorbidity. Scientific research in this field is increasingly published in biomedical journals, with research leadership of Western countries, most notably, the United States. This study contributes to a better understanding in this challenging field and identifies the main areas of research, the publication sources chosen for their scientific dissemination and the major scientific leaders. Advances in several subjects and research areas will allow for use of new theories and models to fundamentally changes in the management of people with multiple chronic diseases.S1 Checklist(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Whether TNF-β induction of inflammatory pathways in primary human chondrocytes (PCH) can be modulated by resveratrol, was investigated. Monolayer and alginate cultures of PCH were treated with TNF-β, anti-TNF-β, nicotinamide (NAM), antisense oligonucleotides against Sirt1 (Sirt1-ASO) and/or resveratrol and co-cultured with T-lymphocytes. We found that resveratrol suppressed, similar to anti-TNF-β, TNF-β-induced increased adhesiveness in an inflammatory microenvironment of T-lymphocytes and PCH. In contrast, knockdown of Sirt1 by mRNA abolished the inhibitory effects of resveratrol on the TNF-β-induced adhesiveness, suggesting the essential role of this enzyme for resveratrol-mediated anti-inflammatory signaling. Similar results were obtained in PCH stimulated with TNF-α. Sirt1-ASO, NAM or TNF-β, similar to T-lymphocytes induced inflammatory microenvironment by down-regulation of cartilage-specific proteins, Sox9, Ki67 and enhanced NF-κB-regulated gene products involved in inflammatory and degradative processes in cartilage , NF-κB activation and its translocation to the nucleus. Moreover, resveratrol reversed the TNF-β-, NAM-, T-lymphocytes-induced up-regulation of various NF-κB-regulated gene products. Down-regulation of Sirt1 by mRNA interference abrogated the effect of resveratrol on TNF-β-induced effects. Ultrastructural and cell viability assay investigations revealed that resveratrol revoked TNF-β-induced dose-dependent degradative/apoptotic morphological changes, cell viability and proliferation in PCH. Taken together, suppression of TNF-β-induced inflammatory microenvironment in PCH by resveratrol/Sirt1 might be a novel therapeutic approach for targeting inflammation during rheumatoid arthritis.While Lymphotoxin α (TNF-β), a product of lymphocytes, is known to play a pivotal role in inflammatory joint environment, resveratrol has been shown to possess anti-inflammatory and chondroprotective effects  In inducurcumin .trans-stilbene), found in the skin of red grapes, cranberries, peanuts and root extracts of the weed Polygonum cuspidatum, functions microbiologically as a phytoalexin protecting plants against fungal infections and UV radiation [+-binding catalytic core domain although they exhibit distinct expression patterns, catalytic activities and biological functions [Disease-modifying anti-rheumatic drugs (DMARDs) such as methotrexate or biological agents such TNF-inhibitors, reduce synovitis and systemic inflammation and improve function . Howeveradiation . It has adiation . One of adiation . The sirunctions . Being aunctions –20.in vitro [Studies from our laboratory have previously shown that resveratrol possesses anti-inflammatory and chondroprotective effects in human chondrocytes in vitro –23. Indein vitro , 25. Furin vitro . Inflammin vitro . Contrarin vitro  and it iin vitro .in vivo [Several lines of evidence have shown that normal cartilage homeostasis requires enzymatically active Sirt1 protein in vivo –32. In fin vivo . Indeed,in vivo , 34–37. in vivo . Furtherin vivo , 39. HowThe purpose of this study was therefore, to examine the effect of resveratrol-induced Sirt1 signalling on suppressing inflammatory pathways activated by TNF-β in primary human chondrocytes as potential novel therapeutic approach for the treatment of inflammatory joint diseases such as RA/OA.Anti-active caspase-3, -MMP-9, -MMP-13, -TNF-α and TNF-α were purchased from R&D Systems, Inc., . Antibodies to phospho-specific p65 (NF-κB)/(Ser536) were purchased from Cell Technology . Antibodies to collagen type II and alkaline phosphatase-linked sheep anti-mouse and sheep anti-rabbit secondary antibodies for immunoblotting were purchased from Millipore . Anti-TNF-β was obtained from eBioscience . Anti-Ki67 and secondary antibodies used for fluorescence labelling were purchased from Dianova . Anti-cyclooxygenase-2 was purchased from Cayman Chemical . Monoclonal anti-β-Actin was obtained from Sigma-Aldrich . Sox9 antibody was purchased from Acris Antibodies GmbH . Polyclonal antibody against Sirt1 was purchased from Abcam PLC .TNF-β and polyclonal rabbit anti-TNF-β were obtained as described . BacteriCell culture growth medium was prepared as previously described . DulbeccPrimary human chondrocytes  were purchased from Provitro . Cartilage samples of knee joint used for chondrocyte isolation were derived from human patient , who provided her full informed consent. Participant gave written informed consent. This study was carried out in accordance with the declaration of Helsinki and local ethics committee approval was provided by the Charité-University Medical School Berlin, Germany. Briefly, during monolayer expansion, chondrocytes were seeded at a density of 300,000 cells/T75 cell culture flask and grown until 70% confluence was reached in cell culture growth medium (10% FBS). Chondrocytes were passaged up to two times, and passages 2 and 3 were used in the experiments. Chondrocytes were washed three times with serum-starved medium (3% FBS) and further incubated for 30 minutes with the same medium before initiation of experiments. All experiments were performed with serum-starved medium. A human T-lymphocyte cell line (Jurkat cells)  was cult5´-GTATTCCACATGAAACAGACA-3´) corresponding to Sirt1 mRNA and control 21-mer sense oligonucleotides (SO) (sequence 5´-TGTCTGTTTCATGTGGAATAC-3´) were synthesized by eurofins . All ASOs and SOs were phosphorothioate-modified to protect them from cell nucleases. Transfection was carried out by incubating the primary chondrocytes in monolayer culture for 24h with 0.5μM ASO against Sirt1 or SO control and 10μl/ml Lipofectin transfection reagent  in serum-starved medium before starting the respective experiments. All transfection experiments were carried out on 50% confluent monolayer cultures.Transient transfection of primary human chondrocytes was performed as previously described in detail , 42. The6/ml), or were treated with 10ng/ml TNF-β, or 10ng/ml TNF-β and 5μl/ml anti-TNF-β, or 10ng/ml TNF-α, or 10ng/ml TNF-α and 5μl/ml anti-TNF-α and then co-cultured with T-lymphocytes for 8h. Additionally, in another set of experiments, primary human chondrocytes were either co-cultured with T-lymphocytes and/or with 5μM resveratrol and/or 10ng/ml TNF-β and/or 10ng/ml TNF-α for 8h, or were transfected with Sirt1-ASO or Sirt1-SO in the presence of Lipofectin (10μl/ml) for 24h prior to co-treatment with T-lymphocytes and 5μM resveratrol and 10ng/ml TNF-β or 10ng/ml TNF-α for 8h. After 8h of co-culture, non-adherent T cells were carefully removed by gentle washing once with Hank´s balanced salt solution  and photographed under a light microscope . The number of adhered T-lymphocyte colonies on chondrocytes was determined by scoring 10 different microscopic fields. This experiment was repeated three times independently and statistical analysis was done to obtain the final values.For investigation of interaction between chondrocytes with T-lymphocytes an adhesion assay was performed as previously described . Briefly6/ml) alone for 12h. Additionally, primary human chondrocytes were co-cultured with T-lymphocytes and either treated with 10ng/ml TNF-β, or with 5μM resveratrol, or with 10ng/ml TNF-β and 5μM resveratrol for 12h. Cells were washed in Hanks’ solution (PBS) before methanol fixation and permeabilization of the cells for 10 min at ambient temperature (AT). This was followed by rinsing 3 times with a mixture of protease-free bovine serum albumin (BSA) and PBS for 10min at AT and then incubated with primary antibodies  in a humid chamber overnight at 4°C. Cultures were washed three times with PBS/BSA before incubation with rhodamine-red conjugated secondary antibody (diluted 1:80 in PBS/BSA) for 1.5h at AT and finally rinsed again three times with PBS. Counter-staining was performed with 4′,6-diamidino-2-phenylindole  to visualize cell nuclei for 10 min. Cells were finally washed once with aqua dest. and covered with fluoromount mountant (Sigma). Samples were evaluated under a fluorescence microscope . Quantification of positively stained cells was performed by scoring 100 cells from 10 different microscopic fields.The technique was performed as previously described in detail . Briefly6/ml) were transferred to alginate cultures, treated as described above and cultured under similar conditions with serum-starved medium for 10 days to examine the effects of TNF-β and/or resveratrol on chondrocytes differentiation potential in a three-dimensional environment. Three-dimensional alginate cultures were prepared as previously described [6/ml) was resuspended in alginate  and slowly added dropwise into a solution containing 100mM CaCl2 at ambient temperature (AT). The alginate beads polymerized in the presence of CaCl2 after 10 min. Subsequently, the CaCl2 solution was removed and the alginate beads rinsed three times with 0.15M NaCl solution and twice with serum-starved medium (3% FBS).The medium was changed every 3 days. The cultures were grown in an incubator at 37°C with 5% CO2 in air. These experiments were performed in triplicate, and the results are provided as mean values from three independent experiments. A detailed description of the culture technique used for transmission electron microscopy has been published [sO4 solution, cells from monolayer or alginate cultures were rinsed and dehydrated in ascending alcohol series. They were embedded in Epon, cut on a Reichert Ultracut  followed by contrasting with 2% uranyl acetate/lead citrate. For inspection a transmission electron microscope  or a Jeol 1200 EXII, Akishima Tokyo, Japan  was used.Serum-starved primary human chondrocytes were either left untreated, treated with various concentrations of TNF-β  for 1h, or co-treated with resveratrol 5μM and various concentrations of TNF-β  for 1h in monolayer cultures. In a second approach, monolayer cultured chondrocytes  alone for 4h, or co-cultured with T-lymphocytes and additionally treated with 10ng/ml TNF-β, or 5μM resveratrol or with 10ng/ml TNF-β and 5μM resveratrol for 4h. In a second approach, serum-starved monolayer cultured primary human chondrocytes were either left untreated or treated with 5μM resveratrol, or 10ng/ml TNF-β, or 10mM NAM, or were co-treated with 5μM resveratrol and 10ng/ml TNF-β or 5μM resveratrol and 10mM NAM for 4h. Additionally, chondrocytes were transfected with 0.5μM Sirt1-SO or Sirt1-ASO in the presence of Lipofectin (10μl/ml) for 24h and either left untreated or co-treated with resveratrol (5μM) for 4h. In another set of experiments, serum-starved chondrocytes in alginate beads were left untreated or treated with various concentrations of TNF-β , or were co-stimulated with 5μM resveratrol and various concentrations of TNF-β  for 14 days. The whole-cell extracts were prepared for immunoblotting as previously described [2 and 0.1M NaCl, specific antigen-antibody complexes were detected using nitro blue tetrazolium and 5-bromo-4-chloro-3-indoylphosphate . Total protein amount was defined according to the bicinchoninic acid system  using bovine serum albumin as a standard. Specific binding was quantified by densitometry using ‘‘quantity one” . Specific β-actin antibody was used for the internal control to normalize the sample amounts.Primary human chondrocytes in monolayer cultures were either left untreated or treated with 5μM resveratrol, or 10ng/ml TNF-β, or 5μM resveratrol and 10ng/ml TNF-β or co-cultured with T-lymphocytes  in an inflammatory microenvironment of primary human chondrocytes and T-lymphocytes We have made the following novel findings: (I) TNF-β-mediated adhesiveness of T-lymphocytes to chondrocytes could be significantly reduced by resveratrol and this effect could be inhibited by knock-down of Sirt1 with ASO, (II) resveratrol suppressed NF-κB activation induced by TNF-β or T-lymphocytes in chondrocytes, (III) resveratrol also down-regulated NF-κB dependent gene products involved in cell proliferation (e.g. Ki67), inflammatory (COX-2), in matrix degrading (MMP-9/-13) and apoptotic (cleaved caspase-3) signaling pathways induced by TNF-β- or T-lymphocytes in the same manner, (IV) this downregulation led to the suppression of apoptosis induced by cytokines and T-lymphocytes, (V) Finally, we showed for the first time, that TNF-β- similar to T-lymphocytes-mediated inflammation was inhibited by resveratrol in a Sirt1-dependent manner, highlighting the crucial role of this enzyme in regulation of TNF-β-induced inflammation signaling in chondrocytes.in vitro model of inflammatory joint environment TNF-β enhanced inflammation in chondrocytes, similar to IL-1β [Rheumatoid arthritis (RA) is an inflammatory joint disease affecting 0.5–1% of adults, with 5–50 per 100 000 new cases annually . Indeed,to IL-1β .et al. have further shown that polymorphisms in the TNF-β gene increase susceptibility to RA [We showed in this study for the first time that TNF-β, similar to TNF-α enhances recruitment and adherence of T-lymphocytes to chondrocytes, underling the potential of TNF-β in stimulating and supporting an inflammatory microenvironment in chondrocytes. Indeed, it has been previously shown that an allelic variant in the TNF-β gene leads to higher TNF-β response in mononuclear cells after stimulation with mitogens . The autty to RA .Additionally, in this study, we evaluated for the first time that co-treatment with the natural polyphenol resveratrol suppressed T-lymphocytes adherence to chondrocytes in TNF-β as well as in T-lymphocytes treated cultures and this effect of resveratrol was inhibited by knock-down of Sirt1 with antisense oligonucleotides (Sirt1-ASO). This demonstrates that inflammation in chondrocytes by TNF-β is indeed mediated similar to inflammation by T-lymphocytes. Further, this indicates clearly, that resveratrol-induced suppression of inflammatory microenvironment by TNF-β or T-lymphocytes in chondrocytes is, at least in part, dependent on activation of Sirt1 pathway. These results are consistent with those from previous studies that showed that the multi-targeted, natural polyphenol compound resveratrol has been linked with anti-inflammatory and chondroprotective properties in chondrocytes –23 and fTo determine the mechanisms by which the pro-inflammatory signaling of TNF-β in enhancing adhesiveness of T-lymphocytes to chondrocytes and the activation of inflammation cascade in chondrocytes is regulated, we next investigated with immunofluorescence, how TNF-β-modulated activation of NF-κB and Sirt1 in comparison to T-lymphocytes effects. Indeed, it has been shown in chondrocytes that activated NF-κB is involved in regulation of adhesion, cell-cycle, apoptosis, survival and inflammatory processes by activating IL-1β, TNF-α, IL-6, Cox-2 and MMPs , 57, 58.We have previously shown that untreated primary chondrocytes have very low TNF-β expression and that stimulation with a pro-inflammatory cytokine like IL-1β induced TNF-β in chondrocytes . HoweverTo examine the underlying downstream signaling pathway, by which resveratrol executes its inhibitory effect on TNF-β-signaling pathway, we investigated whether NF-κB and NF-κB-regulated gene end-products involved in inflammation, proliferation and apoptosis were activated. We found that resveratrol significantly suppressed NF-κB activation induced by TNF-β and/or T-lymphocytes in chondrocytes. NF-κB suppression correlated with reduced p65 phosphorylation and nuclear translocation. Furthermore, resveratrol also down-regulated NF-κB dependent gene end-products involved in cell proliferation (e.g. Ki67), inflammatory (COX-2), in matrix degrading (MMP-9/-13) and apoptotic (cleaved caspase-3) signaling pathways induced by TNF-β- or T-lymphocytes. Interestingly, the inhibition effect of resveratrol was, at least in part, Sirt1-signalling dependent. Resveratrol’s inhibition of the NF-κB activation induced by TNF-β and/or T-lymphocytes suggests that resveratrol/Sirt1 signaling acts at a step common to all of these activators. However, we are the first to report the ability of resveratrol to modulate expression and activation of TNF-β in chondrocytes and TNF-β-induced RA-microenvironment. These results suggest that the effects of resveratrol on TNF-β signaling are, at least in part, through suppression of NF-κB signaling in chondrocytes.We further found that treatment of chondrocytes with ASO-Sirt1, like NAM or TNF-β, caused NF-κB activation and up-regulation of NF-κB-related gene end-products involved in degradation, inflammation and apoptosis  and this was accompanied by the decreased synthesis of cartilage-specific matrix, Ki67, Sirt1 and Sox9 expression. However, co-treatment with resveratrol inhibited NF-κB activation and NF-κB-related gene end-products by NAM, TNF-β or T-lymphocytes and revealed a significant increasing in cartilage-specific matrix expression, Ki67, Sirt1 and Sox9 expression, but not by Sirt1-ASO. These results show clearly that TNF-β-mediated NF-κB activation in chondrocytes is similar to Sirt1-ASO or NAM, indicating that Sirt1 is one of the main downstream signaling molecules for TNF-β-signaling that may be modulated by resveratrol-mediated signaling pathways in RA inflammatory microenvironment. Moreover, these findings clearly indicate that down-regulation of Sirt1 by mRNA interference abrogates the suppressive effects of resveratrol on TNF-β- and NF-κB signaling, highlighting the important role and function of Sirt1 for anti-inflammatory and anti-cytokine pathway of resveratrol in RA/OA inflammatory joint environment.In conclusion, our study here demonstrates for the first time that TNF-β induces a pro-inflammatory microenvironment similar to TNF-α and T-lymphocytes, up-regulating pro-inflammatory signaling and suppressing chondrogenic potential in primary human chondrocytes. Further, we could demonstrate that resveratrol-mediated Sirt1 up-regulation inhibits TNF-β-induced pro-inflammatory and degenerative pathways in chondrocytes . Thus, tS1 Fig(ZIP)Click here for additional data file.S2 Fig(ZIP)Click here for additional data file.S3 Fig(ZIP)Click here for additional data file.


There is a clear tendency for authors of scientific papers to over-cite the papers by their fellow countrymen (and countrywomen) relative to the percentage presence of their papers in world output in the same field. We investigated the Over-Citation Ratio (OCR) as a function of this percentage, and the effects of different scientific fields and publication years. For cancer research, we also compared clinical with basic research. We found that the OCR for a given percentage presence has been decreasing over the period 1980–2010, probably because of better communications. It is greater for fields of relatively more national interest  and less for those of international concern . It may also be slightly greater for basic cancer research than for clinical work. The OCR values given allow other types of citation, such as the references on clinical practice guidelines and papers featured in newspaper stories, to be put in context: are they unusually nationalistic, or typical of normal citation behaviour? This paper is concerned with the practice whereby researchers from a given country tend to over-cite research by their fellow-countrymen . In practice, we investigate how often research by a given country is cited by papers from the same country. One might suppose that if a country’s research in a particular field of science represented (say) 1% of world output, then a “fair” quota of citations from the country would be 1% of the total. In practice it is almost always more than this, so we say that the percentage of the citing papers, divided by the country’s presence in the field, is the over-citation ratio, denoted as OCR.other countries, which could be an indicator of the international impact of the work. This indicator needs to be carefully normalised to take account of the various factors that can influence it, and the paper goes into some detail about these. The second reason for the study is that the OCR for research papers cited by papers in the serial literature provides a benchmark for the evaluation of other types of citations. For example, Grant et al. . It is not meant to provide exhaustive data but rather to describe a methodology that can be applied to a given set of papers. It will also give enough results that the main features of OCR can be discerned, and used to show if any given value is higher or lower than expected. Two hypotheses will be explored: the first, the subjects of relatively local or national interest will lead to higher country OCRs than ones of international interest, and second, that in biomedical subjects, clinical papers will also give higher OCRs than ones on basic research.Self-citation can take place at many different levels. The one most commonly addressed in the literature is that of the individual author, who may be thought to boost her ranking by this means. But it appears that author self-citations may be correlated with higher citations by others  were considered. For each cohort, the citation analysis was provided by the WoS, and the collection of citing papers was then examined. Those from the five years following the publication of the cited papers, including the publication year, were isolated, and the numbers from the given country were counted as a percentage of this total number. This percentage was compared with the country’s percentage presence in the field in the given year to give the over-citation ratio, all the counts being integer country counts of papers. However, only countries with 1% or more of the world total of papers in the given year and with an OCR <100 were retained for our analysis as there would have been too much scatter for countries with smaller outputs.The analysis is based on data from the Web of Science (WoS)The fields of astronomy and birds were defined by means of filters containing a simple list of specialist journals and another list of title words. Examples of title words for these two fields are given below.These two fields were chosen to be as international as possible (astronomy), and relatively local or regional (birds). The filters for cancer and diabetes papers were similarly defined but with much longer lists: that for cancer had 185 specialist oncology journals and 323 title words or phrases. On the other hand, the filters for chemistry and engineering were based on the topic function in the WoS: TS = CHEMISTRY and TS = ENGINEERING. In cancer, a distinction was made between clinical papers and basic ones, based on words in their titles . These graphs were on log–log scales, and a best-fit regression line was added with a power law: OCR = M × nJ. For all these graphs, the correlation was usually very good, typically with r2 ~ 0.9. The multiplier M in the equation giving the best fit was then plotted against time: this represented the OCR for a country publishing 1% of world papers. From the best-fit equation we also calculated the estimated value of OCR for a country publishing 3 and 10% of world papers.The OCR for each of the 20 countries was plotted against the country’s presence in the field in the given year .The least-squares correlation line was given by a power law relationship of the type:Figures There is also a fairly consistent pattern: OCR is highest for two fields with particularly local interests  and lowest for two with a more international outlook . It also appears for the three sets of cancer research papers, see Figs. These three graphs show that there is surprisingly little difference in the over-citation ratio for the three groups of cancer research papers. In the 21st century, for which there are more countries, and many more papers, the pattern suggests that basic research is over-cited by own countrymen slightly more than clinical work, but the differences are much smaller than for the different scientific fields discussed previously. The mean values of M and J shown in Table OCR = M pJ where p is the percentage presence of the country in the field, M is a multiplier between 10 and 30, and J is an index between −0.68 and −0.90;OCR is much greater for countries with small scientific output. There is a negative power relationship of the form, OCR has tended to decrease with time between the 1980s and 2005–2010, probably because of easier (and cheaper) international communications;OCR is higher for scientific fields of more national interest, such as ornithology (and chemistry) and lower for ones that are more universal, such as astronomy and medicine.Even with the large numbers of papers in the WoS in the various subject areas that we have considered, there is rather a lot of scatter in the results. This is seen in the figures where the lines do not form clear patterns either with time or with subject area. Nevertheless, there are three very clear conclusions:The paper describes a methodology that can be applied to any selected field and it gives results that show the extent of own country OCR in scientific papers in the serial literature. These values will provide a baseline with which the over-citation ratio of other document types can be compared to show whether they are more, or less, nationalistic than the countries’ researchers.


Heart failure with preserved ejection fraction (HFpEF) is a major unmet medical need that is characterized by the presence of multiple cardiovascular and non-cardiovascular comorbidities. Foremost among these comorbidities are obesity and diabetes, which are not only risk factors for the development of HFpEF, but worsen symptoms and outcome. Coronary microvascular inflammation with endothelial dysfunction is a common denominator among HFpEF, obesity, and diabetes that likely explains at least in part the etiology of HFpEF and its synergistic relationship with obesity and diabetes. Thus, pharmacological strategies to supplement nitric oxide and subsequent cyclic guanosine monophosphate (cGMP)—protein kinase G (PKG) signaling may have therapeutic promise. Other potential approaches include exercise and lifestyle modifications, as well as targeting endothelial cell mineralocorticoid receptors, non-coding RNAs, sodium glucose transporter 2 inhibitors, and enhancers of natriuretic peptide protective NO-independent cGMP-initiated and alternative signaling, such as LCZ696 and phosphodiesterase-9 inhibitors. Additionally, understanding the role of adipokines in HFpEF may lead to new treatments. Identifying novel drug targets based on the shared underlying microvascular disease process may improve the quality of life and lifespan of those afflicted with both HFpEF and obesity or diabetes, or even prevent its occurrence. Heart failure (HF) is a major public health problem on a global scale. Historically, HF was believed to originate from long standing systolic dysfunction, as assessed by reduced ejection fraction (HFrEF), and much progress has been made in the last several decades in slowing the inevitably fatal progression of this condition with drugs and in some cases implantable devices –5. HowevDiastolic dysfunction or impaired relaxation of the left ventricle (LV) is the common clinical condition of HFpEF and is attributable to both cardiac fibrosis and myofilament stiffness , 20. Con2 are at an increased risk of an adverse outcome , independent of other key prognostic variables  activity –60. IncrCoronary microvascular inflammation is now postulated to play the key role in HFpEF progression, encompassing endothelial dysfunction and impaired nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)—protein kinase G (PKG) signaling and increased collagen deposition Figure  54). In. In54). Involvement of microvascular inflammation in HFpEF with the associated reduction in eNOS-mediated NO generation raises the possibility that enhancing cGMP-PKG signaling could be an efficacious therapeutic approach , 72. Potvia bariatric surgery, which was shown effective in improving left ventricular relaxation and reversing concentric LV remodeling and hypertrophy, might be considered to treat obesity-associated HFpEF in younger individuals; however, the long-term cardiovascular effects of this surgery in obese HFpEF patients would need to be assessed -eNOS pathway  are small non-coding RNAs (~21–25 nucleotides in length) that in animal cells generally bind to the 3′ UTR of mRNA to suppress gene expression by either transcript degradation or translational inhibition. The bloodstream contains multiple types of miRNAs in various types of vesicles and complexes, secreted from both healthy and dying cells of likely all tissues throughout the body . Since mAn emerging area in cardiovascular medicine is the study of long non-coding RNAs (lncRNAs), which are transcripts larger than 200 nucleotides that control gene expression at the epigenetic, transcriptional, and posttranscriptional levels . Single-The drug metformin has proven highly effective in the treatment of type 2 diabetes and is currently recommended as first line treatment. Metformin has beneficial actions by reducing hepatic glucose production and by activating AMPK, which enhances cellular glucose uptake. AMPK activation in cardiac myocytes may also inhibit hypertrophy . PreclinConcerns of increased adverse cardiovascular outcomes, including HF, are associated with the use of sulfonylureas and thiazolidinediones (TZDs) in diabetic patients , 143. Thvia an allosteric site on sGC to synergize with NO in producing cGMP, thereby offsetting decreased NO due to diminished NO synthase activity   –169. Proxcretion , 171. Wh actions , 173. FuAdipose tissue is an endocrine organ that secretes multiple “adipokines” that have broad physiological and pathological impact throughout the body , 175. InThe role of sex as well as race in HFpEF, especially their interaction with comorbidities, is an evolving area of investigation. Early studies reported that HFpEF was more common among women than men , 181. ReObesity and diabetes are not only risk factors for the development of HFpEF but have significant impact on its symptoms and outcome. Therefore, focusing on these comorbid conditions in HFpEF might provide a novel therapeutic strategy. Coronary microvascular endothelial dysfunction with impaired NO-cGMP-PKG signaling is a shared condition that is thought to be the basis for diastolic stiffness, inflammation, oxidative stress, and maladaptive cardiac remodeling. Pharmacological approaches that target this signaling axis offer promise in treating or preventing HFpEF. This would include: (a) NO replenishment (inorganic nitrates/nitrites), replacement (nitroxyl donors), or enhanced generation (β3 AR agonists and AMPK agonist); and (b) enhancers of NO-independent cGMP generation (LCZ696/entresto) or prevention of its breakdown (PDE9 inhibitors). A reappraisal of clinical results supports the utility of inhibiting the mineralocorticoid receptor in treating HFpEF, but additional study is warranted. In addition, given the pronounced side effects of spironolactone at higher doses, an endothelial cell-targeted approach might be judicious. miRNA and lncRNA profiling of HFpEF patients offers the promise of not only prognostic assessment and therapeutic monitoring, but personalized treatment strategies as well. A better understanding of the role of adipokines in obesity- and diabetes-associated HFpEF may open up new pharmacological avenues. Finally, SGLT-2 inhibitors offer great promise for treating or preventing HFpEF in obese and diabetic patients. A better understanding of the physiological and molecular basis for the cardiovascular protective actions of this new drug class should foster the development of even more effective compounds.All authors contributed conceptually to the manuscript. All authors authored sections of the manuscript, contributed to figure design, and approved the final version. All appropriate permissions have been obtained from the copyright holders of any work that has been reproduced in this manuscript.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.


Infection with Kaposi sarcoma-associated herpesvirus (KSHV) is estimated to account for over 44,000 new cases of Kaposi sarcoma annually, with 84% occurring in Africa, where the virus is endemic. To date, there is no prophylactic vaccine against KSHV. KSHV gpK8.1, gB, and gH/gL glycoproteins, implicated in the virus entry into host cells, are attractive vaccine targets for eliciting potent neutralizing antibodies (nAbs) against virus infection. We incorporated gpK8.1, gB, or gH/gL on the surface of virus-like particles (VLPs) and characterized these VLPs for their composition, size, and functionality. To determine which viral glycoprotein(s) elicit the most effective serum-nAbs, we immunized BALB/c mice with gpK8.1, gB, or gH/gL VLPs individually or in combination. Neutralizing antibody assay revealed that sera from mice immunized with the VLPs inhibited KSHV infection of HEK-293 cells in a dose-dependent manner. As a single immunogen, gpK8.1 VLPs stimulated comparable nAb activity to that of UV-inactivated KSHV (UV-KSHV). In contrast, UV-KSHV stimulated higher titers of nAb compared to gB (p = 0.0316) or gH/gL (p = 0.0486). Mice immunized with the combination of gB and gH/gL VLPs had a better nAb response than those immunized with either gB (p = 0.0268), or gH/gL (p = 0.0397) as single VLP immunogens. Immunization with any VLP combination stimulated comparable nAb activity to UV-KSHV serum. Our data provide the first evidence that KSHV gpK8.1, gB, and gH/gL glycoproteins can be incorporated onto the surface of VLPs and used as prophylactic vaccine candidates, with potential to prevent KSHV infection. Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8 (HHV-8), is the etiologic agent of Kaposi sarcoma (KS), an endothelial cell tumor . KSHV isKSHV-associated malignancies predominantly present in people with primary and acquired immunodeficiencies, including acquired immunodeficiency syndrome (AIDS) and iatrogenic immunosuppression in organ transplant recipients , 16. KS Prophylactic vaccines typically stimulate an individual's humoral immune system to produce neutralizing antibodies against the surface glycoprotein(s) mediating the infectious agent's entry , 25. TheLike all other herpesviruses, KSHV enters target cells through a multi-step process that involves interactions between multiple viral envelope glycoproteins and host cell surface molecules that include attachment and fusion receptors , 28. TheWe developed KSHV envelope glycoprotein-based virus-like particles (VLPs) as prophylactic vaccine candidates. VLPs are self-assembling structures composed of one or more viral proteins. They can serve as delivery systems to present antigens from different types of pathogens . The sizin vitro neutralization assays performed using recombinant KSHV tagged with enhanced green fluorescent protein (KSHV-eGFP) showed that antibodies generated by KSHV glycoprotein-based VLP-immunized mice can inhibit KSHV infection in vitro.This study demonstrates that targeting viral entry as a strategy for prevention of KSHV infection and its associated malignancies is feasible. We used the Newcastle disease virus (NDV) platform to generate VLPs bearing KSHV gpK8.1, gB, or gH/gL envelope glycoproteins. We immunized wild-type BALB/c mice and measured neutralizing antibody response. Our results confirm that KSHV glycoprotein-based VLPs are immunogenic and generate a KSHV-specific immunoglobulin G (IgG) antibody response in immunized mice. In addition, To generate KSHV gpK8.1 VLPs, we first designed and synthesized a chimeric KSHV gpK8.1-F construct by fusing the KSHV gpK8.1 ectodomain to the NDV fusion protein (F) heptad repeat 2 (HR2), transmembrane (TM), and cytoplasmic (CT) domains Figure . The conTo demonstrate that chimeric gpK8.1-F is functionally expressed on the surface of cells, we transfected Chinese hamster ovary (CHO) cells with the gpK8.1-F plasmid or relevant controls. Forty-eight hours post-transfection, cells were stained with anti-gpK8.1 mouse monoclonal antibody (mAb), which detects the ectodomain of the protein, followed by secondary anti-mouse antibody conjugated to Alexa Fluor (AF488) fluorochrome. Flow cytometry showed that CHO cells transfected with the pCAGGS-gpK8.1-F chimera robustly expressed gpK8.1 (88% expression), similar to cells transfected with pCAGGS-gpK8.1 full-length (WT) (82% expression), which served as a positive control Figure . Cells tTo assemble and produce gpK8.1 VLPs, equal amounts of gpK8.1-F, NDV-matrix (M), and NDV-nucleocapsid protein (NP) cDNA plasmids were co-transfected into CHO cells Figure . The celTo confirm that gpK8.1, NP, and M were incorporated into gpK8.1 VLPs, we lysed purified VLPs in RIPA buffer and analyzed the lysates for protein composition by immunoblot Figure . As expeTo assess the purity of gpK8.1 VLPs, we used silver stain; NDV lysate served as a positive control, and BSA was used for quantification of proteins. Bands of predicted sizes were detected for gpK8.1 (60-72 kDa), NDV-NP (˜50 kDa), and NDV-M (35-45 kDa) Figure . ElectroTo confirm that chimeric gpK8.1-F proteins incorporated into VLPs were in the right conformation and able to bind cellular surface receptors, purified gpK8.1 VLPs were incubated with human mast cells 1 (HMC-1) susceptible to KSHV infection. Cells incubated with VLPs were stained with anti-gpK8.1 mAb conjugated with AF-594 (red) to detect gpK8.1 VLPs, cholera toxin (green) to detect lipid rafts, and DAPI (blue) to detect HMC-1 nuclei Figure . HMC-1 cUpon successful generation and characterization of gpK8.1 VLPs, we used a similar strategy to generate KSHV gB and KSHV gH/gL VLPs. To generate KSHV gB VLPs, we synthesized a chimeric gB-F construct by fusing the KSHV gB ectodomain to the NDV-F HR2, TM, and CT domains Figure . The conTo assess purity, and to confirm detection of the various components of the gB VLP, we used silver stain. NDV lysate was used as a positive control, and BSA was used for quantification of protein. KSHV gB is synthesized as a 110-kDa precursor protein which undergoes furin cleavage and processing to yield envelope-associated disulfide-linked 75- and 55-kDa polypeptides . Bands oTo generate KSHV gH/gL VLPs, we synthesized chimeric gH-F and gL-HN constructs by fusing the KSHV gH ectodomain to the NDV-F HR2, TM, and CT domains Figure , and theTo assess purity of the gH/gL VLPs, and to confirm expression of the gH and gL protein components, we used silver stain. NDV lysate was used as a positive control, and BSA (1 μg and 2 μg) was used for quantification of protein. Bands of predicted sizes were detected for gH (85 kDa), gL (25 kDa), NDV-NP (˜50 kDa), and NDV-M (35-45 kDa) Figure . To confTo test the ability of individual KSHV envelope glycoprotein-based VLPs to stimulate KSHV-specific antibody responses, three groups of five female BALB/c mice each were immunized intraperitoneally with 10 μg of purified gpK8.1, gB, or gH/gL VLPs suspended in 0.5 ml of TNE buffer without adjuvants. Two groups of five mice each were immunized with TNE buffer alone (negative control) or UV-inactivated KSHV (positive control). To test additive or synergistic effects of two or more KSHV glycoprotein immunogens , we alsoThe mice were immunized at Day 0 and boosted twice, on Days 29 and 54. Mice were tail-vein bled on Days 14, 46, and 84, with a terminal bleed on Day 133 Figure .To demonstrate the ability of KSHV VLPs to induce KSHV-specific antibody responses, we used ELISA to determine the presence of anti-KSHV IgG antibodies in sera of immunized mice. Lysate from lytically induced iSLK.219 KSHV-eGFP-expressing cells, which express all KSHV envelope glycoproteins, served as the binding target. Sera from all bleeding time-points were analyzed. All VLP-immunized mice generated KSHV-specific IgG antibody responses that increased following booster immunizations, compared with TNE-immunized mice (negative control) Figure . The incin vitro, sera collected at Day 133 were used to conduct KSHV infection neutralization assays. HEK-293 cells were used as the host for the neutralization assays, as a model of epithelial cells that are susceptible to KSHV infection Graph Pad Prism 6 Software was used for statistical analyses of data. The differences between the percent inhibition of infection between the immunized and control groups of mice were analyzed using unpaired two-tailed t-tests for independent groups. Statistical significance of the tests was based on a p-value equal to or lower than 0.05.


Instinctive defensive behaviors are essential for animal survival. Across the animal kingdom, there are sensory stimuli that innately represent threat and trigger stereotyped behaviors such as escape or freezing , 2, 3, 4 •Mice instinctively escape to a previously memorized shelter location•Orientation and navigation to the shelter does not require shelter-related cues•Memory of the shelter location is formed in a single trial•Changes in the spatial environment lead to rapid updates of defensive actions Vale et al. show that mice exposed to innately aversive stimuli instinctively escape to a shelter location that was learned during exploration of the environment. Memory of shelter existence and location is formed in a single trial and can be rapidly updated, allowing mice to quickly adapt defensive actions to changes in the spatial environment. When escaping from imminent threat, animals have two general options: to move away from the threat or to move toward safety. These two behaviors have different consequences and are fundamentally distinct in the computations they require. Moving away from threat can be implemented as a simple reaction to the stimulus , but it These data suggest that the goal of the escape behavior is to reach safety. To further test this hypothesis, we reasoned that presentation of the threat while the animal is in the shelter should not cause escape behavior. Indeed, auditory stimuli delivered both in the Barnes maze and in a modified version with a surface shelter did not cause escape behavior, despite the sound pressure level inside the shelter being within 2dB of the arena outside  Act of 1986 (PPL 70/7652) following local ethical approval. Male C57BL/6J mice were used for experiments at 6–12 weeks old and were tested during the light phase of the light cycle. The main behavioral arena used was a modified Barnes maze , consist2) [The auditory stimulus consisted of a train of three frequency modulated upsweeps from 17 to 20 kHz over 3 s , lasting2) . The staR.V. and T.B. designed the study and experiments. R.V. performed all experiments with assistance from D.A.E. R.V and T.B. analyzed the data. T.B. wrote the manuscript with input from R.V. and D.A.E.