Nanosilver particles are used in various clinical settings because of their antibacterial properties. However, their safety evaluation when used for gynaecological disorders has not been established. Nanosilver particles were administrated in the vagina of New Zealand rabbits, and the pathological appearance of the surrounding tissue was examined by hematoxylin–eosin staining and transmission electron microscopy (TEM) after 1 and 3 days of treatment. The nanosilver content was assessed by plasma mass spectrometry, and the presence of particles in the hepatic portal vein blood was assessed by TEM. The results of our study show that the vaginal administration of nanosilver particles caused ultrastructural changes to the vaginal mucosa, urethra and rectum, with accumulation of particles in all tissues. These results demonstrate a new migration route of nanosilver particles following vaginal administration. They also demonstrate, for the first time, that the vaginal administration of nanosilver particles can enter the blood circulation system by examining the hepatic portal vein blood under the TEM which is the most direct visualized evidence. Among the numerous medical nanomaterials, nanosilver has been used extensively because of its unique antibacterial properties. Despite the fact that the safety evaluation of nanosilver particles has not been clearly established, nanosilver products are currently used in various clinical applications in China.in vivo studies conducted in animal models have demonstrated that nanosilver particles accumulated in various organs when administered through the oral, intraperitoneal, intravenous and subcutaneous routes [et al. [et al. [Over the past decade, a large number of s routes . In mices routes . In a su [et al.  showed t [et al.  showed tDifferent nanosilver products have been used for the treatment of gynaecological cervicitis or cervical erosion because of their antibacterial properties. However, the safety of nanosilver products following their vaginal administration has not been evaluated. In the present study, we assessed the ultrastructural morphology of the surrounding vaginal tissues. Our results highlight a new migration route of nanosilver particles, and provide a scientific basis for the risk assessment of nanosilver products in women.The nanosilver particles were purchased from Alfa Aesar China (Tianjin) Co., Ltd. (Batch No. B18Z014). As assessed by transmission electron microscopy (TEM), the nanosilver particles were round in shape, and in ∼90%, the diameter ranged between 20 and 40 nm.The absorption and accumulation of nanosilver particles in surrounding tissue following vaginal administration was assessed in healthy female New Zealand rabbits weighting 2.5–3.0 kg. Based on the published literature, the typical clinical dose and practical circumstances, a dose of 0.1 g/kg of nanosilver particle was administered topically in the vagina of the rabbits.The animals were divided into a control group and two treatment groups. The sampling time points were: (i) pre-administration (control group), (ii) 1 day (24 h) following treatment and (iii) 3 days (72 hours) following treatment. At each time point, the animals were killed, and tissue samples were taken from the vaginal mucosa near the end of the urethra, the vaginal mucosa near the end of the rectum, the urethra and the rectum. All samples were washed with normal saline, and 1/3 of each tissue samples were immersed in a 10% formalin fixation solution for hematoxylin–eosin (HE) staining, 1/3 were immersed in a 2.5% paraformaldehyde and 2.5% glutaraldehyde fixation solution for TEM sample preparation, and 1/3 were used for the detection of nanosilver particles following digestion. At each sampling time point, blood samples from the hepatic portal vein were drawn for TEM analysis.All tissue samples were prepared according to standard HE staining techniques, after which their appearance was evaluated for pathological changes using an optical microscope.All samples were placed in a digestion tube filled with 3 ml of nitric acid, and left to be digested for 24 h. The samples were then placed in a fume cupboard and heated at low temperature until the solution liquid turned clear. The samples were heated until almost dry and left to cool. The residue in the digestion tube was dissolved in ultra-pure deionized water, and diluted to 25 ml. The amount of silver in each solution (i.e. the silver content in each sample) was analysed using plasma mass spectroscopy. The specific particle mass per unit (mg) within each different type of tissue was calculated .Each sample was fixed in a 2.5% paraformaldehyde and 2.5% glutaraldehyde solution at 4°C for 2 h. The samples were then washed twice in PBS for 10 min before being placed in a 1% osmic acid fixation solution for 2 h at 4°C. Afterwards, the samples were washed three times in ultra-pure deionized water for 10 min, and dehydrated with graded ethanol. The samples were then subsequently placed in an epoxy propane–resin solution (1:1) for 1 h at room temperature, an epoxy propane–resin (1:4) for 1 h at room temperature, and a pure resin infusion for 2 h at room temperature. The samples were embedded in an Epon812 epoxy resin and cut into 1 µm semithin sections. Each section was stained with methylene blue-azure and analysed under an optical microscope. Ultra-thin sections were stained with uranyl acetate-lead citrate and analysed against a copper grid , 8.About 3.0 ml of blood were drawn from the hepatic portal vein, centrifuged for 5 min at 3000 rpm, and prepared using the same method as for the TEM tissue samples.Under a standard optical microscope, the epithelial cells in the innermost part of the vaginal columnar epithelium of the mucosa were orderly and complete. Below the mucosa, cells were loosely connected to tissue, with the presence of small blood vessels surrounded by minor oedema. Surrounding the mucosa was overlapping ring-shaped smooth muscles, which contained blood vessels. The outer membrane did not feature any conspicuous inflammation.The cross-sections of the urethra of the control animals revealed the following characteristics: some of the epithelial cells in the stratified transitional mucosa epithelium were loose and detached. Below the mucosa, cells were loosely connected to tissue, which contained blood vessels and fibrocytes. The surrounding tissue presented slight oedema, but was not conspicuously inflamed. Outer to the mucosa were intertwined smooth muscles, which also contained blood vessels. The outer membrane of the mucosa did not present any conspicuous changes.The cross-sections of the rectum of the control animals revealed the following characteristics: between the epithelium and the lamina propria were multiple intestinal glands. The lamina propria contained blood vessels, as well as a few lymph nodes that were scattered around the blood vessels and which invaded cells. Outside the lamina propria were smooth muscles arranged in circular rings on the inner layer and longitudinally in the outer layer. Outside these muscles was the placenta percreta and fatty connective tissue, with no conspicuous changes .Figure At 1 and 3 days following treatment, the nanosilver content in the vaginal mucosa, urethra and rectum had all increased as compared with the control group. Because the nanosilver content remained stable 3 days after treatment, it suggests that it caused a localized accumulation in surrounding tissue .Table 1An assessment of the ultrastructural changes of the tissue surrounding the vagina was performed at 1 and 3 days following treatment with nanosilver particles. At 1 day post-treatment, several high density round nanosilver particles were observed in the tissue of the vagina mucosa, the urethra and the cells of the rectum . At 3 daThese results demonstrate that ultrastructural changes occur in the vaginal mucosa, the urethra and the rectum during the first 3 days following the administration of nanosilver particles. In addition, high density round nanosilver particles  were observed in the tissue of the vaginal mucosa, the urethra and the rectum.As with tissues, analysis at 1 and 3 days after treatment revealed several high density round nanosilver particles in the hepatic portal vein blood. Some nanosilver particles formed conjugates with red blood cell, while others formed conjugates with blood platelet .Figure Most studies have evaluated the biological safety of nanosilver particles through the detection of blood silver content , 10 follThe present study revealed that, following the vaginal administration of nanosilver particles, silver accumulates in the tissue surrounding the vagina which caused ultrastructural pathological changes not only to the vaginal mucosa but also to the urethra and rectum. We also observed the migration of individual or multiple high density nanosilver particles between these tissues, thereby creating a cycle of cytotoxic reactions. Collectively, our results demonstrate new migration routes of nanosilver particles when administered intravaginally.Transmission electron microscopy observations performed at 1 and 3 days following treatment revealed several high density round nanosilver particles in the hepatic portal vein blood, with some nanosilver particles forming bonds with red blood cell and blood platelet. Because the blood from the esophagus, stomach, intestinum tenue, intestinum crassum, pancreas, cholecyst and spleen flows through the hepatic portal vein, it is possible nanosilver particles might accumulate in more distant tissues. We are not aware of other studies demonstrating the formation of nanosilver particle conjugates with circulating blood cells. This is clearly of scientific relevance as our observations were made using TEM, the most direct visualization technique to date. By entering the blood circulation, our results suggest new migration routes of nanosilver particles following vaginal administration, and should prompt further research into their systemic distribution.In summary, our results show, for the first time, that the vaginal administration of nanosilver particles leads to (i) their accumulation in the tissue surrounding the vagina, causing ultrastructural pathological changes, (ii) the potential promotion of cytotoxic reactions and (iii) by entering the blood circulation, it suggest additional migration routes to be elucidated.


Nowadays, S53P4 bioactive glass is indicated as a bone graft substitute in various clinical applications. This review provides an overview of the current published clinical results on indications such as craniofacial procedures, grafting of benign bone tumour defects, instrumental spondylodesis, and the treatment of osteomyelitis. Given the reported results that are based on examinations, such as clinical examinations by the surgeons, radiographs, CT, and MRI images, S53P4 bioactive glass may be beneficial in the various reported applications. Especially in craniofacial reconstructions like mastoid obliteration and orbital floor reconstructions, in grafting bone tumour defects, and in the treatment of osteomyelitis very promising results are obtained. Randomized clinical trials need to be performed in order to determine whether bioactive glass would be able to replace the current golden standard of autologous bone usage or with the use of antibiotic containing PMMA beads (in the case of osteomyelitis). CurrentAfter implantation of S53P4 bioactive glass, surface reactions ensure deposition of a calcium phosphate layer when exposed to (body) fluid. Sodium, silica, calcium, and phosphate ions are released from the surface and increase the local pH and osmotic pressure. Thereafter, a silica gel layer is formed on the glass surface, and amorphous calcium phosphates precipitate on this layer. These amorphous structures then crystalize to natural hydroxyapatite, which starts the activation of osteoblasts for the formation of new bone –9. This 2, 4% P2O5, 23% Na2O, and 20% CaO [Various compositions of bioactive glasses have been developed because of their composition-dependent promising bone forming properties, antibacterial properties, and degradability. This review focuses on S53P4 bioactive glass , with the specific composition (by weight) of 53% SiO 20% CaO , 9. ThisThe first reported applications of S53P4 bioactive glass in clinical practice were performed in craniofacial surgery . ConventSuominen and Kinnunen  reported in vivo. All patients reported being satisfied with the treatment and good clinical outcomes were observed. Observed complications were not related to the use of S53P4 bioactive glass.In frontal sinus obliteration, small size bioactive glass granules (0.5–0.8 and 0.8–1 mm) have been used for treatment in 24 patients suffering from chronic frontal sinusitis . During 2; a thickness of 2 mm). One or two bioactive glass discs were used as graft material in eleven patients and when available crushed autologous cartilage or bone from the operative site was used. The number of patients where cartilage or bone was used was not reported. Treatment with bioactive glass discs was successful in 10/11 [There is no consensus in literature about a gold standard procedure in nasal septal perforation repair . Stoor ein 10/11  and 22/2in 10/11 . In bothin 10/11 , 24. TheTurunen et al.  used bioIn mastoid obliteration for the treatment of chronic otitis, S53P4 bioactive glass granules were used in three different studies –28. StooIn all aforementioned studies, beside good clinical outcomes, cosmetic results were described to be good as well. In orbital wall reconstruction, eyes were in the correct position postoperatively, and in septal perforation repair the septum was straight. Moreover, all studies described the absence of foreign body responses and infections, which are two major advantages of bioactive glass over other synthetic materials used in head and neck surgery . AbsenceOsteomyelitis is an infection of bone and bone marrow. Currently, osteomyelitis is commonly treated with surgical implantation of polymethyl methacrylate (PMMA) beads, mixed with antibiotics, in the anatomical area of osteomyelitis after extensive debridement and pulse lavage . These aRomanò et al. , 30 comp in vitro findings [Others have also reported successful treatment of chronic osteomyelitis in clinical practice. McAndrew et al.  describefindings . The angThe success rate of S53P4 bioactive glass granules in the treatment of osteomyelitis is 25/27 , 3/3 3232, and 9Limited clinical results are available for use of S53P4 bioactive glass in instrumented posterior spondylodesis with transpedicular screw fixation. In the treatment of degenerative spondylolisthesis, autogenous bone grafting is still the gold standard procedure . BecauseSince the bioactive glass granules show low fusion rates, it is needed to be concluded that bioactive glass granules, in its current form, cannot be used as a stand-alone solution for posterolateral fusion. Reasons for the bioactive glass not to perform as expected could be the high rotational and compressional forces that are present in the spine. Those forces are not present in other reported applications . High rotational forces in spinal indications have so far not been studied in depth.Similar to the spine, the tibial plateau is a mostly compressive but still multidirectional load-bearing bone structure. The standard treatment for depressed tibial plateaus is treatment with autologous bone grafts . Only onIn the grafting of bone defects that are a result of tumour removal, autologous bone grafts are the standard . Because21 of the 25 patients of a study by Lindfors et al.  were folThe reported findings by Lindfors et al. –38 showeThis review of the clinical evidence for the use of S53P4 bioactive glass showed good results in various clinical indications reporting a follow-up up to fourteen years postoperatively. One of the major advantages of S53P4 bioactive glass compared to autologous bone grafts (the gold standard treatment in most of the discussed applications) is its “off-the-shelf” nature and excellent bone healing capacity. Additionally, it can protect against bacterial adhesion and colonization on its own surface and possesses antimicrobial properties by hampering bacterial growth. In contrast to the two-stage osteomyelitis treatment with antibiotic containing beads, S53P4 bioactive glass offers a one-step treatment solution that eliminates an additional operation procedure which will cost extra time, tools, and added risks for the patients . MoreoveS53P4 was the only bioactive glass in this review because this composition of bioactive glass is most commonly used in clinical practice to date . HoweverAccording to long-term findings, S53P4 bioactive glass degrades slowly with remnants still visible fourteen years after implantation . HoweverIt is noteworthy that in none of the reported papers foreign body reactions or infections were observed in the studied patients, regardless of the clinical application. Some of the studied procedures are susceptible for infections, for example, the treatment of nasal septal perforation. Because of its antimicrobial effects, the treatment of osteomyelitis became a new indication for S53P4 bioactive glass. This is even more beneficial since the current treatment with antibiotics is rapidly becoming problematic due to the rise in antibiotic resistant bacteria. With clinical success rates at least comparable to the gold standard procedure , 31, 47,Results obtained in spondylodesis procedures did not match the expectations based on other clinical indications , 34. Cur in vivo angiogenic potential of S53P4 bioactive glass has not been published, to the knowledge of the authors. As indicated in literature, angiogenesis is a very important process in bone repair [To date, evidence on thee repair . Especiae repair . The posAnother factor that should be investigated is the influence of granule size in the various applications. In a rabbit model, it has been reported that bone growth was significantly more abundant in bone defects filled with granules of 0.68–0.8 mm than with 0.2–0.25 mm sized granules . HoweverThis literature review provides evidence that S53P4 bioactive glass can be an effective treatment of bone defects in various clinical situations. Long-term follow-up studies reported excellent results. Although resorption is usually not completely accomplished, the application of S53P4 bioactive glass in craniofacial surgery applications and grafting of benign bone tumour defects could be beneficial, especially when autologous bone grafting is risky or impossible.Treatment of osteomyelitis with S53P4 bioactive glass is safe and effective even in one-stage treatment options, without the addition of local antibiotics. Adequate debridement, proper defect filling, and adequate containment of the bioactive glass granules are essential. More clinical and health economic cost-effectiveness data is needed to determine if S53P4 bioactive glass can replace antibiotic containing PMMA beads as the current standard of osteomyelitis, most preferably utilising randomized controlled trials.


Astyanax mexicanus), which exhibit both an eyed and eyeless morph. Although eyed Astyanax surface fish have bony sclera similar to other teleosts, the ossicles of blind Astyanax cavefish generally do not form. We first sampled cavefish from multiple independent populations and used ancestral character state reconstructions to determine how many times scleral ossification has been lost. We then confirmed these results by assessing complementation of scleral ossification among the F1 hybrid progeny of two cavefish populations. Finally, we quantified the number of scleral ossicles present among the F2 hybrid progeny of a cross between surface fish and cavefish, and used this information to identify quantitative trait loci (QTL) responsible for this trait. Our results indicate that the loss of scleral ossification is common–but not ubiquitous–among Astyanax cavefish, and that this trait has been convergently lost at least three times. The presence of wild-type, ossified sclera among the F1 hybrid progeny of a cross between different cavefish populations confirms the convergent evolution of this trait. However, a strongly skewed distribution of scleral ossicles found among surface fish x cavefish F2 hybrids suggests that scleral ossification is a threshold trait with a complex genetic basis. Quantitative genetic mapping identified a single QTL for scleral ossification on Astyanax linkage group 1. We estimate that the threshold for this trait is likely determined by at least three genetic factors which may control the severity and onset of lens degeneration in cavefishes. We conclude that complex evolutionary and genetic patterns underlie the loss of scleral ossification in Astyanax cavefish.The sclera is the tough outer covering of the eye that provides structural support and helps maintain intraocular pressure. In some fishes, reptiles, and birds, the sclera is reinforced with an additional ring of hyaline cartilage or bone that forms from scleral ossicles. Currently, the evolutionary and genetic basis of scleral ossification is poorly understood, especially in teleost fishes. We assessed scleral ossification among several groups of the Mexican tetra ( The vertebrate sclera is the tough outer covering of the eye that provides structural support to the eye and helps maintain intraocular pressure. In humans and other mammals, the sclera is made up of a thick layer of fibrous collagen ; howeversonic hedgehog , indian hedgehog , and bone morphogenic protein 2  have all been implicated [Most of our understanding of scleral development and ossification comes from chicks (reviewed in ). In chiplicated . Followiplicated , and groMore recent research, lead primarily by Franz-Odendaal and colleagues, has focused on the scleral cartilage and ossicles of teleost fish . Like cAstyanax mexicanus, or the Mexican tetra, is a natural model system for the study of eye development, including scleral ossification [Astyanax surface fish have two scleral ossicles, which appears to be the ancestral state for this group [The teleost fish fication ,17,18. Tfication . Like mois group ,14. Scleis group . Startinis group .Astyanax appears to have a genetic basis. SF and CF inherit the presence/absence of scleral ossicles with fidelity from generation to generation [1 hybrid progeny of SF and CF exhibit scleral ossicles [Astyanax, and possibly other teleosts as well. For example, the loss of scleral ossicles is coupled with the degeneration of the embryonic lens in CF, and when tissues from the optic vesicle or lens of SF are transplanted into developing CF, the CF form ossicles [The presence/absence of scleral ossicles in neration , and theossicles , suggestossicles . Conversossicles . Lens reossicles . Since tossicles .Astyanax as a model system for ocular development and evolutionary genetics, the intraspecific variation in scleral ossicle number observed in this species makes it ideally suited to elucidate the evolutionary and genetic factors influencing scleral ossification in teleosts and other vertebrates. But, despite the wealth of recent research on scleral ossicle development in Astyanax described above  mapping. QTL mapping employs the statistical analysis of phenotypic and genotypic distributions in hybrids to identify regions of the genome responsible for variation in some trait of interest [Astyanax , but thinterest . This stx e.g., –26). QTL. QTLAstyAstyanax. First, we assess the distribution of scleral ossicles and ossification among different CF lineages by sampling seven different populations, including several from independently evolved “Old” and “Young” lineages [2 intercross of Astyanax SF and CF to elucidate the genetic basis of scleral ossicle number and identify QTL for scleral ossification. The results of this study suggests that the loss of scleral ossification has evolved convergently at least three times among Astyanax cavefish; however, the skewed inheritance of this trait in SF x CF F2 indicates that this seemingly simple phenotype is the result of an underlying genetic threshold with a complex genetic basis.In the present study we assess three aspects of scleral ossification in lineages . Second,This study was approved by the University of Maryland, College Park Institutional Animal Care and Use Committee (IACUC Protocol R-12-53 to WRJ). All fish were euthanized using a lethal dose of 250 mg/L buffered MS-222 following our approved UMCP IACUC protocol.Astyanax cavefish populations and their hybrids in order to assess the extent of scleral ossification and its genetic basis in this group. For the population analysis, we selected several members from the “Old” cavefish lineage–Chica (n = 3), Curva (n = 2), Sabinos (n = 3), Pachón (n = 3), and Tinaja (n = 4) caves–as well as two members from the “Young” cavefish lineage–Molino (n = 1) and Rio Subterráneo (n = 2) (1 hybrid progeny (n = 4)  . For our (n = 4) . For bot2 hybrid progeny (n = 196) resulting from a cross of Astyanax SF from Texas and CF from Pachón cave in order to identify QTL that contribute to scleral ossification. The details of this cross and the resulting genetic map have been described elsewhere [For our third analysis, we examined scleral ossification among the Flsewhere ,29. The To assess the extent of scleral ossification in all three analyses, we stained fish for bone and cartilage following standard protocols using alizarin red and alcian blue . For theFor the quantitative genetic samples, we stained only the left eye from each fish. We dissected the left eye using micro-dissecting scissors, working carefully to avoid piercing the eye or damaging the scleral ring. Once removed from the head, we re-fixed all eyes in 4% paraformaldehyde for 2 hours at room temperature. After dissecting the eyes, we stained them all at once in three 96-well boxes used to hold 1 mL pipette tips. To keep track of the samples, we placed each eye in an individual staining cradle created from a 1 mL pipette tip. We cut the nozzle from each pipette tip to the 200 μl mark, heated the sheared end under a Bunsen burner for 3–5 seconds until just soft, and then fixed a piece of nylon-webbing to the tip to allow stain to enter the cradle. After making these cradles, we placed one eye inside each individually-labeled pipette tip, placed each tip into the 96-well frame, and then placed the whole frame back inside the pipette tip box for staining. We stored all stained sclera in a solution of 1x PBS and 0.5% sodium azide at 4°C until further imaging. All sclera were imaged within one week of staining.2 eye cornea-down in a solution of 1x PBS and photographed the coronal (back) portion of each eye using a Zeiss Discovery v20 Stereomicroscope. This position gave us the best estimate of the degree of ossification surrounding the entire eye. After photographing the eye, we used the image analysis program ImageJ v 1.47t [total = π× eye diameter. We then used the broken segment line tool of ImageJ to measure the circumference of the eye that was occupied by bone (Cbone). Dividing Cbone by Ctotal provides the proportion of the circumference of the eye occupied by scleral ossicles. In cases where the sclera was mostly ossified, we simply measured the circumference of the eye that was unossified, and subtracted this from Ctotal before dividing to get the proportion of the circumference of the eye occupied by scleral ossicles. Finally, to estimate the relative width of the scleral ring, we used the line segment tool of ImageJ to measure the width of the sclera ring in the ventral portion of the eye. We then divided the width of the ring by the total diameter of the eye.For the population and complementation samples, we qualitatively assessed scleral ossification by dissecting the stained specimens and visually noting the presence/absence of ossified elements within the sclera. The presence of any ossifications within the sclera was interpreted as evidence of scleral ossification. For the quantitative genetic samples, we measured three aspects of scleral ossification: (1) the number of scleral ossicles present within the sclera ; (2) the proportion of the circumference of the eye occupied by scleral ossicles, following a modified form of the method used by Franz-Odendaal et al. ; and (3) v 1.47t ,33 to fiAstyanax CF and SF populations via Maximum Likelihood ancestral character state reconstruction [Astyanax mexicanus SF populations and five of the seven CF populations used in this study  [not assess sclera ossification in any of the surface fish populations included in this phylogeny, but instead assumed that all exhibit ossified sclera. We feel this assumption is justified since all SF so far examined possess scleral ossicles, and the development of these structures in SF have been described in detail [Astyanax surface fish, most other Characiform fishes also posses two scleral ossicles, as do approximately half of all teleost fish species so far surveyed [Astyanax mexicanus. We reconstructed ancestral states for scleral ossification via Maximum Likelihood assuming unordered characters with an equal probability of transition between ossification and loss of ossification. We performed this reconstruction with the program Mesquite v2.75 [To determine the number of losses of scleral ossification among the different cavefish populations sampled, we traced the evolution of the qualitative presence/absence of scleral ossification among truction . Our rec Tinaja) . For the Tinaja) ,37; howen detail ,18,20. Asurveyed ,38. Thuste v2.75 .1 progeny would provide evidence of complementation and suggest that different mutations–and, hence, convergent evolution–are responsible for the loss of scleral ossification in these two CF populations. A similar strategy has previously been used to demonstrate convergence in eye loss [Astyanax cavefish.In addition to our test of independent evolution via ancestral state reconstruction, we also tested this hypothesis via genetic complementation. We assessed scleral ossification in the F1 hybrid progeny of two independently derived cavefish populations as described above. Complementation occurs when the hybrids of two independently derived mutant strains exhibit the wild-type condition , which indicates that the mutations responsible for the mutant phenotype are different in each strain. The presence of ossified sclera in any of the Feye loss  and pigmeye loss  among distepwiseqtl in the program R/qtl [th percentile of genome-wide maximum penalized LOD scores for each phenotype using 1,000 random permutations of the genotypic and phenotypic data using the R/qtl function scantwo. These permutations were performed in two stratified groups. One group included 141 F2 that were genotyped at 235 microsatellite markers [2 that were genotyped at an additional 463 SNPs [Our analysis of quantitative trait loci (QTL) contributing to scleral ossification followed our previously published protocols ,29. Brieam R/qtl ,40. We cam R/qtl , we incl markers  and the Astyanax cavefishes  exhibited small, pigmented eyes with ossified sclera on both sides of their head . We observed a range of phenotypes, from eyes with no scleral ossicles to those with two fused ossicles that occupied the entire circumference of the eye  and overall standard length  of the each fish, hence their appropriate use as covariables in this analysis. Unfortunately, the limited variation caused by the skewed distribution of scleral ossicles, as well as the presence of significant covariables, likely limits our power to detect QTL for scleral ossification in this cross.For our third analysis, we quantified scleral ossification from the left eyes of  the eye . Surpris the eye . Only 4  the eye . Of thes2 in order to test the hypothesis that any QTL for scleral ossification overlap existing QTL for eye or lens size. All three eye phenotypes were normally distributed (results not shown), which is consistent with numerous previous analyses of eye and lens size in Astyanax hybrids [In addition to scleral ossification, we also quantified eye diameter, eye area, and pupil area in 115 F hybrids ,26,44.Astyanax genome for quantitative trait loci (QTL) associated with scleral ossification while controlling for age and standard length as covariates. We initially found no significant QTL for scleral ossification when treated as a continuous trait, although we did find two marginally non-significant QTL at the significance threshold P < 0.07 . At the peak QTL position of 59.5 cM, F2 hybrids with two CF alleles have a smaller proportion of their eye occupied by scleral ossicles than individuals with one or more SF alleles . This effect is consistent with CF possessing fewer scleral ossicles. The second QTL, on Astyanax linkage group 2 from 0.00–78.1 cM, is associated with width of the scleral ring relative to total eye diameter. At the peak QTL position of 0.00 cM, F2 hybrids with one or more CF alleles have wider scleral rings relative to the total diameter of their eye in the ventral portion of their sclera  had one or fewer ossicles, and only 2% (4/196) had no ossicles as in most Astyanax cavefish . CoupleAstyanax cavefish? The results of Yamamoto et al. [Astyanax, including the presence of a phenotypic and genotypic threshold. Yamamoto et al. [What might be the underlying genetic cause of a threshold for scleral ossicle development in o et al.  and Dufto et al.  both sugo et al.  found tho et al.  extendedo et al. ), most io et al.  found th2 progeny with (1+) and without (0) sceral ossicles to the ratios expected for a trait controlled by one or more loci. Our observed ratio of 48:1 (192:4) is consistent with a genotypic threshold caused by three loci, but not with thresholds caused by one, two, or four loci  [2 hybrids are only expected to exhibit scleral ossicle loss in 3/196 individuals, or 0.253 = 1.56% of the F2 population. This expectation is clearly met in our study, as we observed scleral ossicle loss in only 4/196 = 2.04% of the F2 population  ,43. Indeneration , and sev of 48:1 2:4 is coneration . Given tAstyanax and other teleosts. For example, since the results of Yamamoto et al. [Astyanax [2 hybrids to exhibit the cavefish phenotype of reduced scleral ossicles. However, a better design would be a backcross or testcross, which would greatly increase the number of mutant progeny for analysis, thus improving the power of future studies to detect QTL for ossicle development. Additionally, our study assessed scleral ossification in adult fish ranging in age from 2–5 years old. Given that age is significantly associated with degree of scleral ossification in teleosts   1, 2, 7, 11, 18, and 23. Red line indicates P = 0.05; any value above that threshold is considered statistically significant. Shaded fields indicate the 95% Bayesian confidence interval for the location of each QTL. Colored boxes at the top of the chart indicate the position of eye, lens, retina, or bone QTL found in other studies. a = Protas et al. [b = Protas et al. [c = O'Quin et al. [Detailed view of the eye or pupil size QTL on s et al. , b = Pros et al. , c = O'Qn et al. . Two QTL(PDF)Click here for additional data file.S1 Table(CSV)Click here for additional data file.S2 Table(CSV)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Conclusions: in the studied sample, lymphocytic foci on salivary glands were the only histological parameter associated to the diagnosis of SS. Early indication of labial minor salivary gland biopsy to patients under investigation of SS could limit the effects of immunosuppressive therapy on the histological features associated with the evolution of salivary gland involvement in SS.Diagnosis of Sjögren’s syndrome (SS) is complex and the usefulness of labial minor salivary glands biopsy in this process remains controversial. Objectives: to evaluate the clinical and laboratorial profile and histological features on labial minor salivary glands from patients under investigation of SS. Study Design: clinical charts from 38 patients under suspicion of SS and submitted to labial minor salivary glands biopsies were reviewed. Clinical and laboratorial data were retrieved from the clinical files and the HE-stained histological slides were reviewed under light microscopy. Results: mean age of the patients was 56.5 years and 97% were females; histological analysis showed that 42% of the cases showed ductal dilatation, lymphocytic foci were found in 52.6% and, from this group, 80% of the cases presented a foci/lobules ratio above 0.8. Acinar/ductal ratio was considered diminished in 39.5% of the samples. Thirty six (95%) and 32 (84%) patients, respectively, complained about xerostomia and xerophthalmia. A study of the time interval of the symptoms that led to SS investigation showed a mean of 116 months. Moreover, sixty-six percent of the patients had already been submitted to immunosuppressive therapy prior to the labial minor salivary gland biopsy. Age of the patients, scintigraphic alterations on salivary function, antinuclear factor (ANF), anti-Ro and anti-La did not show statistical significant association with the histological features. Lobules/foci ratio above 0.8 was the only histological parameter statistically associated with Sjögren’s syndrome diagnosis ( Key words:Sjögren syndrome, minor salivary glands, biopsy, lymphocytic foci. Sjögren’s syndrome (SS) is a chronic autoimmune disease characterized by xerophthalmia and xerostomia, functional alterations in both salivary and lacrimal glands and the presence of specific and unspecific serum autoantibodies in the affected patients -4. DiagnAll specimens from biopsies directed to the removal of labial minor salivary glands performed in patients under investigation for Sjögren’s syndrome registered in the Oral Pathology laboratory, School of Dentistry, State University of Rio de Janeiro, from 2006 to 2009 were reviewed. Histological slides containing hematoxylin and eosin (HE)-stained 5µm sections were analyzed under light microscopy , according to the following criteria: number of salivary gland lobules; percentage of adipose tissue on the salivary glands (less than 10% or more than 10%); absence or presence of ductal dilatation, acinar atrophy and sclerosis of the glandular connective tissue; acinar/ductal ratio ; the number of lymphocytic foci (1 focus was composed of at least 50 grouped lymphocytes); and the ratio number of lobules/number of foci. The established ratio as the limit between the groups considered “compatible” and “non-compatible” with the diagnosis of Sjögren’s syndrome was 0.8, which means the presence of at least 5 lymphocytic foci in 4 minor salivary gland lobules. This criterion was used in addition to the absolute number of lymphocytic foci due to the fact that in some cases the submitted specimens were constituted by less than 5 salivary gland lobules.p<0.05). This study was approved by the Ethics Committee, State University of Rio de Janeiro (COEP 059/2010).After selection and review of all HE-stained histological slides, clinical and laboratory information was retrieved from the clinical records of all patients on the Rheumatology service at Pedro Ernesto University Hospital, School of Medicine, State University of Rio de Janeiro. Data included age, gender, symptoms of xerostomia and xerophthalmia, results from the Schirmer tests and major salivary gland scintigraphy, history of recurrent parotitis, pulmonary involvement and presence of other concomitant rheumatoid autoimmune diseases. Serum laboratory data retrieved from the clinical records included autoantibodies, such as rheumatoid factor (RF), ANF, anti-Ro and anti-La. Time interval with the symptoms that led to the investigation of Sjögren’s syndrome and history of previous/present immunosuppressive treatment before the labial minor salivary gland biopsies were also retrieved. Patients treated or under follow-up in other institutions, without clinical records presenting sufficient data for analysis, and showing HE-stained slides presenting no sufficient available tissue for histological analysis were excluded from the final studied sample. Data were tabulated and analyzed with the aid of the Statistical Package for Social Science  with statistical significance level of 5%  and 37 patients were females (97%). Histological analysis showed that 55.3% presented more than 10% of glandular adipose tissue and 42% of the cases showed ductal dilatation. Acinar atrophy and connective tissue sclerosis were found in 15.8% and 10.5% of the cases, respectively. Lymphocytic foci were found in 52.6% of the cases and, from this group, 80% of the cases presented a foci/lobules ratio above 0.8. Acinar/ductal ratio was considered diminished in 39.5% of the samples. Figure Thirty six (95%) and 32 (84%) patients, respectively, complained about xerostomia and xerophthalmia. Schirmer test was performed in 29 patients (76%), of which 18 (62%) showed results compatible with Sjögren’s syndrome. Eight out of the 38 patients (21%) reported previous episodes of recurrent parotitis and 20 out of the 33 patients (61%) submitted to salivary gland scintigraphy showed altered salivary function. Thirty two patients (84%) reported being diagnosed with other autoimmune diseases, especially rheumatoid arthritis, lupus erythematosus and fibromyalgia, and 5 patients (13%) reported associated pulmonary symptoms. Laboratory exams showed that out of the 32 patients tested for ANF, 17 (53%) showed positive results. In contrast, from the 24 patients who had been tested for RF, only 4 (17%) showed positive results. Anti-Ro and anti-La were requested together for 24 patients and 9 (38%) showed positive results for both. Time interval with the symptoms that led to Sjögren’s syndrome investigation ranged from 1 to 288 months, with a mean of 116 months, and 25 out of the 38 patients (66%) had already been submitted to immunosuppressive therapy prior to labial minor salivary gland biopsy. Time interval from the beginning of the symptoms and the labial minor salivary gland biopsy ranged from 4 to 312 months, with a mean of 91 months.p<0.0001). Out of the 38 patients, 16 (42%) showed lobules/foci ratio above 0.8, out of which 14 were diagnosed with Sjögren’s syndrome after analysis of other criteria. One patient presented a lobules/foci ratio lower than 0.8, but it was diagnosed with the syndrome due to the presence of other clinical and laboratorial parameters. Two patients were not diagnosed with Sjögren’s syndrome, despite presenting a lobules/foci ratio above 0.8, due to lack of confirmation after analysis of the other parameters  during this time interval, and it is believed that some histological parameters could be altered by local and systemic effect of the drugs. Zandbelt  disease . The reset al., which showed that only 53% of the patients who presented serological positive results showed lymphocytic foci compatible with SS  and a weak correlation with subjective parameters, such as xerostomia and xerophthalmia . The resIn the studied sample, lymphocytic foci on salivary glands were the only histological parameter associated to the diagnosis of SS. Early indication of labial minor salivary gland biopsy to patients under investigation of SS could limit the effects of immunosuppressive therapy on the histological features associated with the evolution of salivary gland involvement in SS.


Camelus dromedaries) in Khartoum State, Sudan. A cross sectional study was conducted in Khartoum State, Sudan, in a total of 240 camels selected randomly from four localities. Sera sampled were tested for the presence of RVFV-specific immunoglobulin G (IgG) antibodies using a competitive enzyme-linked immunosorbent assay (c-ELISA).Rift valley fever (RVF) is a mosquito-borne viral disease of domestic livestock and wild ruminants. In camels RVF may cause abortion among pregnant camels, but is most often asymptomatic among other camels. In this study, a seroepidemiological survey was conducted to determine the prevalence of RVFV antibodies and to identify the potential risk factors associated with RVFV seropositivity among the Sudanese one-humped camel  and heavy rainfall  were recorded as potential risk factors for contracting RVF.RVFV seropositivity was recorded in 23 out of 240 animals, prevalence rate of 9.6 % among camels in Khartoum State. Age (OR = 8.29, Older age and heavy rainfall were considered as potential risk factors for seropositivity to RVF. Surveillance for RVF among camels and distribution of mosquito vectors should continue to better understand the clinical signs associated with RVFV infection in camels and provide public health authorities an opportunity to anticipate and prepare for a possible RVF outbreak in Khartoum State, Sudan. Periodically, RVFV spreads to other areas, including northward into Egypt in 1977 and eastward across the Red Sea into Saudi Arabia and Yemen in 2000 [Rift Valley fever (RVF) is a mosquito-borne viral disease that typically occurs in various areas of sub-Saharan Africa, where virus activity varies from a low-level enzootic cycle to explosive outbreaks covering large areas . RVF is  in 2000 . RVF in  in 2000 . The fir in 2000 . Serolog in 2000 –15. A re in 2000 . New Hal(Camelus dromedarius) in Khartoum State, Sudan. This study would be expected to reduce the impact on the livelihood of pastoral communities and ultimately avoid disease spread in human population.Currently, little is known about the prevalence of RVF in livestock in Sudan and no information is available in regard to the potential risk factors associated with RVF among the livestock in the country, including the Sudanese dromedary camels. Circulation of RVFV in camels and detection of RVFV-specific antibodies is well documented in the different region of the African continent, including Sudan, Saudi Arabia, Tanzania, South Africa, Mauritania, Kenya, Niger and Nigeria , 17–27. The study was conducted in Khartoum State during the period from October 2014 to March 2015. The State covers an area of approximately 20,971 square kilometers. The State is located between latitudes 15° 8° - 16° 39° N and longitudes 31° 36 - 34° 25 E in the semi desert tropics. It is dominated by semi desert climate, which is characterized by very hot/dry summer and cold winter. The average temperature ranges from 21 °C in the winter to 47 °C in the summer. The mean annual evaporation rate is 7.7 mm/day, and the average relative humidity ranges from 21 % to −38 %. Khartoum State is boarded by the River Nile State in the north; Kassala States in the east, North Kordufan in the west and Elgezera State to the South. The total population of camels in the country is approximately 4.6 million. The camel population of Khartoum State is 6,585 as estimated by the Sudan Ministry of Animal Resources, 2006. A map of the localities included in the study area of Khartoum State is presented in , sex , breed , body condition , and management risk factor attributes including herd size , grazing system , rain fall , mosquito vector (presence or absence), mosquito control (practiced or not), vegetation , water bonds (presence or absence), and the four localities included in the study.A total of 240 blood samples were collected from the dromedary camels in the study area of Khartoum State, Sudan. Serum samples were separated and were kept frozen at -20 °C for detection of RVFV-specific IgG antibodies using competitive enzyme-linked immunosorbent assay (cELISA).A competitive enzyme-linked immunosorbent assay (cELISA) was performed, as described by Hassanain et al. , using aχ2) test. A multivariable model for the outcome variable was constructed using logistic regression analysis with enter method for modeling checking. RVF was considered as the dependent variable and the risk factors as independent variables. Finally, odd ratios and 95 % confidence interval (CI) were calculated, and risk factors with a p-value < 0.05 were taken as significant association to RVF seropositivity.The data collected were entered into a computer on a Microsoft Excel spread sheet. Statistical analysis was performed using SPSS version 16 (for Windows) and double checked before analyses. Associations between the outcome variable (status of RVF seropositivity in camels) and its potential risk factors were first screened in a univariable analysis using Chi-square  compared to young animals (<2 years of age). Heavy rainfall increased the risk of contracting RVF compared to low rainfall . Significant association between RVF and potential risk factors in the final model were shown in (Table Using a cELISA assay, RVFV-specific IgG antibodies were detected in 23 out of 240 camels included in the study. The overall prevalence rate of RVFV antibodies among camels in Khartoum State of Sudan was estimated to be 9.6 %. The highest and lowest rates of RVFV seropositivity were recorded in Sherq Elnile and Omdurman, respectively. The results of the univariate analysis using chi-square test were presented in Table . The finIn recent years, the distribution and nature of RVF has changed substantially. RVF has become of great veterinary concern to dairy producers, wildlife managers and veterinary diagnosticians because of the frequent occurrence of sporadic cases and outbreaks among domestic and wild ruminants , 31–33. Aedes vectors in this region [In epidemiological surveys, high prevalence rates of 45 % and 38.5 % for RVFV seropositivity were reported among camels in Tanzania and Mauritania, respectively , 27. In s region , 36. In s region , 28. Thes region , 27, 37.Heavy rainfall is another potential risk factor that affects RVFV seropositivity among camels in Khartom State. Our result is in agreement with that of the study conducted by other workers who reported association between RVF seropositivity and rainfall , 25. TheIn contrast, the risk assessment studies indicated that there was no significant association between RVF seropositivity and the rest of the individual or management risk factors included in the study. There was no significant difference between localities regarding RVF seropositivity in camels. In addition, there was no significant association between mosquito control and RVF seropositivity among camels suggesting failure of the control program against RVF in Khartoum State. Highest and lowest rates of RVFV infections were recorded in Sherq Elnile and Omdurman localities, respectively.The study provides evidence of circulation of RVFV among camels in Khartoum State, Sudan, as determined by the presence of RVFV-specific IgG antibodies using cELISA. It is recommended that further study should be conducted in sentinel camel herds to determine the incidence of new cases and to conduct RVF virus isolation attempts. Any recovered RVFV strains should be employed to determine the virus whole genome sequence and to identify the genetic lineage of the virus circulating among camels in Khartoum State. Surveillance for RVF in domestic livestock and studies on distribution of the mosquito vectors should continue to provide public health authorities an opportunity to anticipate and prepare for a possible RVF outbreak in Khartoum State, Sudan.


By aligning probe sequences with expressed sequence tags (ESTs) in NHP, inter-species conserved (ISC) probesets, which have two or more probes complementary to ESTs in NHP, were identified on human GeneChip® platforms. The utility of human GeneChips® for the assessment of NHP expression patterns can be effectively evaluated by analyzing the hybridization behaviour of ISC probesets. Appropriate normalization methods were identified that further improve the reliability of human GeneChips® for interspecies (human vs NHP) comparisons.While researchers have utilized versions of the Affymetrix human GeneChip® platforms  were identified for both monkey and chimpanzee. Expression data was generated from peripheral blood mononuclear cells (PBMCs) of 12 human and 8 monkey (Indian origin Rhesus macaque) samples using the Focus GeneChip®. Analysis of both qualitative detection calls and quantitative signal intensities showed that intra-species reproducibility (human vs. human or monkey vs. monkey) was much higher than interspecies reproducibility (human vs. monkey). ISC probesets exhibited higher interspecies reproducibility than the overall expressed probesets. Importantly, appropriate normalization methods could be leveraged to greatly improve interspecies correlations. The correlation coefficients between human (average of 12 samples) and monkey (average of 8 Rhesus macaque samples) are 0.725, 0.821 and 0.893 for MAS5.0 (Microarray Suite version 5.0), dChip and RMA (Robust Multi-chip Average) normalization method, respectively.ISC probesets in each of the seven Affymetrix GeneChip® platforms to assess the expression profiles of NHP for intra-species studies. Caution must be taken for interspecies studies since unsuitable probesets will result in spurious differentially regulated genes between human and NHP. RMA normalization method and ISC probesets are recommended for interspecies studies.It is feasible to use Affymetrix human GeneChip An underlying assumption in such studies is that transcripts of humans and NHP are highly conserved, and probe sequences designed to detect human genes will detect their orthologs in NHP samples. It is estimated that chimpanzees (Pan troglodytes) and humans shared 98.77 % DNA similarity [® for NHP expression analysis.Microarray studies on non human primates (NHP) have been used to address viral pathogenesis ,2, neuromilarity  comparedmilarity ,12, and ® platforms for NHP expression profiling have robustly addressed the quantitative aspects of cross platform performance. Vahey and colleagues [® and demonstrated that there was no significant difference in the dynamic range of the raw fluorescence distribution for equivalent amounts of human cRNA and macaque cRNA hybridized to the chip. Chismar and colleagues [® platform and compared the expression patterns of humans with that of the rhesus macaque. They concluded that the percentage of 'present' calls observed in the transcriptome of macaque brain is lower than that of human brain, and that this is especially true for genes with lower signal intensity. Caceres and colleagues [® platforms for the study of expression profiles in NHP is to employ a sequence analysis approach.Few published studies employing human GeneChiplleagues  used thelleagues  used thelleagues  used the® platforms to assess expression patterns in NHP samples by: a) identifying ISC probesets based on sequence analysis; b) assessing intra (within NHP species)- and interspecies (between NHP and human samples) reproducibility of GeneChip® data; and c) applying appropriate normalization methods to improve interspecies reproducibility.In this study, we address the power of human GeneChip® hybridizes with the transcriptome of a NHP, there are three possible outcomes: 1) it hybridizes with the ortholog of the NHP; 2) it cross-hybridizes with a non-ortholog transcript, or 3) it fails to hybridize due to sequence divergence. In Affymetrix GeneChip® system, a probeset is composed of 11–20 probes and each probe is a 25-mer oligo. We identified probes on the human GeneChip that are complementary to ESTs in NHP postulating that these probes would hybridize most optimally with the transcripts of NHP. We defined a probeset as an ISC probeset if it had at least two complementary probes. The rationale used to define the criterion that defines an ISC probeset is described in the methods section. The procedure used to generate ISC probesets is shown in Figure ® platforms  were generated for both monkey and chimpanzee. Detailed information about each ISC probeset such as probe sequence, GenBank accession and the position and degree of matching is provided in the supplemental materials (Macaca mulatta) than for chimpanzee (Pan troglodytes). This is not because monkey EST sequences are more similar to human sequences than chimpanzee EST sequences, but because we have a much greater amount of EST sequences available for monkey. At the time of writing this manuscript, there were 33,474 monkey ESTs available, while there were only 6,943 ESTs available for chimpanzee. As the number of defined ESTs will increase in the future, additional ISC probesets could be identified for both monkey and chimpanzee using this method.When a probe sequence on the human GeneChipaterials . Table 1®s, there are three probesets  that target the gene CXCR4 at different positions in its transcript. In order to address this redundancy issue, we converted the number of probesets into the number of unique UniGene clusters based on the GeneChip® annotation file provided by Affymetrix Website [® and the U133Plus2.0 GeneChip® have the lowest and highest frequency of redundant probesets for a given gene, respectively.It is not uncommon, especially in the U133Plus2.0 platform, that multiple probesets target the same gene. For example, in the U133A and the U133 Plus 2.0 GeneChip Website . While a® from Affymetrix and covers the human genome most extensively. Figure The U133Plus2.0 is the most current version of human GeneChip® data observed in intra- and interspecies samples. The intra-species reproducibility is displayed in Figure ® for NHP intra-species studies.The qualitative detection call (present / absent) output from MAS5.0 was the initial approach used to examine the reproducibility of GeneChip® platforms are used to compare the expression pattern of humans with those of NHPs, care must be taken in the interpretation of data. If we consider a probeset as being expressed when 50% or more of replicates have present calls, then 3445 (2059+1386) and 2321 (2059+262) probesets are expressed in the PBMC fraction of humans and monkeys, respectively  was created. Probesets that are not expressed in human PBMCs were excluded in this analysis. Pair-wise correlation coefficients were calculated for all 20 samples . The correlation coefficients were visualized using heat spectrum graphs where colors ranging from red to white correspond to correlation coefficients of 0.5 to 1.0, respectively. In figure To assess the intra- and interspecies reproducibility of GeneChip® data variation [® data at the probe level using a non-linear algorithm while MAS5.0 normalizes data at probeset level using linear scaling. Sequence divergence usually leads to one or very few probes in a probeset being problematic while the majority of probes in that probeset may still work reasonably well. If the variation generated from these problematic probes were normalized, the interspecies reproducibility should improve. Figure Different normalization methods have been shown to significantly affect GeneChipariation -17. We cariation -16 and d® expression data as applied to the derivation of meaningful expression profile data from NHP. The utility of the human Affymetrix GeneChip® for the assessment of expression profiles in NHP depends on the experimental design and on the approach to data normalization and analysis. Our observations suggest that: 1) it is feasible to use the human GeneChip® in the evaluation of expression profiles of NHP samples for intra-species comparisons; 2) use of ISC probesets and RMA normalization are recommended for interspecies studies; and 3) with the increasing amount of ESTs of NHP, additional ISC probesets (and perfect probesets) will be identified in the near future.This paper presents a comprehensive analysis of probe sequences and GeneChipMacaca mulatta) and chimpanzee (Pan troglodytes) were downloaded from NCBI website [Affymetrix GeneChip probe sequences were downloaded from Affymetrix website . The EST website .® probe sequences and monkey /chimpanzee EST sequences. The length of a probe sequence is always 25 nucleotides while the number of probes in a probeset varies from 11 to 20 depending on GeneChip® platforms  in comparison with an RT-PCR experiment, the primer length is equivalent to our probe length, and two primers (one forward and one backward) usually generate a unique sequence in a whole genome; 3) a probe sequence on the Affymetrix GeneChip® is a well designed sequence with a single probe hybridizing with a unique transcript in whole transcriptome; and 4) since the EST sequences in NHP are very limited so far, most of them do not cover whole transcript such that a false negative could be generated if we require all the probes in a probeset being complementary to known ESTs. In order to convert probeset IDs to UniGene IDs and map them onto chromosomes, probeset annotation files were downloaded from Affymetrix website [Stand alone BALST program was downloaded from NCBI website . Perl sc website . No animRhesus macaque) was collected and peripheral blood mononuclear cells (PBMCs) were separated by Histopaque-Ficoll (Sigma) gradient centrifugation. RNA preparation, Hybridization, staining and scanning of the GeneChip® was carried out as described by Vahey et al. [®. Signal values and detection calls (present or absent) for all samples were determined by using MAS5.0 . Signal values were scaled to the default target signal intensity of 500). A matrix of detection calls  and a matrix of signal intensities for all samples across all probesets were constructed. A gene must exhibit 50% or more of 'present' calls in all samples to be considered 'expressed'. In this study, an expressed probeset in human is a probeset that has 6 or more present calls among 12 human samples. Similarly, an expressed probeset in monkey means there were 4 or more present calls among 8 monkey samples. The signal intensities output from MAS5.0 were log2 transformed. Model-based normalization was performed using dChip version 1.3 [rma function in the package was used at its default setting, that is, 'RMA' background correction, 'quantile normalization', 'PM only model' and 'median polish summarization'. By default, the signal intensities were already log2 transformed.Briefly, peripheral blood from healthy human and NHP  and set color scales between 0.5 (red) and 1.0 (white).Intra- and interspecies correlation coefficients of signal intensities were calculated by built in function 'n 1.9.0. . VisualiNHP: non human primateEST: expressed sequence tagISC: inter-species conservedRMA: robust multi-chip averageMAS5.0: Microarray suite version 5.0ZW developed the original hypotheses, performed the bioinformatics analyses to test them and drafted the manuscript. MV provided critical input on design and execution of the laboratory experiments and with ZW interpreted the data sets and revised the manuscript. ML conducted all aspects of the animal handling including the harvest of well characterized primary samples. MN and AA are technical staff who extracted the nucleic acid and performed the laboratory portions of the microarray experiments. All authors read and approved the final manuscript.Macaca mulatta) in GeneChip® platforms Plus2.0, U133A and U133B, respectively. Note: All the four additional files are multiple-sheets MS excel files. Within each worksheet of a file, the rows are ISC probesets, the columns are probeset IDs, positions of a probe (x and y coordinates in the chip), GenBank accessions of a matching EST, probe sequence, matched EST sequence and position, BLAST e- values and matching identities, in that order.There are three worksheets in this file. Worksheet 1, 2 and 3 are ISC probesets for monkey (Click here for fileMacaca mulatta) in GeneChip® platforms Focus, FL, U95Av2 and U95B, respectively. Note: All the four additional files are multiple-sheets MS excel files. Within each worksheet of a file, the rows are ISC probesets, the columns are probeset IDs, positions of a probe (x and y coordinates in the chip), GenBank accessions of a matching EST, probe sequence, matched EST sequence and position, BLAST e- values and matching identities, in that order.There are four worksheets in this file. Worksheet 1, 2, 3 and 4 are ISC probesets for monkey (Click here for filePan troglodytes) in GeneChip® platforms Plus2.0, U133A and U133B, respectively. Note: All the four additional files are multiple-sheets MS excel files. Within each worksheet of a file, the rows are ISC probesets, the columns are probeset IDs, positions of a probe (x and y coordinates in the chip), GenBank accessions of a matching EST, probe sequence, matched EST sequence and position, BLAST e- values and matching identities, in that order.There are three worksheets in this file. Worksheet 1, 2 and 3 are ISC probesets for chimpanzee (Click here for filePan troglodytes) in GeneChip® platforms Focus, FL, U95Av2 and U95B, respectively. Note: All the four additional files are multiple-sheets MS excel files. Within each worksheet of a file, the rows are ISC probesets, the columns are probeset IDs, positions of a probe (x and y coordinates in the chip), GenBank accessions of a matching EST, probe sequence, matched EST sequence and position, BLAST e- values and matching identities, in that order.There are four worksheets in this file. Worksheet 1, 2, 3 and 4 are ISC probesets for chimpanzee (Click here for file


This study tested the effectiveness of a COI barcode in discriminating bird species, one of the largest and best-studied vertebrate groups. We determined COI barcodes for 260 species of North American birds and found that distinguishing species was generally straightforward. All species had a different COI barcode(s), and the differences between closely related species were, on average, 18 times higher than the differences within species. Our results identified four probable new species of North American birds, suggesting that a global survey will lead to the recognition of many additional bird species. The finding of large COI sequence differences between, as compared to small differences within, species confirms the effectiveness of COI barcodes for the identification of bird species. This result plus those from other groups of animals imply that a standard screening threshold of sequence difference (10× average intraspecific difference) could speed the discovery of new animal species. The growing evidence for the effectiveness of DNA barcodes as a basis for species identification supports an international exercise that has recently begun to assemble a comprehensive library of COI sequences linked to named specimens.Short DNA sequences from a standardized region of the genome provide a DNA barcode for identifying species. Compiling a public library of DNA barcodes linked to named specimens could provide a new master key for identifying species, one whose power will rise with increased taxon coverage and with faster, cheaper sequencing. Recent work suggests that sequence diversity in a 648-bp region of the mitochondrial gene, cytochrome  c oxidase I might have this potentialCan a single gene be used to identify all different animal species? An analysis of 255 North American birds shows that cytochrome  Archaea, and subsequently to a redrawing of the tree of life. More recently, the polymerase chain reaction has allowed sequence diversity in any gene to be examined. Genes that evolve slowly, like rRNA, often do not differ among closely related organisms, but they are indispensable in recovering ancient relationships, providing insights as far back as the origin of cellular life  has been widely employed in phylogenetic studies of animals because it evolves much more rapidly than nuclear DNA, resulting in the accumulation of differences between closely related species . In factThe general concordance of mtDNA trees with species trees implies that, rather than analyzing DNA from morphologically identified specimens, it could be used the other way around, namely to identify specimens by analyzing their DNA. Past applications of DNA-based species identification range from reconstructing food webs by identifying fragments in stomachs  to recogc oxidase I (COI), might serve as a DNA barcode for the identification of animal species. This gene region is easily recovered and it provides good resolution, as evidenced by the fact that deep sequence divergences were the rule between 13,000 closely related pairs of animal species ; none was shared between species. COI sequences in the 130 species represented by two or more individuals were either identical or most similar to other sequences of the same species. Furthermore, with a few interesting exceptions discussed below, COI sequence differences between closely related species were far higher than differences within species  .Tringa solitaria, Solitary Sandpiper; Sturnella magna, Eastern Meadowlark; Cisthorus palustris, Marsh Wren; and Vireo gilvus, Warbling Vireo). COI sequences in each of these polytypic species separated into pairs of divergent clusters in the NJ tree. The intraspecific K2P distances in these exceptional species were 3.7%–7.2%, 9- to 17-fold higher than the average distance (In most cases the neighbor-joining (NJ) tree showed shallow intraspecific and deep interspecific divergences . Howeverdistance .Larus argentatus, L. canus, L. delawarensis, L. glaucoides, L. hyperboreus, L. marinus, and L. thayeri ; Haematopus bachmani and H. palliatus (Black Oystercatcher and American Oystercatcher); Corvus brachyrhynchos and C. caurinus (American Crow and Northwestern Crow); and Anas platyrhynchos and A. rubripes   .Although species were the focus of this study, we noted that the NJ tree of COI sequences generally matched avian classifications at higher levels, with most genera, families, and orders appearing as nested monophyletic lineages concordant with current taxonomy  and S1.The simplest test of species identification by DNA barcode is whether any sequences are found in two species; none was in this study. Although sequences were not shared by species, sequence variation did occur in some species. Thus the second test is whether the differences within species are much less than those among species. In this study we found that COI differences among most of the 260 North American bird species far exceeded those within species.In order to conservatively test the effectiveness of COI barcodes as an identification tool, our sample must not have underestimated variability within species or have overestimated it among species. Our measures of intraspecific variation could be underestimates if members of a species show sequence divergence across their distribution that our study failed to adequately register. The two to three representatives of the 130 species used to examine this issue were collected from sites that were, on average, approximately 1,080 km apart, suggesting adequate representation of genetic diversity across their ranges. However, to further investigate this issue, we compared sequence differences within species to geographic distances between the collection points for their specimens and found these were unrelated . Based oOn the other hand, our discovery of four polytypic species within a sample of 130 makes it likely there are other North American birds with divergent populations that may represent hidden species. Recent studies have identified marked mtDNA divergences within North American populations of Common Ravens , Fox SpaCalidris mauri and C. pusilla, Fraternicula arctica and F. corniculata, and Empidonax traillii and E. virescens. There were sufficient data to perform this analysis on three of the four polytypic species and on 70 of the 126 remaining species  are split into two by some taxonomists , and theThe importance of sampling multiple individuals within each species is highlighted by a recent review which found evidence of species-level paraphyly or polyphyly in 23% of 2,319 animal species, including 16.7% of 331 bird species . This reT. solitaria PS-1 and T. solitaria PS-2, highlighting a need for further taxonomic study.We expect that the assembly of a comprehensive barcode library will help to initiate taxonomic investigations that will ultimately lead to the recognition of many new avian species. This process will begin with the discovery of novel COI barcodes. Some of these cases will simply represent the first barcode records for described but previously unanalyzed species, but taxonomic study will confirm that others derive from new species. We propose that specimens with barcodes diverging deeply from known taxa should be known by a “provisional species” designation that links them to the nearest established taxon. For example, the divergent clusters of Solitary Sandpiper specimens might be called What threshold might be appropriate for flagging genetically divergent specimens as provisional species? This threshold should certainly be high enough to separate only specimens that very likely belong to different species. Because patterns of intraspecific and interspecific variation in COI appear similar in various animal groups  in the stomachs of predators. A DNA barcode could similarly identify fragments of birds that strike aircraft  and recoThe two essential components for an effective DNA barcode system (and thus a new master key to the encyclopedia of life ) are stab and COI, are rivals for the largest number of animal sequence records greater than 600 bp in GenBank . However, COI coverage for birds is modest; 173 species share COI sequences with 600-bp overlap. As these records derive from a global avifauna of 10,000 species, they provide a limited basis to evaluate the utility of a COI-based identification system for any continental fauna, impelling us to gather new sequences.Existing data can only yield limited new insights into the effectiveness of a DNA-based identification system for birds. Two mitochondrial genes, cyt http://www.barcodinglife.com). Taxonomic assignments follow the latest North American checklist . In cases where this primer pair failed, an alternate reverse primer (BirdR2-ACTACATGTGAGATGATTCCGAATCCAG) was generally combined with BirdF1 to generate a 751-bp product, but a third reverse primer (BirdR3-AGGAGTTTGCTAGTACGATGCC) was used for two species of Falco. The 50-μl PCR reaction mixes included 40 μl of ultrapure water, 1.0 U of Taq polymerase, 2.5 μl of MgCl2, 4.5 μl of 10× PCR buffer, 0.5 μl of each primer (0.1 mM), 0.25 μl of each dNTP (0.05 mM), and 0.5–3.0 μl of DNA. The amplification regime consisted of 1 min at 94 °C followed by 5 cycles of 1 min at 94 °C, 1.5 min at 45 °C, and 1.5 min at 72 °C, followed in turn by 30 cycles of 1 min at 4 °C, 1.5 min at 51 °C, and 1.5 min at 72 °C, and a final 5 min at 72 °C. PCR products were visualized in a 1.2% agarose gel. All PCR reactions that generated a single, circa 750-bp, product were then cycle sequenced, while gel purification was used to recover the target gene product in cases where more than one band was present. Sequencing reactions, carried out using Big Dye v3.1 and the BirdF1 primer, were analyzed on an ABI 377 sequencer. The electropherogram and sequence for each specimen are in the “Birds of North America” file, but all sequences have also been deposited in GenBank .Click here for additional data file.Sequences described in


Molecular detection of herpesviruses DNA is considered as the reference standard assay for diagnosis of central nervous system infections. In this study nested PCR and real time PCR techniques for detection of Herpes simplex virus type 1 (HSV-1), Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in cerebrospinal fluid of HIV patients were compared.Forty-six, 85 and 145 samples previously resulted positive for HSV-1, CMV and EBV by nested PCR and 150 randomly chosen negative samples among 1181 collected in the period 1996–2003 were retrospectively reassessed in duplicate by real time PCR and nested PCR.Samples giving positive results for CMV, HSV-1 and EBV with nested PCR were positive also with real time PCR. One of the negative samples resulted positive for HSV and one for EBV. Real time PCR showed comparable sensitivity and specificity vs nested PCR.Real time PCR proved to be a suitable method for diagnosis of herpesvirus infections in CNS, showing comparable sensitivity and being less time consuming than nested PCR. Opportunistic infections as well as tumors and vascular and metabolic disorders are common in HIV-infected patients -3. GenerAlthough the rate of CNS complications is relatively low, if compared with the high prevalence of herpesviruses in the population, these viruses represent the most important pathogens associated with viral encephalitis and meningitis ,8, beingOne-step or nested polymerase chain reaction (PCR) has rapidly replaced immunological assays based on virus specific Ig antibodies in CSF for laboratory diagnosis of Herpesvirus infections, even if serological methods are considered an additional tool for defining clinical diagnosis.Although nested PCR is considered the method of choice in terms of specificity ,13,14, sIn the last years, introduction of real time PCR has markedly increased the ease and the speed in the virology laboratory due to the relevant technology that permits rapid temperature cycling within a close system. Considering the importance of relationship between viral load in CSF and severity and outcome of disease, an additional advantage of Real Time PCR is the capability to perform simultaneous qualitative and quantitative analysis.Here is reported our experience gained in the diagnosis of herpesvirus infections of the CNS in HIV patients by means of nested PCR and Real Time PCR, which has been recently applied in our laboratory.A total of 1181 CSF samples collected in the period 1996 – 2003 from HIV patients attending at Luigi Sacco Teaching Hospital of Milan  affected by acute encephalitis or meningitis or encephalopathy or other neurological syndromes were considered.Particularly, they consisted of 684, 954 and 933 CSF samples previously tested for HSV-1, CMV and EBV, respectively, by means of nested PCR. Of these, all the positive samples and 150 negative samples randomly chosen were retested by means of nested PCR and real time PCR. Each sample was re-extracted and run in duplicate.CSF positive and negative samples were stored at -80°C until analysis.Clinical data of patients with positive samples were available only in the 20% of the total. These patients generally recorded meningoencephalitis signs such as central motor or sensory alterations, consciousness loss, seizures defects.Spin-column based QIAamp Mini Kit  protocol extraction for CSF was used as indicated by the manufacturer.This procedure allowed for the rapid purification of DNA from 200 μL of CSF and comprised four successive steps carried out using QIAamp Spin Columns in a standard microcentrifuge. Purified DNA was concentrated at a final volume of 20 μL.glycoprotein D gene of HSV-1 [gp58 of CMV [Bam HI-W region of EBV [Nested PCR was carried out in a 50 μl mixture containing 40 μl of first amplification mix -, 2U/μl Taq DNA polymerase (Roche Diagnostics-Germany) and 5 μl of purified DNA. Primer pairs selecting for of HSV-1 , for the8 of CMV , and forn of EBV  are showAfter an initial 2 min denaturation at 94°C, 35 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec were carried out, followed by a 5 min extension at 72°C using a thermal cycler . The reaction mixture for the second amplification round was the same as for the first one, except for the "inner" primers used instead of the "outer" primers. In the second amplification round 44 μl of amplification mix and 1 μl of the first amplification round PCR product were used. The thermal cycling was repeated as for the first amplification round but using 30 cycles after the initial 2 min denaturation. Each amplification run contained a negative control, consisting of water and a positive plasmidial control.Analysis for the PCR products was performed by means of 4 % agarose gel electrophoresis followed by visualization with ethidium bromide (0.4 μg/mL) staining and UV illumination to confirm the expected products.For diagnostic real time PCR Taq polymerase RT PCR Kit  was used. Target regions for HSV and CMV were the same as in nested PCR, while EBNA-1 gene was amplified for EBV  as shownThe RT PCR was performed in 25 μl mixture containing 20 μl of amplification mix  and 5 μl of purified DNA.The amplification program included an initial decontamination with uracile N'-glycosilase at 50°C for 2 min, followed by denaturation at 95°C for 10 min and 45 two steps of 15 sec at 95°C and 1 min at 60°C.t) values were calculated by determining the point at which the fluorescence exceeded a background limit of 0.04. Each analytical session comprised also a negative control (distilled water).The RT PCR products were detected by measuring fluorescence with passive reference dye in Sequence Detection System ABI Prism 7000 (Applied Biosystem). Control threshold  and its concentration determined spectrophotometrically. Then, plasmidic DNA was serially diluted in a stabilizing buffer to the final desired concentration. Amounts of copies/mL in each sample were determined by means of a quantification software , by considering an extraction recovery of 80%.Quantification was carried out by analysing four positive plasmidial standards at 10Of 954 CSFs previously examined for CMV by means of nested PCR, 85 samples resulted positive. Among the 684 CSFs tested for HSV-1, 46 samples were found positive, while, 145 of 933 CSFs tested resulted positive for EBV.Reassessment of these positives and of 150 negative samples confirmed results previously obtained with the same method, with the exception of one HSV-1 positive sample, which resulted negative when retested, as shown in Table Results from real time PCR are reported in Table By considering nested PCR as gold standard ,13, sensIntroduction of PCR into routine diagnostic has rapidly gained a pivotal role for diagnosis of a wide range of diseases, supplanting, in many cases, other methods, such as the classical serodiagnosis. This is particularly true for diagnosis of herpes virus infections in immunocompromised patients, where diminished or suppressed virus-specific antibody responses do not reflect possible reactivated herpes mediated aethiologies .Real time PCR has represented a further step forward, since it allows for quantitative detection of target DNA in a single sample over a large range, remaining possible qualitative detection. Even if contradictory results have been found -21, quanIn the last years several real-time PCR methods have been developed for detection of herpesviruses in different biological specimens -27. RealIn the present work, we compared a real time PCR panel for detection of herpesvirus DNA in CSF with methods employing nested PCR. Our results indicate an overall agreement between the two methods, as reported by other authors ,27,28. DSince extraction panels and primers for HSV and CMV were uniform for the both types of assays, inhibitors present in the DNA preparations could not explain the different results obtained for these samples. Moreover, being nested PCR generally considered as the gold standard for diagnosis of herpes virus in CSF, we tried to use similar primers, chemistry and amplification conditions in order to limit differences for a better comparison of the performance of the two methods. Thus, discrepancies could be likely attributed to the different detection of amplification products, although real time PCR has been reported to be as sensitive as nested PCR . This suData obtained in this study confirms the validity of real-time PCR method for detection of herpesvirus DNA in CSF specimens of HIV patients, being sensitive, rapid and quantitative. Since specific and rapid diagnosis is the main target in the case of CNS infections, real time PCR could be considered the method of choice, due to its high specificity, sensitivity and rapidity, once proper quality controls will be available.The company Amplimedical SpA-Bioline Division-Italy will pay the article processing fees for the manuscript.LD conceived of the study and participated in its design and coordination, AL carried out real time PCR, EDV participated in data analysis and drafted the manuscript, GG performed statistical analysis, RB carried out nested PCR, MRG participated in design and coordination of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Great apes are threatened with extinction, but precise information about the distribution and size of most populations is currently lacking. We conducted orangutan nest counts in the Malaysian state of Sabah (North Borneo), using a combination of ground and helicopter surveys, and provided a way to estimate the current distribution and size of the populations living throughout the entire state. We show that the number of nests detected during aerial surveys is directly related to the estimated true animal density and that a helicopter is an efficient tool to provide robust estimates of orangutan numbers. Our results reveal that with a total estimated population size of about 11,000 individuals, Sabah is one of the main strongholds for orangutans in North Borneo. More than 60% of orangutans living in the state occur outside protected areas, in production forests that have been through several rounds of logging extraction and are still exploited for timber. The role of exploited forests clearly merits further investigation for orangutan conservation in Sabah. A new aerial method for surveying orangutan densities provides robust estimates of their number and could be used for large- scale surveying of great ape populations in Asia and Africa Pongo abelii in Sumatra and Pongo pygmaeus in Borneo, are threatened with extinction in the near future [The two orangutan species, r future ,2. A prer future . Densitir future ,5. In orr future . In addir future .The latest estimates available for orangutan populations in the Malaysian state of Sabah (North Borneo) range from 20,000  to less 2R = 0.86, n = 13, p < 0.001), by the second observer , by both observers , and with the average value obtained for both observers at each site .Ground densities estimated with Distance 3.5 and aerial densities predicted with our model are given in We recorded 2,708 orangutan nests during ground surveys  and 6,936 nests from the helicopter . The size of the sampling areas ranged from 0.001% to 1% (ground survey) and from 1.8% to 16.9%  of the total size of each forest surveyed .Our surveys confirmed that orangutans were patchily distributed throughout their range in Sabah , occurri2 locally) were identified in the semi-inundated lowland forests of Kinabatangan (population 13) and Segama floodplains (population 16), Kulamba (population 12), and Tabin (population 14). Most of these forests were highly disturbed, fragmented, and located at the edge of newly established oil palm plantations.The highest nest abundances were recorded in lowland forests below 300 m asl, although we recorded a few nests as high as 1,300 m asl, which appeared to be the upper altitudinal limit for the species in Sabah. The highest orangutan densities  than in areas where more conventional practices were implemented : Mann–Whitney U test, U = 5.5, p = 0.03. Hunting pressure was low in all these forests  yielded higher orangutan densities in old exploited areas and in areas that were exploited under sustainable logging practices  were present in Sabah at the time of our surveys . Two majAerial surveys are widely used for estimating animal abundance and population trends in open and semi-open landscapes . In SabaNest detectability from the helicopter depends on observers and canopy structure. Ideally, specific models for deriving nest densities from aerial indexes (number of nests detected per kilometer of flight) should be designed for different human observation skills and for different habitat types. However, observer bias can be avoided if the same team of skilled people conducts the entire survey. The second source of bias could be overcome with the design of several habitat-specific models. Before these types of models are designed, ground-truthing must be conducted in different habitat types in order to validate a baseline model and to determine habitat-specific correction factors when necessary.Nest parameters used for obtaining the final orangutan density estimates  are a major source of inaccuracy in aerial and ground nest surveys , and theP. p. morio subspecies [Our survey shows that there are currently about 11,000 orangutans present in Sabah, making the state the main stronghold for the bspecies . Howeverbspecies . This debspecies .P. p. morio found in Borneo (population 16).The current network of protected areas in Sabah harbors about 4,000 orangutans, representing about 40% of the total number found in the state. About 60% of the total number of orangutans survives in commercial forest reserves subjected to timber extraction, and these forests harbor the largest unfragmented population of the subspecies The impacts of forest exploitation on ape abundance and ecology depend on several factors, such as (1) the forest types that existed initially and the quality of the regrowth forest , (2) typOur results tend to indicate that the mosaic of habitats found in the semi-inundated mixed dipterocarp forests that were originally occurring in the floodplains of east Sabah could potentially still harbor a significant number of animals following high disturbance levels . However, we can assume that the very high orangutan densities documented in some of the areas located close to oil palm plantations partly result from the influx of newcomers following recent land conversion to agriculture ,30. We cLess diverse habitats (dry lowland dipterocarp forests) located in the interior of the state appear to maintain fewer orangutans, particularly following conventional, nonsustainable logging practices (populations 15 and 16). In the extensive tracts of dry lowland dipterocarp forests exploited for timber (populations 15 and 16), our data suggest that, when hunting pressure is low, orangutan abundance is directly related to the degree of logging and associated damage. For these two populations, the highest orangutan densities were identified in Deramakot, a commercial forest reserve (part of population 15) implementing sustainable logging practices , suggestP.p. morio are not necessarily valid for other Bornean orangutan subspecies and for the Sumatran species [Possible inter- and intraspecific differences in general ecology and feeding behavior of orangutans may also influence population responses to habitat disturbances , and the species ,33,34,35All great ape species require large forest areas to survive. An ecological network combining protected areas with seminatural landscape elements and production forests could be seen as an option to conserve biodiversity, while also providing opportunities for the sustainable use of natural resources ,37. HoweFinally, aerial nest surveys may also be of use in Africa, although it may be difficult to detect nests of African great apes from a helicopter because they tend to be lower in the canopy and it may be impossible to distinguish between gorilla and chimpanzee nests in those areas where the two species are sympatric .2 in the northern portion of the island of Borneo. It is one of the 13 states in the federation of Malaysia. Approximately half of the total land mass is covered with forests , some emergent trees; (3) recently exploited forests (timber extraction was conducted less than 15 y ago): logging roads not entirely colonized by pioneer tree species; few emergent trees; (4): active exploitation: logging activities were ongoing at the time of the survey.All major forest blocks in Sabah were identified from 1:50,000 vegetation maps, and these were divided into 16 different blocks. For each block, we determined a systematic stratified sampling using equidistant parallel line transects, the location of the first line being randomly selected . BecauseFrom the back seats, two observers looked for orangutan nests from either side of the helicopter. All visible nests were recorded. It was impossible to estimate the impact of nest age on nest detection, and the observers acknowledged that a few days-old fresh nests (still green in color) and nests at their latest stages of decay (just a few branches visible) were difficult to detect in the canopy. These nest categories are likely to have been underdetected. The two observers indicated all sightings to a nest recorder seated between them. The nest recorder noted the number of nests detected by the observers per each 30-s period. All crew members were in constant radio contact during the flights. After the flight, data collected by the copilot and the nest recorder were matched in order to determine the location of all sightings along the aerial line transect precisely. The same team of observers conducted all aerial surveys in order to avoid the observer bias.For technical reasons, it was impossible to fit external devices to the helicopter to estimate the distance of the nests to the aerial transects. This prevented us from determining the detection function from our data alone . TrailinBecause the proportion of the actual nest population existing in the forest that was detected from the helicopter was unknown, it was impossible to directly estimate nest densities from our aerial results . We thusth percentile). Heaping was assessed from histograms, and data were grouped where necessary [Nest densities and their variances were estimated by ground line transects using distance sampling ,12. A seecessary ,14. The ecessary . The adeecessary . Finallyecessary . Resultsecessary .D^ was a linear function of both the logarithm of the aerial index AI and the observer effect obs, plus their interaction, in order to include possible differences between observers. We weighted the general regression by the estimated variances of ground nest densities, thus giving a greater emphasis to precise density estimates. The least squares method was used for model fitting by incorporating weights 1/σ^i)log(D^where σ^i)log, and X was the matrix of covariates:where N26, where Σwas a 26 × 26matrix with σ2 · diag(var(logD^i))in the diagonal and zeroes elsewhere.βwas a 4 × 1 vector of parameters to be estimated, and ɛ was a 26 × 1 vector of errors with multivariate normal distribution D^ rewritten using the quantities log (D^)w = W · log(D^), wX = W · X, and ɛw = W · ɛ, with W = diag(σ^−1i)log was w has a more familiar distribution N26, allowing the use of linear regression tools to estimate model parameters via least square theory. We used the backward model selection procedure [p value and greater than a 10% cutoff were then removed one by one, and each new model was retested until all p values of the remaining covariates were less than the cutoff value. We assessed the goodness of fit of the best model by computing the coefficient of determination 2R. The best model supported by the data considered only the aerial index effect  withUnlike ɛ, ɛrocedure  to selec0,AI recorded during helicopter flights, asUsing this model (5), we predicted an orangutan nest density from any new aerial index values, . This mowithX, the matrix defined above, once the observer effect had been removed, s2 the residual mean square up to a constant that was an estimate of σ2, and t23,0.025 the appropriate two-sided t-distribution percentile [and rcentile . Followircentile , this inwhereandA numerical application gaveThese intervals are shown in t-test, n = 5, t = −1.738, p = 0.157; 95% confidence interval of the difference: −110 to 25; ratio between ground and aerial nest densities = 0.94). This result showed that nest detectability was similar in forests that had been exploited for timber in Sabah and in Kinabatangan. Thus, we used the baseline model without any correction for all recent and old exploited forests of the state. Exploited swamp forests had a very open canopy, and predicted aerial nest densities were higher than estimated ground densities, although the difference was not significant . However, in order to not overestimate the final densities, we applied a correction factor of 0.54 to aerial indexes obtained in two areas of extensive exploited swamp forests (parts of populations 12 and 14). In primary lowland dipterocarp forest, the predicted aerial nest density was lower than the estimated ground density in the only site that was tested . Aerial indexes obtained for Danum (part of population 16), the only site of primary lowland forest with a significant orangutan population assessed during our survey, were multiplied by a correction factor of 1.5.Model (5) was obtained with results from the old and recently exploited semi-inundated mixed lowland dipterocarp forests of Kinabatangan. We tested its validity by comparing nest densities predicted from aerial data and estimated from ground line transects at several sites. We found no significant differences for five sites of old and recently exploited dry lowland dipterocarp forests . This interval was then back-transformed to obtain a confidence interval for D^whereThe numerical application gavewithThe results of the ground and aerial surveys were processed with a geographic information system , using a combination of administrative maps and satellite images. When necessary, we stratified each forest block according to (1) disturbance type: no disturbance, old or recently exploited forests, ongoing exploitation; (2) altitude: lowland, below 500 m above sea level (asl); upper land, 500–1,000 m asl; lower mountain, 1,000–1,500 m asl; mountain, above 1,500 m asl; and (3) habitat type: swamp forests, semi-inundated mixed lowland dipterocarp forests, dry lowland dipterocarp forests. We then determined the percentage of habitat actually occupied by orangutans as the ratio between the total length of aerial transects and the length flown over areas with no visible orangutan nests . This percentage was applied to the total size of each forest block determined from maps in order to estimate the final size of “habitat occupied by orangutans.” We then multiplied the estimated orangutan densities by the estimated size of orangutan habitat occupation to obtain overall population estimates.A confidence interval for the population estimate was computed via parametric bootstrapping for the whole population in Sabah . We assuOver a 2-y period (2002–2003), ground surveys  and aerial surveys (72 “man days”) were conducted in all major forests of the state . Recce w