For ≈ 24 years the AIDS pandemic has claimed ≈ 30 million lives, causing ≈ 14,000 new HIV-1 infections daily worldwide in 2003. About 80% of infections occur by heterosexual transmission. In the absence of vaccines, topical microbicides, expected to block virus transmission, offer hope for controlling the pandemic. Antiretroviral chemotherapeutics have decreased AIDS mortality in industrialized countries, but only minimally in developing countries. To prevent an analogous dichotomy, microbicides should be: acceptable; accessible; affordable; and accelerative in transition from development to marketing. Already marketed pharmaceutical excipients or foods, with established safety records and adequate anti-HIV-1 activity, may provide this option.Fruit juices were screened for inhibitory activity against HIV-1 IIIB using CD4 and CXCR4 as cell receptors. The best juice was tested for inhibition of: (1) infection by HIV-1 BaL, utilizing CCR5 as the cellular coreceptor; and (2) binding of gp120 IIIB and gp120 BaL, respectively, to CXCR4 and CCR5. To remove most colored juice components, the adsorption of the effective ingredient(s) to dispersible excipients and other foods was investigated. A selected complex was assayed for inhibition of infection by primary HIV-1 isolates.HIV-1 entry inhibitors from pomegranate juice adsorb onto corn starch. The resulting complex blocks virus binding to CD4 and CXCR4/CCR5 and inhibits infection by primary virus clades A to G and group O.These results suggest the possibility of producing an anti-HIV-1 microbicide from inexpensive, widely available sources, whose safety has been established throughout centuries, provided that its quality is adequately standardized and monitored. The global AIDS epidemic has proceeded relentlessly for ≈ 24 years with no promising prophylactic intervention in sight. In 2003 there were 5 million new HIV infections, and 3 million AIDS deaths . To dateAnti-HIV-1 vaccines applicable to global immunization programs are not expected to become available for many years. Thus, other prevention strategies are urgently needed. This includes educational efforts and application of mechanical and/or chemical barrier methods. The latter correspond to microbicides, i.e. topical formulations designed to block HIV-1 infection  when applied vaginally  before intercourse ,5-7. ConTreatment with anti-retroviral drugs has decreased mortality from AIDS in industrialized countries but has had a minimal effect so far in developing countries . To avoi®; Sadaf® Foods, Los Angeles, CA; additional ingredients: fructose, citric acid); PJ4 (Cortas Canning & Refrigeration Co. S.A.L., Beirut, Lebanon); PJ5 ; PJ6 ; PJ7 . Starches used were: PURE-DENT® B815 Corn Starch NF, PURE-DENT® B816 Corn Starch USP, Spress® B825 Pregelatinized corn starch NF, Spress® B820 Pregelatinized corn starch NF, INSTANT PURE-COTE™ B792 Food starch-modified, INSCOSITY™ B656 Food starch-modified (Grain Processing Corporation, Muscatine, IN); PURITY® 21 corn starch NF and PURITY® 826 corn starch NF ; Remyline AX-DR Waxy rice starch and Remy DR native rice starch, medium grind (A&B Ingredients, Fairfield, NJ); ARGO® corn starch ; STALEY® pure food powder starch (Tate & Lyle, Decatur, IL); STARCH 1500 pregelatinized starch NF (Colorcon, West Point, PA). The following polymers were used: polyethylene glycols (PEG) 1000 NF, 1500 NF and 8000 NF; and hydroxypropyl methylcellulose, 50 cps, USP (Spectrum, New Brunswick, NJ); Carbopol 974P-NF (B. F. Goodrich Co., Cleveland, OH); Carbophil, Noveon AA1 (Noveon, Inc., Cleveland OH); and Pharmaburst B2 (SPI Pharma, New Castle, DE). Fattibase was from Paddock Laboratories, Inc., Minneapolis, MN.Pomegranate juices (PJ) were purchased in local New York City stores; their origin is given in parentheses: PJ1 (Madeira Enterprises Inc., Madeira, CA); PJ2 was prepared from fresh ripe pomegranates in our laboratory; PJ3  and BaL (2.47 × 1010 virus particles/ml) were from Advanced Biotechnologies, Inc., Columbia, MD. Primary HIV-1 isolates, MT-2 cells, HeLa-CD4-LTR-β-gal and U373-MAGI-CCR5E cells (both contributed by Dr. Michael Emerman) and Cf2Th/synCCR5 cells (contributed by Dr. Tajib Mirzabekov and Dr. Joseph Sodroski) were obtained from the AIDS Research and Reference Reagent Program operated by McKesson BioServices Corporation, Rockville, MD. CEMx174 5.25M7 cells, transduced with an HIV-1 long terminal repeat (LTR)-green fluorescent protein and luciferase reporter construct, expressing CD4 and CXCR4 and CCR5 coreceptors [Recombinant proteins employed were: HIV-1 IIIB gp120, biotinyl-HIV-1 IIIB gp120, CD4, and biotinyl-CD4 (ImmunoDiagnostics, Inc., Woburn, MA); HIV-1 IIIB BaL gp120 and FLSC (a full length single chain protein consisting of BaL gp120 linked with the D1D2 domains of CD4 by a 20 amino acid linker) (produced in transfected 293T cells [eference ) were preference . Monocloeference  was synteceptors , were obeceptors -26. PBMCeceptors .® 21 corn starch, NF grade (S21) was selected for further studies, and the corresponding PJ complex was designated as PJ-S21. PJ-S21 was freeze dried and used to prepare the following formulations: PEG suppositories (50% PJ-S21, 45% PEG 1000, 5% PEG 1500); Fattibase suppositories (50% PJ-S21, 50% Fattibase); and mucoadhesive instantly dispersible tablets (50% PJ-S21, 20% HPMC, 20% Pharmaburst, 7.5% Carbopol 974P and 2.5% Carbophil).In attempts to separate gp120-CD4 binding inhibitory activity from most other ingredients of PJ, 200 mg of distinct starch preparations were added per ml of PJ1. After mixing for 1 h at 20°C, excess juice was decanted, and the pellets resuspended in 1 ml of distilled water. Based on results of enzyme linked immunosorbent assays (ELISA), PURITY50 of primary HIV-1 strains in the absence or presence of PJ-S21 at graded concentrations for 3 days at 37°C. The experiments were done in triplicate. Infection was quantitated by measuring luciferase activity [Inhibition of infection by HIV-1 IIIB and BaL, respectively, was determined relying on a β-galactosidase readout system . The enzactivity  using a CD4-HIV-1 gp120 binding and its inhibition were measured by ELISA. Wells of 96-well polystyrene plates (Immulon II, Dynatech Laboratories, Inc., Chantilly, VA) were coated with 100 ng/well of either gp120 IIIB or gp120 BaL, and post-coated as described . Dilutio50) between 1/16 and 1/64]. Consistently, PJs from distinct geographical areas had the highest inhibitory activity , grapefruit, honey, lemon, lime, pineapple, pomegranate and red beet (10% reconstituted dry powder)] were assayed for inhibition of infection by HIV-1 IIIB of cells expressing the CD4 and CXCR4 receptors and coreceptors. Most juices (4-fold diluted) had no inhibitory activity, except blueberry, cranberry, grape and lime juice, respectively [endpoints for 50% inhibition of infection (EDBlocking virus entry is a primary target for microbicide development ,8-11. ThTo delineate sites on gp120 blocked by the PJ inhibitor(s), the inhibitory effect of PJ on binding to gp120 IIIB of antibodies to peptides derived from the amino acid sequence of gp120 was studied. The binding of antibodies to peptides (102–126), 303–338), (306–338), 361–392), 386–417), (391–425), (411–445) and (477–508) was significantly (≥ 50%) inhibited  from PJ. After striving intermittently for over four years to accomplish this, it was discovered that the inhibitor(s) of gp120-CD4 binding can be adsorbed effectively (≥ 99%) onto a selected brand of corn starch Fig. , resultiPJ-S21, to the same extent as the original PJ, inhibited the binding of gp120 IIIB-CD4 complexes to cells expressing CXCR4, as determined by flow cytometry Fig. . SimilarTo be considered as a topical microbicide, PJ-S21 must be formulated to withstand storage in a tropical environment. Accelerated thermal stability studies revealed that a water suspension of PJ-S21 maintained only 4, 11, and 33%, respectively, of its original activity (measured by inhibition of gp120-CD4 binding) when stored for 30 min at 60°C, and one week at 50°C or 40°C. On the other hand, a dried PJ-S21 powder remained fully active after storage at 50°C for 12 weeks . Consequently, anhydrous formulations should be preferred for further development.Three such formulations were prepared: two kinds of suppositories, melting at 37°C, and a tablet (for compositions see Methods section). The inhibitory activity of PJ-S21 was fully preserved after 12 weeks storage at 50°C within tablets, and at 30°C within the suppositories (the highest temperature considered to prevent melting). Data showing the inhibition of infection by HIV-1 IIIB and BaL respectively, by PJ-S21 and its formulations  are sumA microbicide can be considered potentially successful, only if it has antiviral activity against primary virus isolates belonging to distinct virus clades and phenotypes. PJ-S21 meets this requirement since it inhibited infection by primary HIV-1 strains of all clades tested having R5 and X4R5  phenotypes  interfering with gp120 binding to CD4 Fig.  blocked residues ; and othex vivo [Blocking of CD4 binding sites on HIV-1 gp120 by monoclonal antibodies or a CD4-IgG2 recombinant protein has been shown to be sufficient to inhibit HIV-1 infection of human cervical tissue ex vivo  and in pex vivo . TherefoThe application of PJ-S21 as a topical anti-HIV-1 microbicide requires reasonable uniformity among batches produced at distinct times and locations. Similarities in gp120-CD4 binding inhibitory activity among distinct freshly prepared and commercial juices stored for unknown periods , presumably, because of irreversible binding of the PJ inhibitors. Interestingly, there are only a few references available regarding the use of starch as an adsorbent for different compounds: flavors [Particular attention must be devoted to the selection of starch, a pharmaceutical excipient generally used in vaginal formulations , for eff flavors ,60, dyes flavors -63, low- flavors , lipids  flavors ,66, prot flavors  and iodi flavors .in vitro  i.e. ≥ 100-fold higher than the dose needed for blocking HIV-1 infection hallenge . This quIn an alternative approach to formulation development, PJ-S21 can be incorporated into a water dispersible film (similar to the widely available "breath control" strips) or into water dispersible sponges  which arPJ-S21 can be classified as an AAAA candidate microbicide: Acceptable; Accessible; Affordable; and Accelerative in transition from development to marketing. Thus, PJ-S21 would be expected to circumvent some problems associated with antiretroviral drugs and possibly some of the other candidate microbicides, i.e. uncertainty related to potential side effects, investment and time needed to establish inexpensive large scale production, and monopoly of supply.50(90), effective dose(s) for 50% (90%) inhibition of infection; ELISA, enzyme linked immunosorbent assays; FBS, fetal bovine serum; FLSC, a full length single chain protein consisting of BaL gp120 linked with the D1D2 domains of CD4 by a 20 amino acid linker; HIV-1, human immunodeficiency virus type 1; HRP, horseradish peroxidase; LTR, long terminal repeat; PBMCs, peripheral blood mononuclear cells; PBS, phosphate buffered saline; PEG, polyethylene glycols; PJ, pomegranate juice; S21, PURITY® 21 corn starch NF grade; SF, seminal fluid.AIDS, acquired immunodeficiency syndrome; BSA, bovine serum albumin; EDThe authors declare that they have no competing interests.ARN developed the concepts representing the basis of the manuscript and designed most experiments. NS contributed to the development of experimental techniques and carried out experiments other than infectivity assays. YYL did all the tissue culture work and viral infectivity assays. AKD did all the molecular modeling studies and contributed to the development of cell based enzyme linked immunosorbent assays.The pre-publication history for this paper can be accessed here:


Each of the cells in our body is a complex machine. Within each cell, thousands of proteins and other molecules interact to produce the highly organized cellular events that are needed for life. However, there is no cellular line manager telling the individual proteins where to go and what to do. It's rather like an ant colony. No one tells the individual ants how to build a nest. They just do it. Similarly, no one tells the individual actin molecules, for example, to get together and polymerize to form the cytoskeleton that is essential for cell movement and other cellular processes. It just happens.Ant colonies and actin polymerization are both examples of emergent behavior. Broadly defined, emergent behavior is when a collection of individuals interact without central control to produce results that are not explicitly programmed. Scientists are only just beginning to understand how complex behaviors of this type can emerge from a myriad of individual interactions, many of which are well-defined experimentally.Listeria monocytogenes—in their study of the complexities of cellular actin polymerization. This rod-shaped microbe, which lurks in well-ripened brie and other unpasteurized cheeses, can cause the sometimes fatal disease listeriosis, particularly in young babies or people with weakened immune systems.Jonathan Alberts and Garrett Odell have turned to an unlikely ally—the bacterium Listeria microbes in food reach a person's gut, they penetrate the cells lining the gut, reproduce, and then spread from cell to cell without ever exposing themselves to the extracellular environment. In this way, they avoid the host's immune system. One particular protein produced by Listeria is central to this sneaky intracellular lifestyle: ActA. Expression of ActA allows the microbes to hijack the machinery in the host cell that controls the growth of actin networks. Some of the cellular actin, instead of forming the cytoskeleton of the cell, polymerizes around the bacterium, forming a dense “comet tail” of actin that generates a force to move the bacterium around the cell and push it into neighboring cells. This clandestine use of our cellular machinery for actin polymerization is far simpler to understand than the elaborate use our cells normally make of it, so the study of Listeria propulsion provides a scientific stepping-stone to understanding the involvement of the actin cytoskeleton in normal cell movements.When Listeria cells do not move smoothly. Instead, the microbes move jerkily, with runs interspersed with pauses. The simulation faithfully reproduces this type of movement and offers a mechanistic explanation for it.Alberts and Odell have used established data on the biochemical and mechanical details of actin polymerization and the physiological concentrations of the proteins involved in the process to build a computer simulation of how an actin network assembles around a moving, rod-shaped bacterium. Their “in silico” reconstitution, which considers the behavior of individual actin filaments and requires a cluster of 80 computer processors to be run for several days at a time, produces realistic bacterial motion in terms of speed and persistence, and models the actin tail shape. The model also reproduces smaller scale emergent behavior. Real The approach described by Alberts and Odell can now be used to investigate many other, more complex cell mechanics problems, such as how the cellular movements involved in cell division are achieved. With the availability of detailed biological information and powerful computers, we may at last start to solve the mystery of how interactions among maybe 100,000 gene products can produce the organized cellular processes that cell biologists have been watching under the microscope for years.


Cyclin-dependent kinases (CDKs) are a large family of proteins that function in a variety of key regulatory pathways in eukaryotic cells, including control over the cell cycle and gene transcription. Among the most important and broadly studied of these roles is reversible phosphorylation of the C-terminal domain (CTD) of RNA polymerase II, part of a complex array of CTD/protein interactions that coordinate the RNAP II transcription cycle. The RNAP CTD is strongly conserved in some groups of eukaryotes, but highly degenerate or absent in others; the reasons for these differences in stabilizing selection on CTD structure are not clear. Given the importance of reversible phosphorylation for CTD-based transcription, the distribution and evolutionary history of CDKs may be a key to understanding differences in constraints on CTD structure; however, the origins and evolutionary relationships of CTD kinases have not been investigated thoroughly. Moreover, although the functions of most CDKs are reasonably well studied in mammals and yeasts, very little is known from most other eukaryotes.Trypanosoma or Giardia. Kinases involved in CTD phosphorylation, CDK7, CDK8 and CDK9, all are recovered as well-supported and distinct orthologous families, but their relationships to each other and other CDKs are not well-resolved. Significantly, clear orthologs of CDK7 and CDK8 are restricted to only those organisms belonging to groups in which the RNAP II CTD is strongly conserved.Here we identify 123 CDK family members from animals, plants, yeasts, and four protists from which genome sequences have been completed, and 10 additional CDKs from incomplete genome sequences of organisms with known CTD sequences. Comparative genomic and phylogenetic analyses suggest that cell-cycle CDKs are present in all organisms sampled in this study. In contrast, no clear orthologs of transcription-related CDKs are identified in the most putatively ancestral eukaryotes, The apparent origins of CDK7 and CDK8, or at least their conservation as clearly recognizable orthologous families, correlate with strong stabilizing selection on RNAP II CTD structure. This suggests co-evolution of the CTD and these CTD-directed CDKs. This observation is consistent with the hypothesis that CDK7 and CDK8 originated at about the same time that the CTD was canalized as the staging platform RNAP II transcription. Alternatively, extensive CTD phosphorylation may occur in only a subset of eukaryotes and, when present, this interaction results in greater stabilizing selection on both CTD and CDK sequences. Overall, our results suggest that transcription-related kinases originated after cell-cycle related CDKs, and became more evolutionarily and functionally diverse as transcriptional complexity increased. Cyclin-dependent kinases (CDKs) belong to a large protein family with 13 members described so far in human cells including CDKs1-11, along with PCTAIRE and PFTAIRE kinases named after conserved domain sequences -4. Gener1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 [Among the most important and broadly studied roles of CDKs in transcription is the reversible phosphorylation of the C-terminal domain (CTD) of the largest subunit (RPB1) of RNAP II. The CTD consists of multiple repeats of an evolutionarily conserved heptapeptide with the consensus sequence Tyrro6-Ser7 . The numro6-Ser7 ,16 with ro6-Ser7 ,17,18. Bro6-Ser7 ,20. Morero6-Ser7 . Throughro6-Ser7 -24.in vitro; they all have been referred to as 'CTD kinases' [in vivo, CDK1 and CDK2 are primarily cell-cycle related kinases [Drosophila in mammals and its role in Tat-dependent HIV-1 transcription is still unclear [in vitro, the role of the CDK1 in mRNA synthesis in vivo is not, as yet, clearly understood. It has been proposed as a candidate for mitotic RNAP II inactivation by inhibition of CDK7 CTD-kinase activity [To date at least five of the CDKs (CDK1, 2, 7, 8 and 9) have been shown to phosphorylate the CTD kinases' -28. Bothkinases' . The CDK kinases . CDK2 ha unclear ,28. Althactivity .In animals and yeasts, interactions between the CTD and CTD-specific kinases have become a focal point of biochemical and genetic investigations of RNAP II transcription and transcription-linked mRNA processing ,26,30. HTrypanosoma, Plasmodium and Giardia), to explore the evolutionary relationships between the CTD and CTD kinases. We also provide a phylogenetic distribution of CDKs from a wide range of organisms, suggesting new hypotheses regarding the emergence and evolution of different members of the CDK family.Here we present a comparative genomic analysis of CDKs, using complete genomes from members of the "CTD clade" , as well as from other diverse eukaryotic organisms lacking a canonical CTD  and Vandepoele et al. (2002) [We identified 133 CDK family members, 123 from animals, plants, yeasts, and four protists from which genome sequences have been completed, and 10 additional CDKs from incomplete genome sequences of organisms with known CTD sequences Table , only 10. (2002)  (Table 1Drosophila and Caenorhabditis), yeasts, both plants (Arabidopsis and Oryza), Encephalitozoon and Giardia  [In this unrooted tree the highly diversified cell-cycle kinases defined in humans, CDKs1-6, fall into a large cluster with 69% Bayesian support. This grouping includes CDKs from all organisms examined in the study. Among these putative cell-cycle CDKs, some plant and protistan kinases can be assigned with reasonable confidence to specific CDK groups. For example, apparent orthologs of human CDK1 are found in other animals (s (2000) , CDK5 anTrypanosoma brucei and Giardia lamblia. This includes strongly supported clades of presumed orthologs of human CDKs7-11 respectively. A well-defined CDK7 family is recovered, including sequences from yeasts, the microsporidian, plants, and animals. These are the primary groups that make up the "CTD-clade," in which the RNAP II CTD is invariably conserved  [Plasmodium, suggested previously to be a CDK7 [Plasmodium kinase. The a priori hypothesis that PfMRK belongs in the core CDK7 group is strongly rejected with our data set in a likelihood paired-sites test.In contrast to cell-cycle kinases, our phylogenetic results failed to identify a clear ortholog of any transcription-related CDKs from two of the complete genomes examined, ved Fig. . CDK7 shs (2003) ; howevere a CDK7 , does noGiardia, based on nearest sequence similarity to Kin28 in a more limited comparison to CDK sequences from fission yeast [Giardia sequence. The a priori hypothesis that GlCAKlike belongs in the core CDK7 group also is strongly rejected in a likelihood paired-sites test.Likewise, GlCAKlike (gi: 292497120) has been proposed as a CDK7 from on yeast . In our Encephalitozoon is a member of the RNAP II "CTD clade," TBlastN searches of the complete genome of Encephalitozoon found six CDKs but none show a phylogenetic affinity to CDK8.A robust CDK8 family is recovered with strong support values in both distance bootstrap and Bayesian inference. Like CDK7, this family includes putative orthologs only from members of the "CTD-clade," specifically yeasts, animals and plants. Although the microsporidian Encephalitozoon, animals, plants and Plasmodium. This group is divided into two well-defined sub-clades. One of them consists of BUR1 from yeast along with CDK9 orthologs from animals; the other contains CTK1 from yeast, CDC2L5 and CrkRS from human, and apparent orthologs from Drosophila and Caenorhabditis, both plants, and Plasmodium. A putative CDK9 also is found in Encephalitozoon, but falls at the base of the larger CDK9 grouping and does not associate clearly with either subgroup  appear to be transcription-related kinases, PfCrk1 groups with the CDK10/11 family, and PfCrk3 with CDK9.In an effort to understand the broader evolutionary history of CDKs, three deep-branching protists with complete genomes, nce data , a relatDictyostelium discoideum that has a canonical RNAP II CTD, and Leishmania major, Cryptosporidium parvum and Entamoeba histolytica, which all lack a CTD (see The phylogenetic distribution of protistan kinases indicate that cell-cycle related kinases are present, or at least their functions are more strongly conserved (see discussion above regarding CTD/CDK co-evolution), in a more diverse array of eukaryotes than are transcription-related kinases. This pattern also is seen in a more widely-sampled analyses including CDKs from a number of organisms with incompletely sequenced genomes, including  CTD see . Thus, tTrypanosoma [Drosophila and C. elegans. The CDK5 family has undergone expansion in metazoans, including PFTAIRE and PCTAIRE kinases, and putative orthologs of CDK5 are detected in Plasmodium, Trypanosoma and Giardia. Interestingly, no CDK from plants associates strongly with the CDK5 group, while the CdkB-type kinases, which are specific to plants, branch as sister to a broader CDK1/CDK5 clade. Our overall results suggest that cell-cycle kinases have undergone extensive and independent evolutionary diversification in different eukaryotic lineages, and it may be difficult to classify many of them based on orthologous relationships in phylogenetic analyses. It may be that functional homologies, once established experimentally, will prove to be more consistent criteria for designating CDK groups.Our analyses support well-defined groups for cell-cycle kinases CDK1, CDK4/6 and CDK5. An ortholog of either CDK1 or CDK5 is found in all of the organisms in our study, and these two families appear to be closely related. TbCrk3 was proposed as a functional homolog of CDK1 in panosoma ; here itArabidopsis (AtCdkF), and its apparent orthologs from animals, branching as a sister group. In addition to their role as CTD kinases, members of the CDK7 family in plants, animals and fission yeast can function as a CDK-activating kinase (CAK) [Arabidopsis [Drosophila and Caenorhabiditis, exhibits only CAK activity but no CTD kinase activity. Consistent with the phylogenetic relationships recovered in our analysis, human CCRK and other animal orthologs were recently shown to have CAK activity [Arabidopsis have diversified substantially, and may be regulated in different ways from those in yeast, animals, and even rice.Clear orthologs of CDK7 from animals, plants, yeasts and Microsporidian are strongly supported as a core family, with CDK-activating kinase from se (CAK) ,42. Unlibidopsis . AtCdkF activity . In contactivity . ApparenScCAK1 and SpCSK1 from yeasts also have CAK activity; however, despite their functional similarity to kinases in the CCRK group, they do not group with animal or plant CAKs Fig. ; howeverEncephalitozoon. Our further blast results (unpublished data) failed to identify any of the units of the CDK8/SRB10 (SRBs8-11) sub-module in the Microsporidia suggesting a loss of CDK8/SRB10 unit from these highly reduced parasites.CDK 8 (SRB10 in yeast) is a component of the multi-subunit Mediator complex, which transduces signals from cis regulatory elements to RNAP II; it is proposed to inhibit transcription initiation by phosphorylation of the CTD. CDK8/SRB10 and its partner cyclin C/SRB11, together with SRB8 and SRB9, form a specific sub-module that is variably associated with the RNAP II holoenzyme, and potentially with the free mediator complex . ApparenEncephalitozoon suggests CDK8/SRB10 function is absent from the Microsporidia. The loss of CDK8 from Microsporidia, along with absolute conservation of CDKs7 and 9 in all members of the "CTD-clade" . The latter is the only apparent ortholog of a CTD-directed kinase (CDKs 7, 8 or 9) identified in our analyses from any organism outside the "CTD-clade." It remains to be determined whether PfCRK3 possesses the P-TEFb function of CTK1, since it is the only protistan sequence present in either CDK9 sub-group, and the RNAP II CTD has not been conserved in apicomplexans or their closest relatives  [Arabidopsis and 8 from Oryza) (Table Arabidopsis and Oryza (Oryza is extremely similar to OsCdkC1 and very likely a second copy of CdkC (CDK9) from Oryza. There is no evidence of biological functions for these kinases as yet, but our results indicate that they are part of a large CDK9 complex specific to plants.In addition to the two previously identified copies of CDK9 (CdkC1 and CdkC2) from  (CdkC1) ,34, our nd Oryza . Os1562.Drosophila, while CDK11 occurs in human, Drosophila and Caenorhabditis. Three putative CDK11 orthologs were found in plants (two from Arabidopsis and one from Oryza). CDK10 has been implicated in the regulation of the G2/M phase of the cell cycle [S. pombe and PfCRK1 from Plasmodium show close relationships to the CDK10/11 family, but no function has yet been determined for these kinases either.In this group, orthologs of CDK10 are found only in human and ll cycle , but a cll cycle . CDK11 all cycle . The cloThe apparent co-evolution between the CTD and certain CTD-specific kinases suggests an explanation for strong stabilizing selection on CTD structure in some eukaryotes, and its complete degeneration in others. Based on the genomes examined in this study, either the origins of CDK7 and CDK8 in an unknown ancestor of the "CTD-clade," or the canalization of reversible phosphorylation of the CTD in some eukaryotic groups but not others, could account for the variation seen in RPB1 C-terminal structure. In either case, once thoroughly "locked" into RNAP II function, the CTD must have recruited other transcription and processing related proteins into a growing machinery of the "transcriptosome" . Our resThis work also provides a new perspective on the overall evolution CDKs and evolutionary relationships among kinase families. Our combined genomic and phylogenetic analyses suggest that transcription-related kinases originated later than cell cycle-related CDKs. Finally, our results point to potential functions for a variety of previously uncharacterized kinases, based on their apparent orthologous relationships to defined CDKs. Additional completed genomes, particularly those from broadly diverse protists , will be critical to address these questions further. Such comparative analyses will be invaluable in helping to guide experimental studies, which ultimately are required to verify the functional properties of each putative CDK.Representatives of all previously identified CDKs from budding yeast and human were obtained from Genbank, and used as probes in TBlastN and PSI-Blast  against Initially, a number of inferred protein sequences were grouped into six subsets according to clear similarities to specific CDK family orthologs. These subgroups first were aligned in CLUSTAL X , and thea priori trees. Trees were sampled from the posterior probability distribution every 100 generations. The empirical burn-in required for likelihoods to converge was less than 100,000 generations; an additional 400,000 generations were run and the first 100,000 were excluded from analysis of Bayesian posterior probabilities. Thus, a total of 4,000 trees were examined to determine the 50% majority-rule consensus tree and Bayesian support values. In addition, 1000 distance (PROTDIST + NEIGHBOR) bootstrap replicates were performed in PHYLIP 3.573 [Maximum-likelihood (ML) estimates of substitution parameters were made with the program TREEPUZZLE-50  assumingIP 3.573 , also usa priori alternative hypotheses regarding CDK7 evolution were compared by KHT likelihood paired-sites tests [Plasmodium or GlCAKlike from Giardia, which previously have been characterized as a CDK7 orthologs [a priori hypotheses of orthologous relationships to CDK7 were significantly worse than the Bayesian consensus tree.Several es tests . Trees wrthologs ,38, to gZG was primarily responsible for database searching and assembly of CDK genes. ZG and JWS performed phylogenetic analysis. ZG drafted the manuscript and figures and JWS contributed editorial revisions. All authors read and approved the final manuscript.Arabidopsis and Oryza. The phylogenetic weight of these latter plant sequences disrupts inferred relationships among CDK9 orthologs as shown in Fig. 1. Support values are from Bayesian inference and only values above 50% are shown. As in Fig. 1, CDK names in red are from groups in which the CTD is not strongly conserved, those in blue from members of the "CTD-clade." Inferred groups of CTD-directed CDKs 7, 8 and 9 are shown on the tree.The single most likely tree, with branch lengths, recovered from 16,000 ML trees in the posterior probability distributions of four separate iterations of Bayesian inference. Thirty-two additional sequences were added to this analysis, and are indicated in bold in Table 1. They represent CDKs identified in incomplete genomes of organisms from which CTD structure is known, as well as a large amplification of apparent plant-specific orthologs of CDK9 from Click here for fileOriginal protein sequence alignment.Click here for fileEdited protein sequence alignment.Click here for file


Have you ever noticed that long-distance runners and sprinters seem specially engineered for their sports? One's built for distance, the other speed. The ability to generate quick bursts of power or sustain long periods of exertion depends primarily on your muscle fiber type ratio , which depends on your genes. To this extent, elite athletes are born, not made. No matter how hard you train or how many performance-enhancing drugs you take, if you're not blessed with the muscle composition of a sprinter, you're not going to break the 100-meter world record in your lifetime. (In case you'd like to try, that's 9.78 seconds for a man and 10.49 seconds for a woman.)Of course that doesn't prevent those at the highest levels from using the latest performance enhancer to get that extra 1% edge. But wait until trainers hear about the Marathon Mouse. A new study by Ronald Evans and colleagues provides evidence that endurance and running performance can be dramatically enhanced through genetic manipulation.Skeletal muscles come in two basic types: type I, or slow twitch, and type II, or fast twitch. Slow-twitch fibers rely on oxidative (aerobic) metabolism and have abundant mitochondria that generate the stable, long-lasting supplies of adenosine triphosphate, or ATP, needed for long distance.  Fast-twitch fibers, which produce ATP through anaerobic glycolysis, generate rapid, powerful contractions but fatigue easily. Top-flight sprinters have up to 80% type II fibers while long-distance runners have up to 90% type I fibers. Coach potatoes have about the same percentage of both.Endurance training can enhance the metabolic performance of muscle types, and now it appears that training can also induce conversion between fiber types. Specific changes in gene expression trigger this oxidative fiber transformation, but the transcription factor responsible for engineering this shift was unknown. Evans and colleagues suspected that a nuclear receptor called PPARδ—a major regulator of fat burning in fat tissue that is also prevalent in skeletal muscle—might be involved.To investigate this possibility, the authors genetically engineered mice to express an activated form of the PPARδ protein in skeletal muscle. Type I fibers normally express higher levels of PPARδ than type II fibers, and the transgenic mice showed much higher levels of the protein than their normal littermates. The transgenic mice also had much redder muscles than the controls—type I fibers have high levels of myoglobin, the red-pigmented protein that facilitates the movement of oxygen within muscle—and elevated levels of proteins associated with mitochondrial biogenesis and operation. A final line of evidence indicating a type I fiber switch was the elevated level of specialized type I contractile proteins and decreased level of specialized type II contractile proteins in the transgenic mice. Interestingly, these same results were seen when naturally occurring (endogenous) PPARδ levels were stimulated in the normal mice . This suggests that muscle fibers can be transformed into type I endurance fibers by simply activating the endogenous PPARδ pathway.In a weight-conscious world, oxidative fibers are thought to offer resistance against obesity since obese individuals have fewer type I fibers than average-weight individuals. Sure enough, transgenic mice fed a high-fat diet gained far less weight than normal mice fed the same diet, even in the absence of exercise. The transgenic mice had much smaller fat cells, which the authors attribute to enhanced oxidative capacity of the muscle tissue, and improved glucose tolerance.  But what about performance? Remarkably, the marathon mice ran about an hour longer than controls, showing dramatic improvement in both running time and distance—increases of 67% and 92%, respectively.Altogether, these results show that PPARδ drives the conversion of type I muscle fibers by activating pathways that enhance physical performance and protect against obesity. The finding that endurance and running capacity can be genetically manipulated suggests that muscle tissue is far more adaptable than previously thought. Maybe Olympiads can be made after all—but don't give up on training just yet. A full understanding of the molecular basis of muscle fiber determination, including the interactions between PPARδ and its regulatory components, awaits further study.


Modeling the complex development and growth of tumor angiogenesis using mathematics and biological data is a burgeoning area of cancer research. Architectural complexity is the main feature of every anatomical system, including organs, tissues, cells and sub-cellular entities. The vascular system is a complex network whose geometrical characteristics cannot be properly defined using the principles of Euclidean geometry, which is only capable of interpreting regular and smooth objects that are almost impossible to find in Nature. However, fractal geometry is a more powerful means of quantifying the spatial complexity of real objects.s) surface fractal dimension  geometrical complexity of tumor vascular networks, and their behavior during computer-simulated changes in vessel density and distribution.This paper introduces the Ds significantly depends on the number of vessels and their pattern of distribution. This demonstrates that the quantitative evaluation of the 2-D geometrical complexity of tumor vascular systems can be useful not only to measure its complex architecture, but also to model its development and growth.We show that form of branched anatomical structures, in an attempt to define more appropriate methods of describing them quantitatively. This knowledge can be used to predict the aggressiveness of malignant tumors and design compounds that can halt the process of angiogenesis and influence tumor growth.Studying the fractal properties of neovascularity induces reflections upon the real significance of the complex  Under tendothelial cells which, together with pericytes (supportive vascular smooth muscle cells), carry all of the genetic information necessary to form tubes, branches and entire capillary networks.The cyclic nature of the microvascular bed in the corpus luteum provides a unique experimental model for examining the discrete physiological steps of angiogenesis in the life cycle of self-limited; however, this is not true of tumor angiogenesis which, once begun, continues indefinitely until the entire tumor is eradicated or the host dies. Without blood vessels, tumors cannot grow beyond a critical size (1–2 mm) or metastasize to another organ.However, many human diseases (including solid tumors) are driven by persistently up-regulated angiogenesis . In someAngiogenesis is one of the most complex dynamic processes in biology, and is highly regulated by a balance of pro- and anti-angiogenic molecules. It is now widely accepted that the "angiogenic switch" is "off" when the effects of pro-angiogenic molecules is balanced by that of anti-angiogenic molecules, and "on" when the net balance is tipped in favor of angiogenesis ,3. Pro- promotion, mediation and inhibition of angiogenesis, very little is known about its underlying complex dynamics. Vasculature and more generally tubular organs develop in a wide variety of ways involving many cell processes [Although considerable advances have been made in our molecular and cellular knowledge of the rocesses -8.non-linear dynamic system that is discontinuous in space and time, but advances through qualitatively different states. The word state defines the configuration pattern of the system at any given moment, and a dynamic system can be represented as a set of different states and a number of transitions from one state to another over a certain time interval [In mathematical terms, angiogenesis is a interval ,10.a) endothelial cells are activated by an angiogenic stimulus; b) the endothelial cells secrete proteases to degrade the basement membrane and extra-cellular matrix; c) a capillary sprout is formed as a result of directed endothelial cell migration, d) grows by means of cell mitoses and migration, and e) forms a lumen and a new basement membrane; f) two sprouts come together to form a capillary loop; and g) second-generation capillary sprouts begin to form [At least seven critical steps have so far been identified in the sequence of angiogenic events on the basis of sprout formation:  to form ,11,12 , which is used to allow a histological assessment of tumor angiogenesis. The results of studies carried out over the last decade have suggested the value of using tumor MVD as a prognostic index in a wide variety of solid cancers, and it has also recently been assumed that MVD may reveal the degree of angiogenic activity in a tumor. On the basis of these assumptions, the quantification of MVD is thought to be a surrogate marker of the efficacy of anti-angiogenic agents as well as a means of assessing which patients are good candidates for anti-angiogenic therapy. However, MVD has a number of substantial limitations, mainly due to the complex biology characterizing tumor vasculature , and thesurface fractal dimension (DS) as a quantitative index of the 2-D geometrical complexity of vascular networks and their behavior during computer-simulated changes in vessel density and distribution.However, quantitative descriptors of its geometrical complexity can be usefully abstracted from the fractal geometry introduced by Benoit Mandelbrot in 1975 ,21. We hThe human vascular system can be geometrically depicted as a complex fractal network of vessels that irregularly branch with a systematic reduction in their length and diameter .irregularity of their shape; b) the self-similarity of their structure; c) their non-integer or fractal dimension; and d) scaling, which means that the measured properties depends on the scale at which they are measured [Fractal objects are mainly characterized by four properties: a) the measured .self-similarity. Unlike geometrical self-similarity, which only concerns mathematical fractal objects in which every smaller piece is an exact duplicate of the whole (e.g. Koch's snowflake curve, Sierpinski's triangle and Menger's sponge), statistical self-similarity concerns all complex anatomical systems, including tumor vasculature. The smaller pieces constituting anatomical entities are rarely identical copies of the whole, but more frequently "similar" to it and, in such systems, the statistical properties of the pieces are proportional to the statistical properties of the whole [One particular feature of fractal objects is that the schemas defining them are continuously repeated at decreasing orders of magnitude, and so the form of their component parts is similar to that of the whole ,21: thishe whole .Dimension is a numerical attribute of an object that does not depend on its process of generation, and has been defined in two ways. The first is the topological or Euclidean dimension : 0 to a point (defined as that which has no part); 1 to a straight line (defined as a length without thickness), 2 to a plane surface (defined as having length and thickness, but no depth); and 3 to three-dimensional figures (a volume defined by length, thickness and depth). The second was introduced by the mathematicians Felix Hausdorff and Abram S. Besicovitch, who attributed a real number to every natural object in E lying between the topological dimensions 0 and 3 . The Dγ and D of all Euclidean figures are coincident (Dγ = D), but this is not true of fractal objects in which D is always >Dγ.Benoit Mandelbrot uses the symbol 2 (plane surface) and 3 (volume), and any two-dimensional section of a vascular system  has a dimension lying between 0 (the dimension of a single isolated point) and 2 when the sectioned vessels entirely fill a plane surface  of an irregular object differ when inspected at increasing resolutions that reveal an increasing number of details [scaling window of an experimentally established measure of length ε1-ε2 : i.e. the fractal dimension remains invariant at different magnifications [The irregularity and self-similarity underlying entities -26. In o details . Over th-ε2 Fig. , within ications -27.We have developed a computer model to simulate the geometrical complexity of a histological two-dimensional section of a tumor vascular tree that automatically generates an unlimited number of images with a changeable density of vessels irregularly distributed on a planar surface.a) the number of vessels; and b) their distribution in the surrounding environment. The vessel distribution patterns were randomly generated using different time-dependent seeds for random number function generation.In order to simplify the model, we considered all of the vessels as rounded, unconnected objects of equal magnitude Fig. . As the DS were estimated using the box-counting method [One thousand images were automatically generated for each vessel density (from five to 50 vessels, with the number being increased by five in each group), and their g method .DS was automatically estimated using the equation:ε is the side-length of the box, and N(ε) the smallest number of boxes of side ε required to completely contain the irregular object      DS = dd is the slope of the graph of log [N(ε)] against log (1/ε), in a fixed range of side-lengths (ε 1-ε 2) empirically evaluated by visualization [where lization -29.t as required for parametric variables. p values of less than 0.05 were considered statistically significant.All of the data are expressed as mean values ± standard deviation, and the results were analysed using the Statistica software package (StatSoft Inc. Tulsa, USA). Unvaried analysis was performed by means of the Student DS values can be obtained for images with the same vessel density  when higher vessel densities were considered in the system  and functions . Complexity is a real quality of organized biological matter that is mainly manifested in the living world as diversity and organization. No two anatomical systems are exactly alike because of the enormous variability not only between the different members of a population, but also between the component parts of an organism. The word complexity has long been used descriptively in order to describe, for example, a large number of genes or cellular interconnections [structure of a system (i.e. an intricate architecture or the existence of many different component parts with varying interactions) or its non-linear functions  [One of the most important and distinctive characteristics of biological systems is the nections , but comer time) .space and time, and greatly biases any quantitative method that tends to idealize it as a smooth and regular Euclidean object.The vascular system is a complex network consisting of branched tubes of different sizes that are irregularly settled in the surrounding environment ,7. This fractal objects because of their irregular shape , self-similar structure, non-integer dimension and dependence on the scale of observation  [However, both normal and tumor vasculature can more properly be considered  effect) ,35-37.DS as a quantitative index of the 2-D spatial complexity of the vascular tree, in order to provide a closer-to-reality measure of this complex anatomical entity  as a subset of the range of theoretically possible morphologies [The theory underlying gularity ,21. The hologies .DS also comes from the fact that, like any other complex biological system, the vascular tree cannot be correctly quantified by measuring its individual properties (i.e. micro-vessel density, MVD). DS is a parameter that depends on: a) the number of vessels; b) the spatial relationships between the vascular components; and c) the interactions between the vascular components and the surrounding environment. In other words, its estimate is "ecologically" important because it provides a quantitative index of the "habitat structure".The significance of DS increased with the number of vessels making up the system  Ds can be an estimate of the 2-D geometrical complexity of the vascular system. As 2-D vascular complexity depends on the number of vessels and their distribution pattern, the use of MVD quantification alone to measure the angiogenic dependence of a tumor is strongly biased because the number of vessels does not reflect the number of tumor cells that can be supported by a vessel. Moreover the metabolic needs of cancer cells vary with the tissue of origin and change with tumor progression [gression .b) DS depends on the degree of vessel contiguity and continuity. These two geometrical properties determine what is called the intercapillary distance, and are not only involved in the spatial complexity of tumor vasculature, but also reflect the inviolable demand of a growing tumor for sufficient levels of nutrition and oxygen exchange. Inter-capillary distances are locally defined by the net balance between pro- and anti-angiogenic molecules in each microtissue region, as well as by non-angiogenic factors such as the oxygen and nutrient consumption rates of tumor cells. In normal tissue, vessel density fairly accurately reflects cell metabolic demands because evolutionary pressures have led to close and efficient coupling between vascular supply and metabolic needs. In tumors, the close coupling between vascular density and oxygen or nutrient consumption (i.e. the environment) may be loosened [loosened , thus alloosened ,38.c) DS falls between 0 (corresponding to the Euclidean dimension of a point) and 2 (the dimension of a plane). The more DS tends towards 2, the more the analyzed vascular configuration tends to fill a 2-D space and the greater its geometrical complexity.complex geometry of tumor vasculature and its well-known biological characteristics [In conclusion, the present study indicates that the eristics  mean thaeristics ,39-41.Scientific knowledge develops through the evolution of new concepts, and this process is usually driven by new methodologies that provide previously unavailable observation. The potential broad applicability of the proposed quantitative index makes it possible to explore the range of the morphological variability of vasculature that can be produced in nature, thus increasing its diagnostic importance in cancer research.Ds, Surface fractal dimension; 2-D, two-dimensional; VEGF, Vascular-endothelial growth factor; bFGF, basic fibroblastic growth factor; MVD, microvessel density.The author(s) declare that they have no competing interests.FG conceived, coordinated and designed the study and drafted the manuscript; CR, PC, BF, EEF, EC, MCI participated in designing the study and drafting the manuscript. All of the authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Understanding how the brain processes and stores information depends in large part on knowing which neurons are involved in a particular process and how they're organized into functional networks. Each of the 10 billion or so neurons in the brain has thousands of connections to other neurons, sending (via axons) or receiving (via dendrites) the signals that allow us to think. Each neuron can transmit signals to both local and distant neurons, and it is by mapping these networks that neuroscientists can discern correlations between neural connectivity and physiological responses and ultimately unveil the computational algorithms underlying brain function. Since the beginning of cellular neuroscience at the end of the 19th century, neuronal connections have been explored by tracing axons and dendrites under the light microscope. But even with the resolution of state-of-the-art light microscopy, this approach works only if a small subset of neurons is stained and thus leaves most of the network hidden.Electron microscopy, on the other hand, can provide the spatial resolution necessary both to resolve processes in densely packed neural “wire bundles” and to identify synapses faithfully, but individual electron microscopic images are restricted to two dimensions. Transmission electron microscopy provides cross-sectional images through tissue, while scanning electron microscopy typically provides the appearance of 3D but in reality maps only the specimen surface and is thus blind to the connections within. It's possible to wrest 3D information from the transmission electron microscope by using tilt-series tomography, but sections can't be much thicker than 1 micron (a millionth of a meter). Data from thicker volumes can be obtained, but the process so far has been so painstaking and time-intensive—it involves, among other labor-intensive tasks, manually reconstructing serial sections—that few undertake it.It should, however, be possible to get similar data with “serial block-face imaging,” which involves repeatedly cutting section after section from a plastic-embedded block of tissue and photographing what's left. Scanning electron microscopy is needed for this task, but sample preparation methods are like those used for transmission electron microscopy, albeit with a few additional steps to enhance contrast.This is exactly what Winfried Denk and Heinz Horstmann have done to obtain “truly 3D datasets” using a method they call “serial block-face scanning electron microscopy” (SBFSEM), for which they constructed a “microtome” that goes inside the scanning electron microscope chamber. The resolution achieved is sufficient to reveal “even the thinnest of axons” and identify synapses. The SBFSEM method can generate stacks of thousands of ultra-thin sections, 50–70 nanometers (a nanometer is a billionth of a meter) thick, generating 3D datasets to reconstruct the topology and circuitry of neurons in brain tissue.The authors' custom-designed microtome holds the tissue block in a way that ensures image alignment and maintains focus; all the while the specimen surface is positioned close enough to the objective lens to allow high-resolution imaging.Denk and Horstmann expect that with this method they might ultimately be able to cut sections thinner than the 50 nanometers that their current setup manages. This then would allow them to cut sections even thinner than what is routinely possible in conventional transmission electron microscopy. While the authors doubt that the lateral resolution will ever reach that of transmission electron microscopy, they also argue that such high resolution may not actually be needed to trace neuronal connectivity. On the other hand, the method accelerates 3D electron microscopic data collection “by several orders of magnitude” by obviating the need for the labor-intensive adjustments to correct alignment and distortion required by other methods, an advance that is crucial for large-volume neuroanatomy and might, in addition, open up many hitherto inaccessible problems to ultra-structural investigations.


The causes of PBIS are well known, consisting of various decisions about reporting that are influenced by the data. But its impact is not generally appreciated, and very little attention is devoted to it. What attention there is consists largely of rules for statistical analysis that are impractical and do not actually reduce the bias in reported estimates. PBIS cannot be reduced by statistical tools because it is not fundamentally a problem of statistics, but rather of non-statistical choices and plain language interpretations. PBIS should be recognized as a phenomenon worthy of study – it is extremely common and probably has a huge impact on results reported in the literature – and there should be greater systematic efforts to identify and reduce it. The paper presents examples, including results of a recent HIV vaccine trial, that show how easily PBIS can have a large impact on reported results, as well as how there can be no simple answer to it.PBIS is a major problem, worthy of substantially more attention than it receives. There are ways to reduce the bias, but they are very seldom employed because they are largely unrecognized. A study is more likely to appear in the literature, and thus be indexed and publicly available, if it shows a strong, statistically significant, or otherwise "better" result. A study that does not show such results has a greater chance of remaining hidden in a file drawer, either because the author (or funder) does not think the result is worth mentioning, or because journals are less interested in publishing studies that find "nothing". This form of publication bias, wherein readers have access to only a biased sample of the full distribution of results, is well studied, particularly in the literature on meta-analysis and other systematic reviews where the problem is most apparent (a primer on the topic can be found at the Cochrane Collaboration webpage ). Even wwhich results to publish produces bias. As with traditional publication bias, the tendency is to analyze data and choose to present results which are statistically significant, further from the null, or closer to what the researchers believe is the true value. The implications for the literature as a whole, though, are much the same as the file drawer bias. I label this "publication bias in situ" (PBIS) because the biased reporting of study findings exists within each individual research report . Despite the similarities between the file drawer bias and PBIS, there are fundamental differences. In particular, the bias from some studies having no published findings exists only at the level of the whole literature (no particular study can be said to be biased), while PBIS exists within the results reported from a single study (and thus exists in the literature as a whole by aggregation). More practically, PBIS is substantially more difficult to even identify, let alone correct.Far less attention is paid to the bias that occurs when some results from a study are published, but the choice of This paper defines and describes PBIS and identifies some of the choices that create it. The purpose of the paper is not to make novel technical or statistical claims. Indeed, there is probably no single statistical observation here that will not be clear to a skilled data analyst (or, indeed, that could not be explained to anyone competent in middle-school-level math). The literature includes many observations about issues relating to PBIS. Yet the challenge to the validity of the entire health science literature posed by PBIS – arguably a greater issue than conflict of interest, traditional publication bias, or any other commonly discussed threat to the integrity of the literature – has not received the attention it deserves.ex ante protocols were stated and followed, and is thus put on par with many protocol violations that could be considered mere technicalities. For example, a few years ago, a highly-publicized meta-analysis [In the literature about meta-analysis and systematic reviews there is substantial attention to file-drawer publication bias . But there is considerably less attention to possible picking and choosing which study results to report or statistical methods to use. To the extent that this is considered, it is often bundled with questions about whether analysis  called iThe literature and pedagogy related to observational inference, where the potential for PBIS is considerably greater than for well-designed experiments, seems to pay even less attention. Epidemiology textbooks typically discuss some of the methods that can reduce PBIS (e.g., well-defined protocols), but say little or nothing about the possible bias. Indeed, many study and data-analysis methodologies (some of which are noted below) that are typically taught in classes or by apprenticeship seem designed to create PBIS. Despite this, highly-trained experts summarize claims reported in the literature without mention of the likely bias, indicating an unawareness of the major implications of PBIS.In a notable exception, Hahn and colleagues ,4 discusIt is not clear how common PBIS is or how large the resulting bias, but a few efforts to find it suggest that the potential is great enough that it deserves much more attention.  Only by systematically addressing the problem are we likely to substantially affect it. Moreover, as will be discussed, statistical and research methods that ostensibly address some of the sources of PBIS are unsatisfactory, and a systematic attempt should be made to find better solutions.Reviewers (systematic or otherwise) of the literature can only see what researchers choose to report and highlight in their publications, and that choice can be biased in a number of ways. Researchers have great freedom in deciding exactly what to analyze, how to analyze it, and what to report. All research results are derived from data that can be used to measure many associations. Even the most narrowly focused clinical trial can be analyzed with the endpoint defined in different ways, stratified by age, etc. As with traditionally defined publication bias, the analyses that are deemed unworthy of publication are largely invisible to the scientific and policy community.Among the dimensions of freedom researchers have in deciding what to analyze and report are three choices illustrated by examples in this paper:(1) Which exposures and outcomes to consider in datasets with many variables.(2) Which functional forms to use to represent variables (e.g., how to divide continuous variables into categories).(3) Whether to conduct separate analyses by subgroup, and which subgroup results to emphasize.some question), there is no clearly wrong choice, and thus no clear way of concluding that a particular choice was biased. Fortunately, as should become apparent from the following analysis, PBIS results less from the choices made and more from what the choices are based on (which can often be inferred) and, to an even greater extent, the generally overlooked issue of how the results are presented (which can be easily observed).Making such choices is a legitimate part of research. Indeed, the choices must be made. But when those choices are primarily driven by what produces stronger (or otherwise "better") results, bias is created. This creates a difficult challenge: It is easy to recognize traditional publication bias . But since there is no clearly correct option for any of the above choices , creating a bias from what would be unbiased random variation. Systematic errors (confounding, measurement error, etc.) and methods for correcting for them create many additional opportunities for PBIS; however, for simplicity, systematic errors are ignored in this paper.Choice (1) in the above list has probably received the most attention in previous literature (for example, debates over whether to correct for multiple hypothesis testing and the appropriateness of data dredging). Despite the disproportionate attention, this choice is probably not the major source of PBIS, but it provides a familiar starting point.Many epidemiologic datasets are characterized by thousands or even millions of possible combinations of exposures, endpoints, and covariates. It is frequently assumed that statistical science tells us the "right" way to deal with this challenge, but current practice (not to mention the confusion of students coming out of epidemiology and biostatistics classes) makes clear that there is substantial disagreement among viewpoints about how to apply statistical methods when dealing with multiple hypotheses or measurements. Further consideration makes it clear that statistical rules cannot actually provide clear answers.At one extreme are viewpoints such as, "We must correct measures of statistical certainty  or confidence interval widths) whenever multiple comparisons are made using the same dataset" and even, "statistical analysis can only be legitimate for a short list of pre-specified hypotheses." At the other extreme are viewpoints such as, "regardless of how many comparisons are examined, each can be considered and statistically tested as if it were the only one," and, "it does not matter at all if a hypothesis was proposed after looking at the data." The impasse in this debate seems to stem from both sides attacking straw men, without recognizing that each side has a stronger case some of the time. This can be illustrated with examples.Helicobactor pylori infection contains data that is later used to look at the relationship of household crowding and performance in school. It is difficult to understand why we would make an adjustment when doing the second analysis because we have already done the first . A logical extension of that argument would be to consider the dataset that contains all quantitative human knowledge , and declare that we have to adjust for every statistical analysis ever done, effectively precluding further statistical analysis.A cohort dataset originally used to report the relationship of drinking water source and the occurrence of H. pylori infection. The dataset contains dozens of different measures of food and nutrient intake, as is usually the case for nutrition data. This, plus multiple diagnostic tests for H. pylori which are sometimes discordant, creates a large number of statistical comparisons, any of which could be described as supporting the plain language claim, "poor nutrition affects H. pylori status." A typical approach is to find individual comparisons that support the hypothesis, presenting only these comparisons with statistical tests as if each were the only analysis conducted. The claim about the relationship between the particular measure of nutritional status and the particular measure of H. pylori status is accurate, as are the test statistics reported for that association. But the plain language conclusion (which would probably be drawn) was very likely to be supported by some relationship in the data by chance alone, even in the absence of any true underlying association. This fact is obscured by the reported unadjusted tests statistics or confidence intervals.Researchers investigate the hypothesis that poor nutrition increases the risk of in situ within a single published article, making it a bias of much greater magnitude than the file-drawer effect).As the second example illustrates, unrestricted picking and choosing of comparisons leads to publication bias. A lot of associations that were not deemed worthy of reporting never appeared in the literature, while the few that were "interesting" did. This problem is well known , correcting for multiple comparisons does not affect the reported biased estimates of effect size; changing test statistics and confidence interval widths does nothing to reduce the bias. This alone shows that the standard statistical corrections for multiple tests do nothing to solve the problem. The other standard method for trying to reduce PBIS, rules that limit analyses to pre-specified hypotheses and protocols, will throw away a lot of potentially valuable findings and is nearly impossible to operationalize because detailed protocols require advanced knowledge that may not exist and can never be specified unambiguously.The solutions offered by statistical rules – corrections for multiple hypothesis testing or restricting analysis to Frequentist statistical theory cannot offer a solution to this problem because PBIS, like the file-drawer problem, is not a matter of statistics. The second example illustrates where the problem primarily lies: in the plain-language reporting of results. The statistics that describe the relationship between a particular exposure and outcome measure could be exactly right, but the claim about good nutrition (as a generic concept) and infection status (as if we had a gold standard measure), which will likely be emphasized in the paper and its title (and press releases) and will likely stick readers' minds, might be misleading. Consider how the result would be interpreted if there were a table reporting every analyzed comparison, most of which showed little or no association. Most scientifically literate readers would realize the result was not so convincing, even though those same readers seldom think to object when – as is typical – only one or a few results are reported. By contrast, if the researchers in the first example reported the result of the previous study, it would be unlikely to change most readers' assessments.ex ante hypothesis or having "used up" the α), but without creating the PBIS that would result otherwise. Indeed, this appears to summarize the most obvious generic rule to reduce PBIS : publish everything.This suggests the simplest partial solution to the problem. By reporting a table of results from other comparisons considered, researchers could report their interesting result , and can include dozens or thousands of alternative analyses in appendices or links to data or software that allow the reader to review still more results. Of course, this opportunity is beneficial only if authors choose to take advantage of it (or editors and reviewers demand that they do).The implications of choice (2), the functional form for variables, can be clearly illustrated with a simple example. Consider a study with an exposure variable measured as 10 ordered categories . Assume researchers wish to analyze the association of a disease endpoint and a dichotomous definition of exposure. If there is no clear cutpoint for defining exposure, there are many options. There are 9 cutpoints that divide the observations into two categories, defining those above the cutpoint to be exposed and those below unexposed. Other options include comparing a group of highest categories to a group of lowest categories, leaving out the middle, such as 8–10 versus 1–3, yielding an additional 36 possibilities.How will the researchers choose a definition of exposure? A typical procedure is to let the data inform the choice: The cutpoint that provides the clearest contrast between the exposed and unexposed is judged to be the right one, the "most sensitive" to the presumed effect. It should be immediately obvious that this procedure will bias the result away from the null.To illustrate this, consider a case-control study with 200 subjects . Calculations for this example and others are based on Monte Carlo simulation of different realizations of the data based on the assumed underlying relationship. All simulations were performed using Crystal Ball (Decisioneering Inc., Denver, Colorado, USA).Assume that each of the 10 exposure categories is equally likely for cases and non-cases. If the researchers consider only the 9 cutpoints that dichotomize the data and choose the cutpoint for "exposed" to get the largest odds ratio (OR), the median result will be 1.5. Since the exposure and disease are not associated, this is clearly an upward bias. For those inclined to focus on statistical significance, the chance of observing a significant positive association at a one-tailed significance level of .025 is 13%. (Of course, for any single definition of exposure, the median OR is 1.0 and the chance of seeing a significant relationship is about 2.5%.)Even if researchers do not analyze their data in every possible way and report the strongest association, any decision to report results that is based on associations in the data (e.g., choosing between a cutpoint at 5 or at 6 based on which produces a stronger association) will create bias. Although this observation should be obvious to anyone with an understanding of statistics, letting the data have some influence on the choice is probably more the rule than the exception among researchers. It is often defended on the grounds that there was no way to know what the "right" cutpoint was before doing the study, and the study data is the only existing answer to the question. This is a legitimate point, but it does not reduce the resulting bias. Less scrupulous researchers – who are trying to support a preferred answer to further a policy agenda or advance their careers – need make no such explanation and can intentionally choose the extreme results.As with the file-drawer effect, results in the literature will tend to show effects greater than the true value. Most important, whichever analysis is reported, the plain-language result will be "we found an association between the exposure and the disease," and so collections of studies that each report an exposure-disease comparison with a greater-than-average association, and will seem to be confirming the same result, even though the comparisons are not the same.Extending this example to illustrate how PBIS can compare to traditional publication bias, assume now that there is a positive association between the exposure and disease. In particular, non-cases are still equally likely to be in each of the ten categories, while cases have respective probabilities for each category of (.069, .072, .077, .084, .093, .102, .112, .121, .131, and .139). These values were chosen so that the true OR is similar, whichever of the 9 cutpoints is chosen (for those interested, the numbers follow a logistic curve). True ORs round to 1.5 for all cutpoints.Consider a collection of studies of varying sizes, with fewer larger studies, as we would typically see in the literature, specifically 100 randomly generated studies (more than would likely exist, but better to illustrate) of random size . Note that to avoid committing the very type of transgression discussed in this paper – repeating an analysis until "good" results are found – the reported results are from the first and only run of the simulation.For a single definition of "exposed" values >5), a typical result appears in Figure , a typicCompare this to the results for the same 100 studies where the cutpoint is chosen based on the largest OR Figure . The disUnbiased random errors, when combined with picking and choosing functional forms, lead to biases in reported results. A solution to this problem is much less obvious than its existence.The commonly proposed solution of only reporting results for pre-specified functional forms is not satisfactory, because it is difficult to enforce  and it forces us to ignore real unpredicted findings. Sticking to pre-specified analyses is especially unrealistic in research studies that collect data on many different risk factors and outcomes. Simply labeling all results that were not pre-specified with the caveat, "hypothesis generating," accomplishes nothing. If such results were actually treated as not yet "real", the problems of determining exactly which results were pre-specified and the loss of important serendipitous findings are reintroduced. Of course, results with the caveat are very seldom treated as less real than any others in the literature. Moreover, the "generated" hypotheses will never be retested in exactly the form reported, so the label is simply disingenuous. In sum, the proposed solutions to this type of PBIS are no more realistic or satisfactory as solutions than trying to eliminate traditional publication bias by requiring that all studies be adequately powered.A better family of solutions would be to establish a standard of reporting results for alternative variable definitions (perhaps in online appendices). Not only does this directly reduce PBIS by publishing more results, but it provides readers with a choice of results if they prefer different definitions (information that would be discarded by a pre-specified hypothesis rule). If results for every cutpoint from the 100 trials in the example were reported, the results, as pictured in Figure In many cases there will not be an obvious short list of variable definitions, but some alternative definitions should be obvious and others could be found in previous literature. A simple, but very useful, improvement would be a standard practice of reporting the closest possible replication of previous published analyses using data from the new study. This would directly address the problem of PBIS resulting from data-driven picking and choosing of functional forms . Repeating whatever analyses that previous researchers happened to choose is somewhat arbitrary, but each round of new research can also add a new preferred functional form. The key is that results based on previously published functional forms cannot be data driven and, unlike the standard practice of new studies that make different comparisons but describe them with the same plain language, would actually replicate (or fail to replicate) previous results.New York Times articles actually contained more complete study results. VaxGen tried to salvage some hope for the drug by pointing out the results for non-whites, suggesting that maybe it held promise for some populations [In 2003, VaxGen Corporation  released results of a large HIV vaccine trial in the United States, one of the highest-profile clinical trials of the year. The disappointing result showed a trivial reduction in incidence among the treatment group compared to the placebo group. But the three non-white racial groups (black, Asian, and "other") each showed a substantial reduction in incidence Table . VaxGen ulations . VaxGen'ulations -12.Given the failure to publish the results in a scientific journal, some might argue that VaxGen was guilty of traditional publication bias – not publishing unfavorable results about its products – a charge that is currently being leveled at many drug companies. However, the company actively released fairly complete study results and accompanying analysis to the press, and the results appeared in forums that are more widely read than all scientific journals combined, so the results were clearly not buried in a file drawer.But despite reporting their results, VaxGen was biasing what they published. Singling out of the result for non-whites is a clear case of PBIS. VaxGen did not give equal emphasis to the apparently harmful effect of the vaccine for whites. The company's subsequent report that the different results for whites and non-whites could not be due to chance  does not4-1 = 15 different hypotheses. To adjust for 15 implicit hypotheses makes it very unlikely that any will pass the statistical test, including the population as a whole, even for substantial associations.Setting aside some controversy that erupted about systematic errors in the data, what can we say about the results from the perspective of PBIS and chance alone? To look at the result for non-whites, with its one-tailed p-value of .002, ignoring the fact that the subgroup definition was clearly data-driven, would overstate the finding, as suggested in the preceding examples. But a naive correction for multiple hypothesis testing would make the opposite error. Setting aside the possibility that other covariates would have been used to stratify the data had they produced subgroups with positive findings, the combination of the 4 racial groups implies a test of 2An alternative is to ask the question, "if the vaccine has no effect, what are the chances of seeing, in any racial group or combination thereof, a result at least as strong as the observed 63% reduction?". Phrased that broadly, simulation shows the answer is about 20%. However, most of the 20% comes from the unstable results for the two smallest groups, Asians and "other". Restricting the analysis to combinations of racial groups that contain black, with or without Asians or other, the probability is only 2.1% .So, what is the right answer? We must return to the observation that there is never a single Right Answer from a study; the quality of an answer always depends on what question was being asked. Did VaxGen find a successful vaccine? Clearly not, as the relative risk for the whole population shows. Should the result for non-whites be considered unlikely to be due to chance ? It depends on whether you consider it the answer to the question "does the vaccine show a result for non-whites?", in which case the answer is 'yes' , or "does the vaccine show a result for any racial group?" in which case the answer is 'it is fairly likely we would see such a result due to chance alone.' It is worth noting how this illustrates a popular fallacy in data analysis: Frequentist hypothesis testing is not the objective exercise that some think it to be; it depends on subjective decisions about what to test.ex ante hypothesis that the vaccine would work only in non-whites or because the multiple-hypothesis correction for hundreds of possible racial and other subgroups makes it non-significant. A general rule requiring us to ignore interesting but surprising findings is a huge waste of information. Requiring a data-driven subgroup analysis to be biologically plausible before reporting it offers no solution, since we can usually construct a story to explain whatever associations appear in the data (it has been speculated that some genotypes get a benefit from the vaccine, and the frequency of those genotypes is strongly correlated with race).We might decide to infer that VaxGen would have emphasized the results from any racial subgroup that showed a positive result , and thex ante plan to analyze racial and other subgroups [ex ante, as if that should change our interpretation of the result, seems particularly absurd.To offer the "hypothesis generating" caveat would make little difference, scientifically or in the securities market. It is unrealistic to suggest that this "generated" hypothesis will be re-examined given the overall disappointing result. Two studies in Thailand  that suggests there is a beneficial effect for some people should equally emphasize any apparent harmful effects for other people (and vice versa). The fact that one result is statistically significant and the other is not should be of no consequence. Indeed, selecting which results to report based on statistical significance guarantees there will be publication bias . The reporting of the multiple subgroups results should be accompanied by statistics similar to those calculated here, instead of standard test statistics, so that readers know the probability that an estimated effect (or test statistic) at least as great as the one found would result from chance alone for any of the subgroup analyses. Such information will allow readers (researchers working on related projects, policy makers, investors) to focus on what they consider to be the answer to their own questions.The opportunities for PBIS, along with the almost universal failure to report research results in ways that avoid it, create the possibility that biased study results are very prevalent in the health science literature. Some of the causes of PBIS are well understood, but the enormity of its implications is largely ignored.PBIS can produce very misleading results, leading to widespread misperceptions and misguided policies. The examples presented here show just a few of the many ways that PBIS can result from random error and researchers'  choices. Neither the problem nor the solution lies in the mathematics of data analysis, so answers will not be found by appealing to statistical theory. The critical issue is the completeness of reporting and the plain-language interpretation of results.The simple solutions offered by statisticians are not satisfying or even realistic. All they really let us do is observe that in almost every research report, "the rules" have been violated. This is not helpful. Rather than a right-vs.-wrong view of proper use of statistics that would condemn most of the literature as invalid, we need a realistic way of addressing this problem. The solutions, like the solutions for traditional publication bias, will generally consist of doing a more complete job of reporting what can be reported.HIV = human immunodeficiency virusOR = odds ratioin situPBIS = publication bias None declaredSingle authorThe pre-publication history for this paper can be accessed here:


Why haven't we developed an HIV vaccine yet? And will the new roadmap from the Global HIV/AIDS Vaccine Enterprise help our efforts towards vaccine development? Ho addresses these crucial questions PLoS Medicine, is a clear and cogent document describing how major funders and stakeholders in HIV vaccine development should move forward in a collaborative fashion [The scientific strategic plan of the Global HIV/AIDS Vaccine Enterprise, published in this month's  fashion . There iWhy is it that we still do not have a protective vaccine against HIV 22 years after its initial identification? Many possible explanations come to mind.In the natural course of HIV infection, the virus wins 99% of the time, showing that specific immunity in an infected person is unable to completely clear the virus. We have also known for over a decade that primary HIV isolates are relatively resistant to antibody neutralization, probably because of a “protective shield” on the viral envelope glycoproteins, consisting of variable loop sequences and extensive N-linked glycosylations. Another explanation is the extreme plasticity of HIV that allows new viral variants to evade immune recognition in the same way that they escape from drugs. Moreover, superinfection by a second viral strain has been documented in a number of individuals who have already mounted immune responses to the initial HIV infection. Yet another problem is that the AIDS research community has yet to uncover the correlates of immune protection in vivo. Lastly, proven vaccine approaches from the past have either failed (whole killed virus and subunit vaccines) or faced seemingly insurmountable regulatory hurdles (live attenuated virus vaccine).Given these daunting obstacles, why have so many continued in the long struggle to develop an HIV vaccine? The answer must lie, in part, in the noble cause at hand. Yet there are also some encouraging clinical and experimental observations . Rare paThe scientific strategic plan of the Enterprise is spot-on in identifying the major roadblocks in HIV vaccine development, as well as in establishing the key scientific priorities as we see them today . It righOne might ask, however, whether there are potential downsides to the plan. In the name of continuing this important dialogue, I would like to offer one general concern. Arguably, the reason for the lack of an effective HIV vaccine today is rooted in the basic problems posed by the virus itself. What we need foremost are new scientific solutions, although a prim and proper “process and structure” in our approach will be helpful. The needed breakthroughs to develop a vaccine will likely emerge from the creativity of scientists doing fundamental research that is free of preconceived biases. It is my contention that great new ideas are as likely to come from curiosity-driven basic studies as from the mission-oriented approach that is represented by the new proposal. Therefore, the leadership of the Enterprise must safeguard against the kind of “group think” that is so pervasive in large collaborative endeavors of this nature. The views of a small number of researchers, no matter how smart or accomplished, must not supersede the collective wisdom of the scientific community at large.No doubt important contributions will be made by scientists working outside of the Enterprise. Measures should be taken to ensure that their views and approaches, even if deemed unconventional, are not stifled by the newly established system. Likewise, their research support should not be compromised because the creation of the Enterprise concentrates the funding into the hands of a relatively small number of designated scientists. To me this is a serious risk given the current “flat funding” at the National Institutes of Health.The authors of the “The Global HIV/AIDS Vaccine Enterprise: Scientific Strategic Plan” have laid out a timely and insightful plan to address perhaps the greatest public-health need of the millennium. This document and its later revisions will serve as useful guideposts for the AIDS vaccine development effort for years to come. To be successful in this mission, our research community will ultimately need a specific “scientific blueprint” for making an HIV vaccine. That day will come only after we get another shot in the arm, infusing us with new knowledge and know-how. Is there any doubt that we need to redouble our investment in basic research on the challenges posed by HIV?


Anal smears are increasingly being used as a screening test for anal squamous intraepithelial lesions (ASILs). This study was undertaken to assess the usefulness and limitations of anal smears in screening for ASILs.The cytomorphological features of 200 consecutive anal smears collected in liquid medium from 198 patients were studied and findings were correlated with results of surgical biopsies and/or repeat smears that became available for 71 patients within six months.Adequate cellularity was defined as an average of 6 or more nucleated squamous cells/hpf. A glandular/transitional component was not required for adequacy. Dysplastic cells, atypical parakeratotic cells and bi/multinucleated cells were frequent findings in ASIL while koilocytes were infrequent. Smears from LSIL cases most frequently showed mildly dysplastic and bi/multinucleate squamous cells followed by parakeratotic cells (PK), atypical parakeratotic cells (APK), and koilocytes. HSIL smears contained squamous cells with features of moderate/severe dysplasia and many APKs. Features of LSIL were also found in most HSIL smears.In this study liquid based anal smears had a high sensitivity (98%) for detection of ASIL but a low specificity (50%) for predicting the severity of the abnormality in subsequent biopsy. Patients with cytologic diagnoses of ASC-US and LSIL had a significant risk (46–56%) of HSIL at biopsy. We suggest that all patients with a diagnosis of ASC-US and above be recommended for high resolution anoscopy with biopsy. For corresponding Editorial, please see Leiman, 2005 The incidence of anal squamous carcinoma and its precursor lesions has increased in recent years particularly among men having sex with men (MSM) . Prior tAnal and cervical lesions share many histological and pathological characteristics including the implication of human papilloma virus (HPV) in the pathogenesis of precursor squamous intraepithelial lesions and invasive cancer . Just asAfter approval from the IRB, 200 consecutive anal smears submitted from 198 patients were retrieved from the files of the pathology department at Cedars-Sinai Medical Center. The samples had all been collected from the anal canal using the Rovers endocervex brush , the Digene cervical sampler brush (Digene Corp. Gaithersburg, MD), or the brush from SurePath sample collection kit (TriPath Care Tech, TriPath Imaging, Inc. Burlington, NC) , parakeratotic cells (PKs), atypical parakeratotic cells (APKs), koilocytes, binucleated and/or multinucleated squamous cells (B/MSCs), and dysplastic cells. The number of cells exhibiting each of these morphologic features was recorded as none, rare (no more than 2 cells/smear), and present (3 or more cells/smear). For this study cellularity was defined as the average number of nucleated squamous cells per 40x high power field (nsc/hpf) calculated by counting 10 hpfs. All of the anal smears had been reported using a modified Bethesda 2001 System terminology recommended for cervical smears . After dRevised cytologic diagnoses were correlated with concurrent and/or follow up tissue biopsies or with repeat anal smears all obtained within six months. Statistical analyses were performed using the Fishers Test. A two sided p value of 0.05 was considered as significant .For purposes of this study we required an average of at least 6 nsc/hpf for cellularity to be considered adequate. This was based on the observation that only smears averaging 6 or more nsc/hpf included abnormal cytologic diagnoses ranging from ASC-US through HSIL whereas smears averaging 5 or fewer nsc/hpf were either NIL or ASC-US. 91% (181) of the 200 smears contained an average of 6 or more nsc/hpf. Of the 19 cases that averaged fewer than 6 nsc/hpf, 17 were designated as unsatisfactory and excluded from the study while two that contained atypical squamous cells were reported as ASC-US and included in the subsequent morphologic analysis. Of note, all of the 7 smears with HSIL and 16/17 smears with ASC-H were cellular with an average of 8 or more nsc/hpf.Anucleated squamous cells were present in smears that were NIL and in smears with diagnoses ranging from ASC-US to HSIL. There were numerous anucleated squamous cells in 7 smears that were reported as unsatisfactory. Among the abnormal smears, neither the presence nor number of anucleated squamous cells correlated with cytologic diagnosis.Three or more groups of G/TZ were present in 56% (103) of the smears while rare G/TZ were seen in an additional 18% (33) smears. Of these smears 68% (93/136) had an abnormal cytologic diagnosis (27 ASC-US, 45 LSIL, 14 ASC-H, 7 HSIL). In comparison 26% (47) of smears contained no G/TZ of which 68% (32) were reported as abnormal (15 ASC-US, 14 LSIL, 3 ASC-H) suggesting that the presence of glandular cells did not facilitate abnormal diagnoses. Again all of the 7 smears with HSIL and 12/17 smears with ASC-H contained 3 or more groups of glandular cells, while two smears with ASC-H contained only rare glandular cells.Parakeratotic cells were observed in 71% (130) of the smears in the study. Parakeratotic cells were observed in negative (63%, 37/58) as well as in abnormal (74%, 93/125) cases. In negative cases rare parakeratotic cells were observed more often (in 72%, 27 cases) whereas in smears with epithelial abnormalities presence of rare parakeratotic cells and frequent (>3) parakeratotic cells were about evenly distributed (46%, 43 cases vs 54%, 50 cases).APKs were found in 40% (74) of the 183 smears. The number of cases showing APKs increased with the severity of the dysplasia. There were 3 or more APKs in 22% (41) of the smears constituting 7% of ASC-US, 41% of LSIL, 53% of ASC-H and 71% of HSIL cases. Rare APKs were found in 18% (33) of the smears that were interpreted as ASC-US or above. APKs were not found in any of the negative smears and were seen in 72% of the SIL smears.Classical koilocytes were infrequent (17%) in all diagnostic categories Figure . Three oB/MSCs were observed more frequently in abnormal smears – 81% (101/125) as compared to 33% (19/58) of the NIL smears  of the smears diagnosed as LSIL contained 3 or more squamous cells with features of mild dysplasia  of the 183 smears constituting 39% (181) of the patients in the study. As shown in Table ASIL presents unique challenges in diagnosis and clinical management. By decreasing deaths from opportunistic infections, widespread use of highly active antiretroviral agents and other therapies have done much to improve survival of HIV infected individuals. However, because these therapies do not impact the incidence of HPV infections or malignancies in these individuals, the increased life span of HIV+ individuals probably provides the primary explanation for the rapid and continuing increase in HPV associated AIN that these individuals are experiencing -12. WithAnal cytology has been shown to be a cost-effective screening method for detection of ASIL in populations at high risk for anal carcinoma. To datePrior experience with anal smears as documented in the literature,17 reveaThere is a paucity of literature regarding criteria for adequate anal cytology samples. The 2001 Consensus Conference in Bethesda  suggesteThe presence of G/TZ was not a prerequisite for adequacy in our study. Smears with and without G/TZ detected the same percentage (68%) of abnormal cases. Although most HSIL and ASC-H smears contained 3 or more groups of G/TZs, absence of G/TZ did not correlate statistically with undetected AIN II/III lesions. Thus we do not consider the presence of G/TZ as essential for adequacy, a situation analogous to cervical Pap smears ,21,22. IOn review of the morphological features of AIN lesions in cytology smears, we noticed some salient features. Dysplastic cells were the most reliable indicators of ASIL/AIN. Typical koilocytes were infrequent, observed in only 17% of SILs, a finding previously observed by Darragh et al  who repoIn ASC-H and HSIL, high grade squamous cells are usually small, found as single cells or small sheets admixed with mildly dysplastic cells and atypical parakeratotic cells. Careful scrutiny is required to not miss these high grade lesions.Our experience with anal cytology also indicates that other infectious agents are rarely diagnosed in anal smears. Candida was present in one case. Herpes or trichomonads were not seen.To summarize, liquid based anal smears provide a sensitive method for screening populations at increased risk for ASIL but have a low specificity for predicting the severity of the lesion. Patients with cytologic diagnosis of ASC-US and LSIL have a significant risk of having HSIL and should be recommended for high resolution anoscopy with biopsy.The author(s) declare that they have no competing interests.SA participated in the acquisition, analysis and interpretation of data and helped to draft the manuscript.AEW participated in its design, in the acquisition, analysis and interpretation of data and helped to write the manuscript.PT participated in its design, in the acquisition, analysis and interpretation of data and helped to write the manuscript.SB conceived of the study, participated in its design, in the acquisition, analysis and interpretation of data and helped to write the manuscript.All authors read and approved the final manuscript.