Polypedates teraiensis during their development and metamorphosis, that is, transfer from an aquatic mode to a terrestrial mode of life. Blood smears of 21 different stages (Gosner stage 26 to 46) of tadpoles were investigated. Population of erythrocytes was heterogeneous in population represented by various forms . Correlation between various morphometric values of erythrocytes was determined with different developing stages of tadpoles. Amongst the leucocytes, the lymphocytes were the most abundant cells followed by neutrophils. Neutrophils and monocytes showed varied morphologic forms. The percentage of lymphocytes and neutrophils showed a negative whereas percentage of eosinophil, basophil, and monocytes showed a positive correlation with the developmental stages of tadpoles. Blood platelets were also observed, which were rounded in shape and found in aggregates.The present paper describes a sequential study of the leukocyte profiles and the changes in morphometry and morphology of erythrocytes in the tadpoles of  Anuran metamorphosis is an interesting physiological phenomenon where the aquatic tadpoles become terrestrial froglets. It is a rapid process to meet the demands of a new life form . During  Rana catesbeiana. Granulopoiesis of basophils and heterophils (neutrophils) in the tadpoles of Rana esculenta [Xenopus laevis [Rana esculenta [Rana catesbeiana [ Rana pipiens [Abundance of different leucocytes under normal metamorphosis  and thyrsculenta  and press laevis  have beesculenta , Rana caesbeiana , and Ran pipiens  have beeRana pipiens [Rana catesbeiana [Hyla crucifer, Hyla versicolor, Rana clamitans, and Bufo americanus has also been described earlier [In larval anurans, two general types of erythrocytes, that is, larval and adult forms, are found, which are differentiated by size and morphology during development –24. The  pipiens  and Ranaesbeiana –28. Thyr earlier . Bufo melanostictus (new name: Duttaphrynus melanostictus) [Rana tigerina (new name: Hoplobatrachus tigerinus) [Polypedates maculatus [ Polypedates teraiensis  (Anura: Rhacophoridae).Sequential study on the leukocyte profile and changes in the morphometry and morphology of erythrocytes in different larval stages of anurans is not well documented. Moreover, blood cell profile of Indian anurans is restricted to few adult species, namely,stictus) –33, Ranagerinus) , and Polaculatus . There iaculatus ) of the Polypedates teraiensis [Amaranthus leaves ad libitum. The foam nests of Dubois's tree frog raiensis  were colraiensis . After h For the study, tadpoles from Gosner stages 26 to 46  were con Tadpoles were anesthetized by immersing them in 1% MS-222 (Tricaine Methane Sulphonate) solution. The blood of tadpoles from stage 26 to 44 was obtained from tail amputation through the middle of the tail. For stages 45 and 46, blood was collected from the heart using a fine syringe. Blood smears were prepared using push slide technique. The dried blood smears were stained with Giemsa's stain and were observed under light microscope (Hund H500). Different types of blood cells present in the smear were identified following Hadji-Azimi et al. , Turner r” was calculated in each case by Karl Pearson's method [The relationship between developmental stages and blood cell profile was assessed by drawing scatter plots. The correlation coefficient “s method .Polypedates teraiensis from Gosner stages 26 to 46 is represented in Tables A cumulative account of the blood cell profiles of tadpoles of The erythrocytes were observed to occur in various forms. They were either elongated or circular (round) in shape. Elongated cells were oval (cells with perfect and smooth curves at both the ends)  and elliThe leucocytes observed in the present study were of five types, that is, lymphocytes , monocytes, eosinophils, basophils, and neutrophils. The first two being agranulocytes and the rest were granulocytes. Most of the lymphocytes were rounded in shape. The nuclei were rounded in shape both in large  and smalBlood platelets were rounded in shape and found in clusters .μm (Gosner stage 35) to 24.24 ± 2.14 μm (Gosner stage 42) and 15.15 ± 0 μm (Gosner stage 30) to 22.14 ± 1.61 μm (Gosner stage 27), respectively (μm (Gosner stage 30) to 12.12 ± 1.11 μm (Gosner stage 27) and 9.09 ± 0 μm  to 12.12 ± 2.14 μm (Gosner stage 31), respectively, in different developmental stages (μm2 (Gosner stage 36) to 403.908 ± 43.16 μm2 (Gosner stage 27), while the nuclear area ranged from 64.720 ± 0 μm2 (Gosner stage 30) to 168.290 ± 18.12 μm2 (Gosner stage 28) in the tadpoles of different developmental stages , breadth , and area  of the erythrocytes. Similar negative correlation was observed for nuclear length , breadth , and area . However, the aspect ratio (length/breadth) of erythrocytes was positively correlated both for cells  as well as nuclei  , basophils , and eosinophils  showed a positive correlation, whereas neutrophil  and lymphocytes  showed a negative correlation with different stages during development  have been reported to be present during anemic conditions [Poikilocytosis of RBC with irregular shapes was observed in patches only during early stages, that is, 26 to 31 Figures . Disappen et al.  have obsnditions , and thenditions . Thus, tHyla crucifer, Hyla versicolor, Rana clamitans, and Bufo americanus [Senile erythrocytes (without nuclei) and schistocytes (erythrocytes fragments) were common in stages 41 and 42. Presence of the senile erythrocytes has been reported during metamorphosis in tadpoles of ericanus . SchistoRana catesbeiana. Comparatively larger erythrocytes observed in stages 42 to 45 of the present study resembled the immature erythrocytes described in the tadpoles of Rana catesbeiana [Apart from this, division of erythrocytes was also observed from stages 39 to 44. This may be due to increase in erythropoietic activity during metamorphosis as reported earlier by Maniatis and Ingram  in Rana esbeiana . Rana catesbeiana [Size (length and breadth) of erythrocytes and their nuclei was observed to be negatively correlated with the developmental stages of the tadpoles Figures . The aspesbeiana , 23, 28.Rana catesbeiana, which declined with the onset of metamorphosis. Neutrophils also showed a negative correlation with the developmental stages (Rana catesbeiana [Rana catesbeiana [The leukocyte population (the nonspecific immune system) is reported to be one of the primary lines of defense against invading pathogens and is made up of five different types of white blood cells, each performing different tasks in the immune process , 54 in al stages . Since, esbeiana . A positesbeiana , 57. Thuesbeiana . Basophiesbeiana , generalPolypedates teraiensis during their development and metamorphosis, that is, their transfer from an aquatic to a terrestrial mode of life. As this is the first study to examine the hematological values of the tadpoles of Polypedates teraiensis, it establishes a baseline data that can be used as general reference values for future investigations involving this species and other anuran tadpoles. Thus, the present study provides information regarding the blood cells of the tadpoles of


FCT = 0.054; mtDNA ΦCT = 0.222): (i) Okhotsk Sea–Norton Sound–Aleutian Islands, (ii) southeastern Bering Sea–western Gulf of Alaska, and (iii) Southeast Alaska. Populations in the Bering Sea and in Southeast Alaska are genetically heterogeneous, but populations in the center of the range are homogeneous. Mitochondrial DNA diversity drops from h = 0.91 in the northwestern Pacific to h = 0.24 in the Southeast Alaska. Bayesian skyline plots (BSPs) indicate postglacial population expansions, presumably from ice-age refugia. BSPs of sequences simulated under a demographic model defined by late Pleistocene temperatures failed to detect demographic variability before the last glacial maximum. These results sound a note of caution for the interpretation of BSPs. Population fragmentation in the Bering Sea and in Southeast Alaskan waters requires population management on a small geographic scale, and deep evolutionary partitions between the three geographic groups mandate regional conservation measures.Observed patterns of genetic variability among marine populations are shaped not only by contemporary levels of gene flow, but also by divergences during historical isolations. We examined variability at 15 SNP loci and in mtDNA sequences  in red king crab from 17 localities in the North Pacific. These markers define three geographically distinct evolutionary lineages (SNPs,  Evolutionary biologists and marine fishery managers are challenged with two questions. First, how do genetic differences arise among marine populations? A prevailing paradigm postulates that populations diverge, in part, because of contemporary processes, including random drift and selection. Despite large potentials for dispersal of planktonic larvae in many marine species, shoreline topography and ocean currents, in some cases, isolate populations and promote divergence . IdeallyParalithodes camtschaticus  in the North Pacific and Bering Sea. This species is the largest and most abundant of the king crabs in the North Pacific and extends from the Sea of Japan  and mitochondrial (mt) DNA markers to define population structure for harvest management of red king crabs and to lay a foundation for possible stock enhancements. We use the analysis of allele frequencies to detect the effects of drift between populations, and the analysis of mtDNA variation to resolve historical events. Maternal inheritance of mtDNA produces lineages that trace the dispersals and colonizations that have shaped a deep phylogeographic structure in red king crabs . We usedIn total, 1501 red king crabs were sampled at 17 locations extending from the Okhotsk Sea, through the Bering Sea and western Gulf of Alaska to Southeast Alaska , Table 1m of both polymerase chain reaction (PCR) primers, and 200 nm of each probe , 1 μm of forward and reverse primers, and 5 U GoTaq Flexi DNA polymerase . PCR amplifications were conducted in ABI 9700 thermocyclers with an initial denaturation of 1 min at 95°C, 37 cycles of 40 s at 95°C, 40 s at primer annealing temperature 41°C, and 1 min at 72°C; the final cycle was at 4°C for 5 min. Cycles were made at a ramp speed of 1°C/s. The PCR amplifications were sequenced in the forward and reverse directions by the High-Throughput Genomics Unit . Sequences were aligned with a published sequence for red king crab (GenBank accession no. AB211303) with the muscle algorithm  universal primers LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198  in arlequin 3.11  or haplotype (mtDNA) frequencies. Additional amovas of mtDNA haplotypes were based on an FST analog statistic (ΦST) that accounts for both haplotype-frequency differences between samples and sequence divergences between haplotypes. We used the amovas. The probability an F statistic was larger than 0.0 was estimated with 50 000 permutations of individuals among samples.Single nucleotide polymorphism genotypes for the 15 loci were tested for fit of genotypic frequencies to Hardy–Weinberg expectations (HWE) in each sample with exact probability tests of 20 batches of 10 000 permutations and a burn-in of 10 000 permutations  computed with NTSYS . Haplotype richness was estimated with HP-rare (π) and haplotype diversity (h) and their standard deviations were estimated with arlequin. A 95% plausible parsimony network of mtDNA haplotypes in 1278 crabs was made with TCS 1.21  can be estimated from the results with Neμg, where μ is the mutation rate and g is generation time (about 5 years for red king crab). However, estimates of μ are problematic, as no temporally anchored phylogenetic estimates are available for Paralithodes. Even so, phylogenetic estimates appear to greatly underestimate contemporary mutation rates and do not appear to be appropriate for BSP calibrations (n = 200) of sequences (bp = 665) with Mesquite 2.74  with st 1.6.1  with MCMbrations . To helpite 2.74 . The scaite 2.74 . We reasodelTest . This emHO = 0.148 (western Gulf of Alaska) to HO = 0.220 , so these samples were combined. SNP allele frequencies differed significantly among the 17 samples . An amova indicated that the greatest heterogeneity occurred among four groups, [1]  [4–11] [12–17] , or among three groups [1–3] [4–11] [12–17] . Samples 1 and 3 were significantly different , but not 1 and 2 . Eight samples from the southeastern Bering Sea and western Gulf of Alaska [4–11] were genetically homogeneous . However, a comparison between samples from the southeastern Bering Sea and western Gulf of Alaska was significant . A significant amount of heterogeneity appeared among the six samples from southeast Alaska [12–17] . A core group of samples  showed no heterogeneity . However, samples 15  and 16  each introduced heterogeneity into this core group.No significant temporal shift in SNP frequencies overall loci was detected between two samples from Deadman Reach  and were pooled. A PCoA of ΦST values revealed the same three groups detected with SNPs, with an addition of the outlier sample from Seymour Canal  and haplotype divergence  corresponds to large differences among groups. Most of this divergence was because of differences among the three regional groups [1–3] [4–11] [12–17]  . Unlike SNPs, samples 1 and 2 differed significantly , but not 1 and 3 . As with SNPs, samples 4 and 5 from the southeastern Bering Sea were not significantly different from each other . The six samples from the western Gulf of Alaska [6–11] were homogeneous . As with SNPs, the addition of  to this group did not produce significant heterogeneity . However, a comparison of haplotype frequencies between samples  and [6–11] was marginally significant , but not for frequencies plus haplotype divergences . In Southeast Alaska, a core group  was genetically homogeneous , but the inclusion of sample 15 from Seymour Canal to the group produced a significant amount of heterogeneity .A significant amount of overall haplotype-frequency heterogeneity ( df = 2) . The comn = 551) had a central haplotype (JF738160) that occurred in 57.2% of the crabs. In the haplotype network for the combined Southeast Alaska samples [12–17], JF738160 occurred in 80.7% of the crabs. Several low-frequency haplotypes, unique to the region, were one substitution from the common haplotype. A unique small lineage appeared in Seymour Canal [15], in which the central haplotype was one mutation removed from the most common haplotype.The haplotype networks  varied cDT was not significant in samples from the western Pacific, Bering Sea, and western Gulf of Alaska, but was significant and negative for each of the samples from Southeast Alaska, indicating excesses of low-frequency mutations  with statistical power of 95%, or greater. However, these markers provide only a small amount of power (10–50%) to detect finer differences among populations (FST = 0.001). Such small differences likely reflect short-term frequency shifts from unequal recruitment among populations, or temporary isolations stemming from North Pacific ocean–climate regime shifts. Hence, our discussion is focused on larger-scale geographic patterns resulting from glacial isolations, postglacial dispersals, and population expansions.Previous genetic studies of red king crabs in the Alaskan waters did not resolve some details of population structure reported here. The SNP and mtDNA markers and samples sizes used in this study can detect low levels of differentiation among populations  that are typical of many marine species , and populations in Southeast Alaska had even lower diversities .A striking feature of red king crab phylogeographic structure is a strong gradient in both mtDNA haplotype and nucleotide diversity across the North Pacific. While some SNPs show allele-frequency clines across the North Pacific, a comparable drop in diversity within populations was absent, most likely because SNPs are assays of specific nucleotides and, hence, are not suitable for surveying geographic patterns of within-population diversity. Western populations have high levels of mtDNA diversity  for the last 800 000 years . Each cyDT, by a unimodal mismatch distribution with a mean number of mismatches <1.0, and by a large number of private haplotypes.The high levels of genetic diversity, ragged mismatch distributions, and long coalescence times in the northwestern Pacific indicate that these populations have remained large over the Pleistocene and likely supplied colonists for extirpated eastern North Pacific areas on long time scales  red king crabs, on the other hand, are at the extremes of a large group of genetically homogeneous populations in the northern Gulf of Alaska. Southeastern Bering Sea populations are tied to northern Gulf of Alaska populations by recent common ancestry and by dispersals from the western Gulf of Alaska. Other species, including flatfishes  and WallThe genetically homogeneous populations in this region may have spread from the Kodiak refuge. Parts of the haplotype network for this group consist of a star-shaped mtDNA genealogy, indicating a recent population expansion. It is uncertain, however, whether the genetic similarity among populations is a legacy of the expansion or whether these populations are connected by high levels of larval dispersal in the fast-moving Alaska Coastal Current ; Fig. 2aOther information from stock assessments may provide insights into genetic population structure. Synchronous responses among red king crab populations to climate variables may reflect genomic similarities. For example, populations in the southeastern Bering Sea and western Gulf of Alaska showed similar recruitment patterns over several decades, but these patterns were significantly different from those in Norton Sound and Southeast Alaska . In receThe results of this study clarify issues in the management of red king crab harvests and possible stock enhancements. The genetically defined groups of red king crabs largely coincide with the State of Alaska’s registration areas. Populations in the northeastern Bering Sea (Q), Bristol Bay (T), and the Aleutian Islands (O) are genetically divergent from one another, because of different biogeographical histories. However, oceanic current patterns or biotic interactions prevent large-scale mixing between these populations. The genetic discontinuity between western Aleutian and southeastern Bering Sea populations coincides with a biogeographical boundary at Samalga Pass in the Aleutians that has been attributed to oceanic features . AdditioFST, which is based on frequencies, but not with ΦST, which additionally incorporates sequence divergences between haplotypes  and western Gulf of Alaska , and possibly the northern Gulf of Alaska . Genetic similarity among these populations likely reflects a postglacial radiation from a common glacial refuge and may not indicate close ties by gene flow. While no overall significance was detected among populations in this group, significant SNP and mtDNA divergence was detected with plotypes  and 3. Lplotypes . These gplotypes , indicatIn contrast, Southeast Alaskan populations show much greater levels of subdivision. These populations may have expanded into Southeast Alaskan waters from a glacial refuge population, but are presently isolated in semi-enclosed fjords that limit larval dispersal. Private mtDNA haplotypes in all of these populations indicate isolation and population self-recruitment. These populations may also have diverged from one another because of adaptive shifts to local environmental conditions. While all of these populations fall into a single registration area, they are best managed on a finer geographic scale, because of their demographic independence.Research is underway to develop hatchery techniques to raise crabs for restoring depressed populations, or for stock enhancement. If stock restoration is warranted because of reproductive limitations, several genetic factors must be considered in devising a stock restoration plan. A close genetic match between hatchery broodstock and the depressed population is desirable. While the results of population genetic studies provide important background information, the molecular markers used in these studies are generally neutral to selection and may not detect important adaptive differences among populations. Patterns of gene flow between local populations and the spatial scale of the adaptive landscape for red king crab are unknown. In addition to broodstock choice, artificial culture may alter the genetic profiles of hatchery-reared crabs, because more larvae survive in a hatchery setting than in the wild, setting the stage for selective or random shifts in genetic profiles. Hybridizations between hatchery-reared crabs and wild crabs may compromise the genetic integrity of the wild populations, and persistent hatchery releases may displace wild populations with crabs of hatchery ancestry .In conclusion, the distributions of SNP and mtDNA population markers portray the effects of ancient isolations and dispersals, contemporary gene flow, and random drift on population structure. These findings provide managers with a sound basis for managing harvests and restorations on a population level. However, the failure to find significant differences among populations in some areas with neutral molecular markers does not necessarily indicate that populations are connected with large amounts of gene flow. Historical expansions from a common ice-age refuge can produce the appearance of panmixia, even though populations may be demographically independent from one another.


The purpose of this study was to assess the beneficial effects of using a far-infrared (FIR) belt on the management of patients with primary dysmenorrhea. This is the first study to determine the efficacy of somatothermal FIR using a parallel-arm randomized sham-controlled and double-blinded design with objective physical evidence and psychometric self-reports. Fifty-one Taiwanese women with primary dysmenorrhea were enrolled in the study. Results indicate that there was an increased abdominal temperature of 0.6°C and a 3.27% increase in abdominal blood flow in the FIR group (wearing FIR belt) compared to those in the control group (wearing sham belt). Verbal rating scale and numeric rating scale scores in the FIR group were both lower than those in the control group. Compared to the blank group (wearing no belt), the average dysmenorrhea pain duration of the FIR group was significantly reduced from 2.5 to 1.8 days, but there was no significant difference in the control group. These results demonstrate that the use of a belt made of far-infrared ceramic materials can reduce primary dysmenorrhea. Dysmenorrhea is pain with menstruation, which may be accompanied by headache, dizziness, nausea, vomiting, diarrhea, cold sweats, or other symptoms , 2. For Clinical research has shown that the main cause of primary dysmenorrhea may be associated with increased production of endometrial prostaglandin, resulting in a high concentration of prostaglandins in blood. The pain-related biomolecular induction of cyclooxygenase (COX-2) and prostaglandin is strongly associated with the severity of primary dysmenorrhea . ExcessiFor women with dysmenorrhea, the application of local heat can reduce muscle tension and relax abdominal muscles to reduce pain caused by muscle spasms. Heat can increase pelvic blood circulation to eliminate local blood and body fluid retention and diminish congestion and swelling, thereby enabling a reduction in pain caused by nerve compression. Previous studies reported that local heat inhibits pain signals and increases proprioception , 15.Heating pads and hot water have long been used as a folk remedy for the treatment of dysmenorrhea . Akin etFar-infrared (FIR) rays are invisible electromagnetic waves, which can produce many biological effects including thermal and nonthermal effects –23. WhenThe use of electrothermal FIR belts at a temperature of 50°C was recently reported to be an effective treatment for primary dysmenorrhea , 31. TheThe clinical trial was approved by the institutional review board (IRB) at HungKuang University, (Project number 98-B-002). The participants were screened by a gynecologist. Inclusion criteria were age 18 years or older which can legally give consent to participate in this clinical trial and history of painful menstruation for at least one year. Physical examination and ultrasonography were used to support diagnosis of primary dysmenorrhea. Patients who had gynecological diseases or who used other FIR products within one month before the study were excluded. Patients were instructed not to use any type of medication, such as NSAIDs or oral pills, to relieve painful menstruation during the study period. In the last analysis, a total of 51 subjects aged 19 to 35 years were enrolled in this study. The investigation was conducted between January 1, 2010 and December 31, 2010.μm, was 10.16 mW/cm2 as determined using an SR5000 spectroradiometer  at the Industrial Technology Research Institute, Taiwan. Two kinds of belts each measuring 15 cm × 70 cm were used in this study. The FIR belt and the control belt were embedded with and without 10 wt% FIR ceramic powders, respectively. As previously reported –23, the n = 26) group or control belt (n = 25) group. Each subject then wore the belt around the abdominal region for 30 minutes. After removing the belt, posttreatment measurements of abdominal temperatures and blood flows were performed. All measurements and procedures in this study were performed in a climate-controlled room at a constant temperature and humidity (23°C and 60% relative humidity) . Before n = 51) completed two questionnaires, the verbal rating scale (VRS) and the numeric rating scale (NRS) [All participants (le (NRS) , 34, to P < 0.05 was considered statistically significant (*) and P < 0.01 was highly significant (**).All data between groups before and after applying belts were analyzed by the Wilcoxon Signed-Rank Test using the SPSS 12.0 software package for statistical analysis. A value of With the rise in body temperature, there was a corresponding rise in abdominal blood flow after using the belts for 30 minutes. The average blood perfusion units measured by the MoorLDI2-IR Laser Doppler Imager were 116.5 and 119.0 before and after intervention in the control group, and 115.9 and 121.4 in the FIR group, respectively. There were increases in blood flow of 2.12% and 4.78% in the control group and FIR group, respectively . Thus, tFigures The results of the second day Figures  and 8 weThe results of the third day Figures  and 10 wFigures Previous research has shown that the dysregulation of endometrial blood flow can cause menstrual disorders . Strong In addition to the aforementioned thermal effects, FIR also exerts nonthermal effects in pain relief of dysmenorrhea. Previous studies showed that FIR could increase generation of nitric oxide (NO) and calmodulin in cells , 20, 36.Ischemia is mainly a consequence of decreased microcirculation or a reduction in local muscle blood flow. This scenario has important consequences for cellular metabolic status, as metabolic and acid-base status is significantly worsened with increased acidosis. During dysmenorrhea, the corresponding regional organic ischemia is associated with increased oxidative stress due to increased levels of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, which are responsible for destructive processes in organic tissues. We demonstrated that the FIR ceramic material exerted an antioxidant effect by increasing hydrogen peroxide-scavenging activity , 22. We also found that FIR induced anti-inflammatory effects by inhibiting prostaglandin E2 (PGE2) , and FIRThese findings of this study showed that the somatothermal FIR belt was effective as a novel alternative therapy for relief of primary dysmenorrhea. The FIR belt provided better therapeutic effects in terms of pain relief as well as greater elevation of skin temperature and promotion of blood circulation compared with those observed in patients wearing the sham belt. Compared with traditional complementary and alternative medicine therapies for pain reduction in patients with primary dysmenorrhea, our method has a number of advantages, which include convenience and ease of use, and no external energy source is required. We believe that this novel, noninvasive, and convenient FIR therapy may be of practical use in a clinical setting and suggest that further studies be conducted to confirm our results.


Enteric fever is a major public health problem, causing an estimated 21million new cases and 216,000 or more deaths every year. Current diagnosis of the disease is inadequate. Blood culture only identifies 45 to 70% of the cases and is time-consuming. Serological tests have very low sensitivity and specificity. Clinical samples obtained for diagnosis of enteric fever in the field generally have <1 organism/ml of blood, so that even PCR-based methods, widely used for detection of other infectious diseases, are not a straightforward option in typhoid diagnosis. We developed a novel method to enrich target bacterial DNA by selective removal of human DNA from blood samples, enhancing the sensitivity of PCR tests. This method offers the possibility of improving PCR assays directly using clinical specimens for diagnosis of this globally important infectious disease.Salmonella Typhi bacteria were collected from the specimen by centrifugation and the DNA extracted with QIAamp DNA mini kit. The presence of Salmonella Typhi bacteria in blood samples was detected by PCR with the fliC-d gene of Salmonella Typhi as the target.Blood samples were mixed with ox bile for selective lysis of human blood cells and the released human DNA was then digested with addition of bile resistant micrococcal nuclease. The intact Salmonella Typhi DNA was enriched in DNA preparations and the PCR sensitivity for detection of Salmonella Typhi in spiked blood samples was enhanced by 1,000 fold.Micrococcal nuclease retained activity against human blood DNA in the presence of up to 9% ox bile. Background human DNA was dramatically removed from blood samples through the use of ox bile lysis and micrococcal nuclease for removal of mammalian DNA. Consequently target Use of a combination of selective ox-bile blood cell lysis and removal of human DNA with micrococcal nuclease significantly improves PCR sensitivity and offers a better option for improved typhoid PCR assays directly using clinical specimens in diagnosis of this globally important infection disease which we believe could be of importance in improving clinical care and providing effective evaluation of novel vaccines. Salmonella enterica serovar Typhi, remains a common problem in many parts of the world where access to clean water is limited. In the regions where enteric fever is common, clinical diagnosis of typhoid fever is inadequate, as the symptoms it causes are non-specific and overlap with those of many other febrile illness including malaria, dengue fever, rickettsioses, leptospirosis and melioidosis [et al. has recently presented the current position in typhoid diagnostics, highlighting the need for technological improvements and potential future approaches [There are an estimated 21million new cases and 216,000 deaths attributed to typhoid fever every year [proaches .Salmonella Typhi cells are used to detect antibodies in blood. Many of the surface antigens of the Enterobacteriaceae of which Salmonella Typhi is a member, demonstrate significant conservation and induce antibodies that are cross-reactive. Consequently, the Widal test has very low sensitivity and specificity, and little or no practical value in endemic areas despite its continued use [The first typhoid diagnostic, the Widal test, was developed in 1896 and is still widely used. The Widal test is dependent on agglutination in an assay in which nued use . Severalnued use ,6, but hnued use ,8.Salmonella Typhi, the presence of bactericidal activity in the blood, recent administration of antibiotics, the type of culture medium used, and the length of incubation period may all affect the sensitivity [Salmonella serovar Typhi also slows its growth in blood culture media. One study found that more than 50% of bacterial cells were present intracellularly in the blood from patients with typhoid fever [Isolation of the causative organism remains the most reliable diagnostic method in suspected typhoid fever and blood has been the main sample used for culture of the organism since 1900 . Howeversitivity ,12. The id fever . In addiSalmonella Typhi (specificity) and should detect even low numbers of live or dead bacterial cells (sensitivity). Many Salmonella Typhi PCR-based assays have targeted the fliC-d gene, utilizing nested primers to improve sensitivity [Salmonella bacteria circulating in the blood of a patient with bacteremia is generally low with the majority of patients having <1 organism/ml of blood [Salmonella Typhi [Given the problems associated with serological methods and blood culture, PCR based methods have been exploited recently because they can theoretically amplify DNA only from sitivity -18 and rof blood . This meof blood ,21. Furtla Typhi .Salmonella Typhi cells remain intact and are collected by centrifugation for the extraction of pure Salmonella Typhi DNA. Micrococcal nuclease, derived from Staphylococcus aureus, is a relatively non-specific endo-exonuclease and can degrade both DNA and RNA to 3´ phosphomononucleotides and dinucleotides in the pH range of 7.0-10.0. The enzyme activity is strictly dependent on Ca2+ and therefore is easily inactivated by EGTA. We investigated further micrococcal nuclease activity in ox bile and describe herein a novel method for the selective enrichment of Salmonella Typhi DNA from whole blood samples using ox bile and micrococcal nuclease, which we have termed “the selective target DNA enrichment method” (STEM). This method does not require blood culture. By applying STEM, we could show a great reduction of the human background DNA in the DNA preparations from blood samples, and consequently the sensitivity in PCR detection of Salmonella Typhi in blood samples is dramatically enhanced. Figure From the discussion above it is clear that an optimised and sensitive assay for PCR diagnosis of typhoid would work directly on clinical samples, release intracellular bacteria to improve access to the bacteria DNA, and finally reduce background human DNA to enrich the target bacterial DNA. This can be achieved by using a combination of selective detergents and nucleases. Ox bile can selectively lyze blood cells, releasing the human DNA which is accessible to enzymatic degradation. Due to its bile-resistance, Salmonella serovar Typhi Quailes strain, obtained from the University of Maryland, was used to spike blood samples in this study. The strain was sub-cultured in tryptone soya broth (TSB) or on tryptone soya agar (TSA)  as needed.Wild-type Salmonella Typhi Quailes strain on TSA was inoculated into 2 ml TSB, and cultured in a 37°C incubator with shaking at 200 revolutions per minute (RPM) for about 2 hours. The culture was first adjusted to an optical density at 600nm of 0.25 and then prepared for serial 10-fold dilutions in TSB. An aliquot of 100 μl each of dilutions was plated onto a TSA plate, and the plates were incubated overnight at 37°C to determine the colony forming unit (CFU) of Salmonella Typhi per millilitre. Blood was obtained from healthy individuals with a sterile syringe and immediately pipetted into tubes containing heparin. 100 μl each of the bacterial dilutions was spiked into a 900 μl blood sample and 200 μl spiked blood samples were used for DNA isolation.A colony of overnight culture of The blood samples used in the study were obtained from laboratory volunteers with written informed consent, in accordance with the university policies - Taking Blood Samples from Colleagues or Students for Research and Teaching OHS Policy Document 1/03 approved by the Central University Research Ethics Committee (CUREC), University of Oxford.Blood DNA was isolated from 200 μl heparinised blood sample using QIAamp DNA mini kit  according to the manufacturer’s instruction, except that the DNA was eluted with 50 μl Buffer AE and incubated at 65°C for 5 minutes before centrifugation. The DNA was stored at −20°C until further use. The aliquots of DNA preparation were run on a 1% agarose gel, stained with ethidium bromide and visualized by UV transllumination. The DNA concentration was determined by UV spectrometry.3 gel units of micrococcal nuclease  in 20 μl reaction volume containing 1 μM CaCl2 and 0, 1%, 3%, 5%, 7% or 9% ox bile. Following incubation at room temperature for 10 minutes, the reaction mixture was run on a 1% agarose gel, stained with ethidium bromide, and photographed under UV illuminator.0.5 μg human blood DNA was mixed with 10Salmonella Typhi bacterial DNA from spiked blood samples was shown in Figure A flow diagram of two selective target DNA enrichment methods (STEM1 and STEM2) used in this study for enrichment of 2, and 1 μl (103 gel units) micrococcal nuclease was added. The mixture was incubated at 37°C for 10 minutes and then centrifuged again at 13,000 RPM for 5 minutes. The pellet contained the enriched Salmonella Typhi bacteria and was re-suspended in 200 μl phosphate buffered saline (PBS) for bacterial DNA isolation.In STEM1, 200 μl spiked blood sample was gently mixed with equal volume of 10% ox bile , incubated at room temperature for 10 minutes, and then centrifuged at 13,000 RPM for 5 minutes. The pellet was re-suspended in 100 μl 0.1 μM CaCl2 and 2 μl (2×103 gel units) micrococcal nuclease, incubated at room temperature for 20 minutes, and then centrifuged at 13,000 RPM for 5 minutes to collect the pellet containing enriched bacterial cells. The pellet was re-suspended in 200 μl PBS for bacterial DNA isolation.In STEM2, 200 μl spiked blood was mixed together with 200 μl of 10% ox bile, 4 μl of 0.1 mM CaClFollowing the re-suspension of the enriched bacterial pellet obtained in STEM1 or STEM2, bacterial DNA was isolated using QIAamp DNA mini kit in the same way as described in the section of Conventional DNA preparation above, except that the mixture of 20 μl proteinase K solution and bacterial pellet suspension was incubated for 5 minutes at room temperature to completely remove any residual activity of micrococcal nuclease before Buffer AL was added.Salmonella serovar Typhi Quailes strain were designed according to the fliC-d gene sequence of Salmonella serovar Typhi (Accession number L21912): H-for (ACTCAGGCTTCCCGTAACGC) and Hd-rev (GGCTAGTATTGTCCTTATCGG) [The PCR primers for TTATCGG) , and synThe PCR reaction was carried out in a 50 μl volume, comprising 0.5 U of Taq DNA polymerase , 1 x Qiagen PCR buffer, 1.5 mM magnesium chloride, 200 μM concentrations of each deoxynucleoside triphosphate, 0.5 μM concentrations of the primers H-for and Hd-rev, and 10 μl of DNA template. The following amplification steps were used: 1 cycle of 95°C for 5 min; 35 cycles of 93°C for 30 sec, 55°C for 30 sec, and 72°C for 40 sec; and 1 cycle of 72°C for 5 min. The PCR amplification product was separated by electrophoresis on a 1% agarose gel, stained with ethidium bromide, and photographed by a UV transilluminator.Human blood DNA was incubated with micrococcal nuclease at different concentrations of ox bile. The extent of digestion of human DNA by micrococcal nuclease was compared, as shown in Figure Salmonella Typhi, as shown in Figure DNA prepared by the 2 different STEMs was compared with that isolated through the conventional method from blood samples spiked with fliC-d amplicons from the different DNA preparations. Using the DNA prepared with the conventional method, the fliC-d amplicons were only seen in the sample with 3x105 CFU per millilitre of blood . However, the fliC-d amplicons were seen in all the samples with 3x105, 3x104, 3x103 and 3x102 CFU per millilitre of blood, with highest sensitivity equal to approximately 6 CFU per PCR reaction. Independent experiments consistently found that STEM enhanced PCR sensitivity by 1,000 fold through selective removal of background human DNA in the spiked blood samples.The sensitivity of PCR was compared using DNA prepared by STEM and by conventional methods from the spiked blood samples. Figure Salmonella Typhi in spiked blood specimens. Using this method we were able to show a substantial reduction in dominating human background DNA in clinical samples and consequently increased the sensitivity of PCR detection of Salmonella Typhi from blood samples by 1,000 fold.We describe a novel method (STEM) that enhances detection of Salmonella Typhi PCR-based assays were studied in last decades, none has been brought to clinical use due to the limitations caused by low bactereamic level in typhoid patients. PCR sensitivity is directly related to the actual number of colony forming units found in the blood [et al. demonstrated that the 5 hour broth culture enrichment improved PCR sensitivity by 10 times for spiked blood, and 100 times for spiked stool samples [et al. recently compared these two protocols for DNA preparation, and found that both were able to substantially reduce the human background DNA in most of the cases but loss of bacterial DNA was also a problem [P. gingivalis in periodontal samples was less than 20%. As mentioned above, the MolYsis protocol uses a guanidine hydrochloride chaotropic buffer for lysis of human/animal cells, and bacteria with a thin or labile cell wall (e.g. members of the genus Treponema) or those exposed to cell wall-active antibiotics and/or human immunosystems or bacteria even devoid of a cell wall  are also lyzed. Therefore, the MolYsis protocol may not be suitable for use in the diagnosis of Gram-negative sepsis. They also found, on the other hand, the Pureprove technology was not efficient in removal of human DNA. This is because it is based on selective binding of bacterial DNA to a DNA binding protein that recognizes unmethylated CpG motifs. CpG islands and motifs are not distributed equally over the entire human genome, and fragments without any 5′-methylcytosin are present in every preparation and will mix with prokaryotic DNA. In addition, as cytosine methylation is negatively correlated with gene expression, in regions of active genes the CpG dinucleotides-motifs are also de-methylated and, if fragmentized, could possibly mix with prokaryotic DNA as well. It should be emphasized that in Horz et al. study, the mean absolute bacterial gene copy numbers was 2.23×1010  and 1.37x1010  [Early diagnosis and prompt treatment are essential for optimal management of typhoid patients, and therefore a sensitive and specific PCR assay is useful. Although many he blood . Typicalhe blood ,19. As a samples . We have samples . But lik problem . For exas fluid) . Such a Salmonella is well-known. This could be exploited for developing typhoid diagnostics as bile can selectively lyze human blood cells and release both human DNA and intracellular bacteria without damaging bacterial cells [Bile is a widely used ingredient in culture media for blood culture and the bile resistance of al cells ,25. In tSalmonella Typhi in blood samples. Therefore, this novel method of sample preparation offers a better option for improved typhoid PCR assays directly using clinical specimens in diagnosis of this globally important infection disease which we believe could be of importance in improving clinical care and providing effective evaluation of novel vaccines.In conclusion, we describe herein a novel method, which we have termed “the selective target DNA enrichment method” (STEM). STEM can enrich bacteria from large volume samples, remove background human DNA, and enhance the sensitivity of PCR detection for LZ and AJP are authors on patents in the field of typhoid diagnostics.LZ and AJP conceived of the study and carried out its design. LZ performed the assays and drafted the manuscript. AJP edited the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/164/prepub


In many reptiles, the thermal regimes experienced by eggs in natural nests vary as a function of ambient weather and location, and this variation has important impacts on patterns of embryonic development. Recent advances in non-invasive measurement of embryonic heart rates allow us to answer a long-standing puzzle in reptilian developmental biology: Do the metabolic and developmental rates of embryos acclimate to local incubation regimes, as occurs for metabolic acclimation by post-hatching reptiles? Based on a strong correlation between embryonic heart rate and oxygen consumption, we used heart rates as a measure of metabolic rate. We demonstrate acclimation of heart rates relative to temperature in embryos of one turtle, one snake and one lizard species that oviposit in relatively deep nests, but found no acclimation in another lizard species that uses shallow  nests. Embryonic thermal acclimation thus is widespread, but not ubiquitous, within reptiles. Most organisms inhabit thermally variable environments, and hence must cope with large temperature fluctuations over diel and seasonal cycles, coupled with marked spatial variation . Additionally, most organisms have little physiological control over their own body temperatures, so that much of this ambient thermal variation generates corresponding variation in body temperatures. The thermal dependence in rates of many fitness-relevant activities  has imposed selection for the ability to flexibly modify physiological responses to temperature, in the frequent situation where an organism cannot control its temperature by behavioural means Thermal acclimation of physiological traits has been extensively reported in the post-embryonic stages of animals embryos of oviparous vertebrates also. These embryos cannot thermoregulate behaviourally, and they develop in nest-sites that can experience highly variable temperatures during incubation We might expect to see acclimation in the Recent technical advances provide such a measure. We can monitor heart rates of embryonic reptiles non-invasively, by using infra-red radiation to detect subtle movements (induced by embryonic heart beat) on the shell surface Chelydra serpentina) is a large freshwater turtle occurring throughout much of North America Sceloporus undulatus is a medium size iguanid lizard (to 80 mm snout–vent length [SVL]) from the eastern United States; we obtained laboratory-laid eggs from gravid females (n = 16) collected from Teasdale, Mississippi  in May 2009. The eastern three-lined skink Bassiana duperreyi is a montane species (to 80 mm SVL) from southeastern Australia. In December 2008, we collected 71 freshly-laid eggs  from field nests in the Brindabella Range  oviparous colubrid snake widespread in Asia The common snapping turtle . The eggs were transferred to an incubator set at 20, 25, or 30°C for two hours prior to being placed individually on a Buddy infrared heart rate monitor placed inside the incubator to record heart rates , and lizard eggs were individually incubated in 64 ml glass jars filled with moist vermiculite (-200 KPa), snake and turtle eggs were incubated at plastic boxes . The jars and plastic boxes were then randomly assigned to different temperature treatments (i.e. acclimation temperature) within the range of 20–30°C  of 15 eggs were measured using closed system respirometry, with the eggs inside glass jars fitted with rubber stoppers and three-way stopcocks as metabolic chambers 2 consumption), such that the ΔPO2 in the chamber before and after the experiment was maintained at <1%  to avoid the potential stress of oxygen suppression on the embryos. A gas sample was taken using a sealed syringe, and introduced into an Oxzilla II differential O2 analyzer  after being drawn through Carbosorb, to absorb CO2 and then through Drierite to absorb H2O. The O2 analyzer was calibrated between each measurement using room air. Output from the analyzers was recorded using ExpeData software, and rates of O2 consumption were calculated using the equations of Vleck 2 consumption were determined. Mean values of O2 consumption rates and heart rates at each temperature were used to analyze the relationship between embryonic heart rate and oxygen consumption.Rates of OGuide for the Principles of Animal Care.All experimental procedures were approved by the Animal Care and Ethics Committee at the University of Sydney (L04/7-2007/3/4665) and Iowa State University (3-09-6695-J), and were conducted in accordance with the NIH P<0.05.We used linear regression to quantify relationships between temperature and heart rate or oxygen consumption, as well as between embryonic heart rate and oxygen consumption. Repeated measures ANOVA was used to test for the effect of incubation temperatures and test temperatures on the heart rates of embryos. Means are presented ± one standard error and significance was assumed if 2 consumption (V̇O2) increased with temperature over the range of 20–30°C (Both embryonic heart rate (HR) and rate of OP<0.0001). EmbryonP<0.0001).C. serpentina, S. undulatus, B. duperreyi, and E. taeniura, respectively. In all four species, heart rate increased dramatically with increases in test temperature over the range of 20 to 30°C (10) for C. serpentina, S. undulatus, B. duperreyi, and E. taeniura of 2.0, 2.4, 2.4 and 2.3 respectively.The mean egg mass was 12.70, 0.41, 0.46 and 20.36 g for  to 30°C . The theB. duperreyi (10) of temperature compensation due to thermal acclimation at test temperature of 25°C were 1.3, 1.3, and 1.2 for C. serpentina, S. undulatus, and E. taeniura (between 22 and 30°C), respectively.Three of the four species also provided clear evidence of thermal acclimation of heart rates. Statistical analysis indicated that incubation temperature affected embryonic heart rate in all species except for uperreyi . In the uperreyi . The coeBassiana duperreyi within the temperature range of 20–30°C, suggesting that heart rate can serve as a valid index of metabolic rate. Unlike oxygen consumption rate , available data suggest that heart rate is relatively constant throughout most of embryonic development in reptiles when the eggs are incubated at a constant temperature Lampropholis delicata), turtles (Chrysemys picta and Graptemys kohnii) and snakes (Elaphe taeniura). Heart rate thus appears to offer a stable and robust index of developmental rate in reptiles, at least for studies of thermal effects on rates of embryonic development.An important assumption of our approach is that heart rate provides a valid index of developmental rate. Consistent with this assumption, studies on birds have indicated that fast-developing  species have a higher daily heart rate than do precocial species whose embryos develop more slowly Chelydra serpentina and the crocodile Crocodylus johnstoni found no significant differences in metabolic rate (or heart rate) between embryos incubated at high versus low temperatures, suggesting metabolic compensation at the low temperature Sceloporus undulatus generated contrasting results, with an initial finding of metabolic acclimation 10 of their heart rates (1.2–1.3) was similar to that of heart rates in adult fish Reflecting logistical difficulties, few previous studies have examined thermal acclimation in reptilian embryos. Studies on the turtle The acclimation effect results in partial compensation of metabolic and developmental rates in response to low temperatures; that is, cold conditions do not slow embryogenesis as much as they would have done in the absence of acclimation. This physiological mechanism is likely to increase egg survival and hatchling fitness, because shorter incubation periods reduce risk that an embryo will be exposed to hazards such as extreme temperatures and predation B. duperreyi) exhibit no thermal acclimation of heart rates? One plausible answer may involve the magnitude of diel fluctuations in nest temperature experienced by this species, compared to the others (B. duperreyi extends to the upper elevational limit for oviparous lizards in Australia, with correspondingly low mean soil temperatures B. duperreyi nests may preclude acclimation.Why did one of the four species that we studied (e others . SnappinS. undulatus clearly show thermal acclimation of heart rates, whereas previous studies on the same wide-ranging species have suggested that this species either does Our results for the fence lizard In summary, our data demonstrate that acclimation of heart rate (and thus, by strong inference, developmental rate and metabolic rate


Atotal of 1200 Tanzanian short horn Zebu (TSHZ) from two farms in twovillages were qualitatively assessed for tick infestations and 40 animals(grouped in 10s from each farm) were separated in their herds andquantitatively examined to establish the tick load. The animals were groupedin treatment regime groups , TxRG 1 being the controlgroup was treated with water. Ecotix® was applied on day 0for TxRG 2, days 0 and 7 for TxRG 3 and days 0, 7 and 14 for TxRG 4 and tickload was monitored for 28 days.This study aimed at determining the extent of tick infestations inextensively grazed cattle and assess the efficacy of EcotixRhipicephalus appendiculatus (55%), Amblyommavariegatum (18%), R. (Boophilus) microplus (12.9%), R.(B) decoloratus (7.1%), R. evertsi evertsi (4.4%) andR. composites (2.6%). The overall mean (mean ± SEM) tickdensity on day zero was 63 ± 30 ticks per animal (ranging from20–160). The mean tick density on day zero was 44.6 ± 25.4, 74.6± 30.3, 55.0 ± 26.2 and 77 ± 33.5 for groups one, two, threeand four respectively. Post-treatment quantitative assessment of tick burdenrevealed that the TxRG 1 animals maintained a tick load throughout the studyperiod. A significant decrease in tick load in animals in groups TxRG 2, 3and 4 (P=0.0001) with increasing frequency of Ecotix®application was recorded. There was however no significant difference intick reduction between TxRG 3 and 4 (P=0.0986). Thus TxRG 3 wouldbe sufficient for the monthly tick treatment and with this regime, thefarmer would save up to 2.5 USD per animal during six months of dryseason.All the animals examined were infested with ticks. The identified ticks were® as pour-on preparation using a two applicationregime per month, the tick control strategy was effective.The study revealed a high tick infestation among the TSHZ kept in extensivegrazing systems in Mvomero district and that when treated withEcotix Rhipicephalus and Amblyomma which are the mostimportant in TBD transmission are distributed in almost all cattle keeping areas inTanzania [Tick infestation, tick-borne diseases (TBDs) and tsetse-transmitted trypanososmosisconstitute are the major problems in livestock production in the Sub-Saharan Africa,2. TBD,Tanzania ,11.FurtTanzania . NotablyTanzania , while tTanzania . TherefoTanzania .Bos taurus dairy cattle”. It is recommended by the“Report of the FAO expert consultation on revision of strategies for thecontrol of ticks and TBDs ” that the bestmethod to control or limit the impact of ticks and TBDs is the enzootic stabilitywhich can be achieved by different means. However, the approach to ticks and TBDscontrol by enzootic stability sounds good but may not apply in Tanzania because theprevalence of ticks and TBDs and their associated losses are still very high[According to Pegram  the inteery high-20. In1The use of acaricides for tick control in Tanzania has a long history. Since theconstruction of the first dip tank at Mpwapwa in 1905, 2041 dip tanks and 91 sprayraces had been constructed all around the country  but most® (2.5% high cis cypermethrin) pour-on preparation andcost implications for control of ticks in Mvomero district in Tanzania.On other hand, synthetic pyrethroids like cypermethrin, deltamethrin and cyhalothrinhave been reported to be more effective against ticks in the tropics ,26,27. TThe study was conducted at Mkula and Melela villages located in the arid areas insouthern part of Mvomero district. The two villages were conveniently selectedbecause most of the inhabitants were Maasai pastoralists who use the extensivegrazing systems. The villages were easily accessible and had large herds ofcattle that had not been dipped for the past ten years. Dipping was not donebecause of the following; (i) breakdown of the dip tanks, (ii) water scarcityand (iii) high costs of tick control due to cessation by the TanzanianGovernment to provide free acaricides to farmers. The important economic lossesthough not quantified which were incurred by farmers due to tick infestationsand TBDs included high morbidity and mortalities, reduced production andreproduction performance in cattle .Mvomero district is located between 8–10º S and 28–37º E inthe Morogoro region, Tanzania. The district varies greatly in topography andclimate. Highlands and lowland rainforest are located in the northwest andcentral-north belt respectively, while the drier woodlands are located in thesouth. Rainfall distribution in the district is bimodal, with a long wet seasonfrom March to May and a short wet season from October to December. The dryseasons in Mvomero district are experienced for six months . The northern area has a humid to sub-humidclimate, and annual rainfall ranges from 1500 to 2000 mm while the southern partof the district is much drier, with annual rainfall of up to 600 mm. Averageannual temperatures in Mvomero district ranges from 20–30 °C. Thestudy was conducted for four weeks during dry season (July to August) of 2009.During periods of dry seasons, the tick infestations associated with TBDs inMvomero district are relatively higher as compared to rainy season .The study animals were the Tanzanian short horn zebu (TSHZ), which were managedunder extensive grazing system. Four herds located at a distance of about10–15 km apart, were conveniently selected from the study villages (twoherds in each village). The selection criteria were based on the owners’willingness to participate in the study. The selected herds had an average of300 cattle per herd. All the adult animals in the herds were qualitativelyexamined for the tick infestation. Each animal was examined for presence ofticks on the head, ears, neck, belly, back, legs, perineum and tail. Preliminaryidentification of ticks (based on body morphology and colour) was done while theticks were still attached on the animal body. Presence of one or more ticks onan animal was recorded as a positive case.® treatment trials. The animals were selected basedon the following criteria: (i) had many visible ticks on the skin (positivecases of tick infestation), (ii) the age was one year and above, (iii) had aweight range of 100 to 200 kg and, (iv) there was no history of application ofacaricide. Animals with the stated criteria were ear tagged and separated fromthe rest in each study herd except the control group (TxRG 1). A total of 40animals were selected and allocated in treatment regimes groups (TxRG 1–4)according to their herds of origin. The 10 animals in each of the treated groups were separated from the rest and were being grazed separatelyduring the day time but kraaled with rest of the animals in the herd during thenight. The experimental animals in all the four groups were being grazed interrain of pasture of similar characteristics since the interval from one studyvillage (Melela) to the other (Mkula) was almost 15 – 20 km which did notgive any significant differences in climate and general weather conditions.Animals in TxRG 1 and 4 were from the herds in Mkula village while those ingroup 2 and 3 were from Melela village. All the selected animals were female(cows) since cows constituted more than 95% of the animals in the studyherds.After the animals were examined for presence of ticks, 10 adult cattle from eachherd were selected for quantitative tick assessment and subsequently were usedin the EcotixFor quantitative tick assessment, individual animals in each group wererestrained, cast down and examined for ticks. Each animal was examined bythorough checking and where required combing, all the body regions beginningfrom the head and ears, followed by the neck, belly, udder, back, limbs,perineum and tail. Quantitative examination of each animal involved tickcounting on the whole body ,31. With® pour-on preparation treatment trials. Ecotix® contains 2.5% of high-cis cypermethrinactive ingredient. The treatment trial was conducted as described by Sajid etal. [® according tomanufacturer instructions while animals in TxRG 1 were treated with water(served as a negative control). Briefly, Ecotix® was appliedalong the spinal from the head to the tail at the recommended dose (1 mL per 10kg body weight). Day 0 was the day that the cattle were first examined for tickinfestation and Ecotix® was applied to the animals in groupsTxRG 2, 3 and 4. The acaricide was applied on day 0 for TxRG 2, days 0 and 7 forgroup 3 and days 0, 7 and 14 for TxRG 4. Tick load in the control group wasmonitored along-side with the treatment TxRG. Tick load on animals wasquantitatively monitored through physical counting on the whole body[The selected 40 animals (TxRG 1–4) were involved in theEcotixd etal.  with somole body,31.TickThe prevalence of ticks was determined using the following formula :Population at risk during that specified time period® spreadsheet. Aftervalidation the data were then transferred to Stata  for statistical analysis. A mean tick count inbox graph plot was used for graphical analysis. Because of differences in thenumber of ticks that were observed on animals on day 0 in different groups,before plotting, all means for tick counts were normalized by computingpercentage of number of ticks counted relative to tick counts on day 0 in eachgroup. A robust Poisson regression model was used to show the effect of thedifferent treatment regimes on the total tick count.The data was entered in Microsoft ExcelThe permission to carry out this study was granted by the Morogoro municipallivestock officer and ethical approval for the study was given by the EthicalCommittees of Sokoine University of Agriculture . Verbalconsent was obtained from each of the participating heads of households afterexplaining the purpose and importance of the study prior to commencement of theexperiments.Rhipicephalus appendiculatus. Other identifiedtick species included A. variegatum, R. (B) microplus , R. (B) decoloratus , R. evertsi evertsi and R.compositus. The overall mean (mean ± SE) tick density was 63± 30 ticks per animal with a count range between 20 and 160. The mean tickcount on day 0 between the animal TxRG was as follows: 45 ± 25, 75 ±30, 55 ± 26 and 77 ± 34 for groups one, two, three and fourrespectively. All the examined animals n=1200) from the 4 study herds were infested withdifferent species of ticks. However, not all the tick species identified, werepresent in all the animals. The tick species identified on the animals are shownin Table 200 from ®pour-on preparation on day 0 in TxRG 2, tick count decreased from 100% to 5%, inTxRG 3, the tick counts decreased from 100% to 4% while in TxRG 4, the tickcounts decreased from 100% to 10% by day 28  maintained a tick infestation throughout the studyperiod with a slight increase recorded on day 10 and 14 but later normalizedcompared to the single treatment regime group (TxRG2)  and triple (TxRG 4)Ecotix® treatments were compared (double vs triple treatmentP=0.0986). However, a significant difference (P=0.0002) was observed when singlevs double treatment and single when compared to triple treatment (P=0.0001).Since all the three test regimes; single treatment (TxRG 2), double treatment(TxRG 3) and triple treatment (TxRG 4) received the same initial concentrationof Ecotix) Figure . The mea® pour-on to cattle per month. Basedon the quantitative treatment trials, a simple economic evaluation which appliesfor these geographical settings was computed extrapolating the analysis to allsix months of dry season which has a relatively high tick challenge is describedbelow: Doses of 1 mL of Ecotix® pour-on applications per 10 kgbody weight are required. The average weight of the TSHZ that met our selectioncriterion for study inclusion weighed 150 kg, so 45 mL would be needed in amonth making a total of 270 mL per animal during the 6 months of dry season. Thecurrent selling price of 250 mL bottle of Ecotix® in Tanzanianshillings (Tshs) is 10, 000=  so each mL costs 40shillings and the cost per animal during the six months of heavy tickinfestation would be 10,800= (7.5 USD). From our findings, if TxRG 3 is used,only 180 mL of Ecotix® would be used per animal per six monthscosting 7, 200 shillings. In economic terms, the farmer saves Tshs 3, 600/= (2.5USD) per animal per six months by adopting double Ecotix®application per month . Note that thiseconomic analysis applies only in areas like southern parts of Tanzaniaincluding Mvomero district which have similar climatic conditions and tickchallenges especially during the dry season.During the high tick challenges, the manufacturer recommends a regime of threetimes applications of EcotixR.appendiculatus, a well-known vector for Theileria parva whichcauses East Cost Fever in cattle. R. appendiculatus inhabits a wide rangeof East African environments [R. appendiculatus in alocality. Almost all the above listed factors are favourable in Morogoro and thismay account for R. appendiculatus predominance. Predominance of R.appendiculatus may also be due to the fact that cattle are widely believedto be the primary host for this tick [A. variegatum(vector for Cowdria ruminantium), R.(B) microplus (vector forBabesia spp and Anaplasma spp) R. (B) decoloratus(vector for Babesia spp.) and R. evertsi evertsi (vector forAnaplasma spp.) occurred at a relatively low numbers suggesting thatthey are less abundant under field conditions. Our findings on the general spectrumof ticks were similar to those of other authors in Tanzania [This study demonstrates that different species of single and multi-host tick specieswere prevalent in the TSHZ cattle which were managed under extensive system. Thestudy shows five different species of ticks, which were predominated by ronments  and hasronments ,21. The ronments reportedhis tick ,38. The Tanzania ,11,25,38R. (B) decoloratus, A. variegatum and R.appendiculatus, in the absence of tsetse or any other barrier to cattledistribution, R. (B) decoloratus and A. variegatum tend to becentered on the same foci as R. appendiculatus[Furthermore, studies reports that there is a co-existence between the geographicdistribution of diculatus,11,25. Tdiculatus.R. appendiculatus, the vector of ECF was the main tick in theexamined herds, this could prevent the setting up of enzootic stability. Indeed, astudy by Silayo et al. [Lack of tick control measures on animals grazed in infested areas and the poor animalhusbandry practices may be other factors for the observed high tick loads. Moreover,the limited scope of awareness regarding the impact of ticks, non existence ofacaricide uses and lack of adequate veterinary infrastructure for access by pastoralsociety in the area may also account for the widespread existence of tick species inthe area. This high prevalence of ticks predisposes the animals to TBDs which wouldultimately lead to losses due to reduced productivity and increased animalmortality. Indeed, a study by Swai et al.  in Maasao et al.  reportedo et al. ,42. In ao et al. -20,43.Therefore, the results of present study warrant for immediate tick control measures.The frequent use of efficacious acaricides as a control measure may avert majorcattle losses through deaths and loss of productivity which were reported by thefarmers during the study period. Also, there should be an effort to educate thefamers about the significance of the tick infestations and the best approach tocontrol measures.® as a pour-on preparation showedthat it has high acaricidal effects as it had cleared off almost 95% of the widerange of hard multi-host ticks in cattle in the period of 10 days post-treatment.The second treatment (Figure ® to cattle at two weeks interval gives a morebetter results in controlling ticks as it showed a significantly low tick loadscompared to single treatment. However, the spontaneous decrease in tick countobserved in TxRG 2 which had received single Ecotix® could notwarrant recommending a single treatment and this needs further investigations. Theobserved slight decrease of tick load in TxRG 4 post third Ecotix®treatment and thereafter a spontaneous increase of tick count as from day 17 to 10%by day 28 could be not be accounted for but may be caused by extent of tickchallenge and management factors. Generally, these results are also in agreementwith work done by Msolla [R. sanguineus eggs from hatchingwhile it had 100% larval, nymphal and adult tick mortality. We therefore recommendthe use of Ecotix® pour-on preparation in the routine control ofticks and TBDs in Tanzania.According to the Tanzania Ministry of Agriculture , an acary Msolla  who founy Msolla  reported® a pour-on preparation wasable to control ticks by double treatment per month different from the threetreatment recommended by the manufacturer. For economic reasons, double treatment iscost effective for control of ticks. The high-cis cypermethrin are reported to havea persistent effect and are generally considered to be an improvement over otherinsecticides relative to their environmental impact when applied at insecticidaldoses [Furthermore, the study found that Ecotixaldoses .® a pour-onpreparation, the farmer can save up to 2.5 USD per animal per each six months of dryseason when the extent of tick infestation is relatively high. Based on the commonaverage herd size of up to 5,000 owned by a pastoral household in Tanzania, thefarmer may save up to 12,500 USD in six months of dry season.Economically, with double treatment regime of Ecotix® treatment regime, the farmer is able to save 2.5 USD peranimal during the six months of dry season.From the findings of this study, it is concluded that cattle in Mkula and Melelavillages in Mvomero districts were infested with different species of ticks atvarying intensities. A 2.5% high-cis cypermethrin pour-on showed high efficacyagainst tick infestations in TSHZ cattle managed under extensive system in thisarea. Furthermore, 2.5% high-cis cypermethrin preparation can be applied to cattlegrazed under extensive management system twice per month at two weeks intervalrather than thrice per month as recommended by the manufacturer. With thisEcotix® pour-on preparation.This study was funded by Farm base Ltd of Dar es Salaam, Tanzania which is themanufacturer of EcotixHEN: Developed the concept, wrote the protocol, collected the data during field work,drafted the manuscript and final proof reading of the manuscript before wassubmitted to the journal. AM: Statistical analysis of all the data, interpretationand description of analysed data, read and corrected the manuscript on technical andlanguage part. Edited the manuscript before was submitted to the journal.RHM:Planned and set the experiment, facilitated in design and coordination of theexperiments during field work, advised on the manuscript formatting, technicaladvice on different kinds of acaricides and their use in Tanzania. Edited themanuscript before was submitted to the journal. All authors read and approved thefinal manuscript.


Diabetic foot ulcers are serious complications for diabetic patients, yet the precise mechanism that underlines the treatment of these diabetic complications remains unclear. We hypothesized that dietary antioxidant supplementation with vitamin C, combined either with vitamin E or with vitamin E and NAC, improves delayed wound healing through modulation of blood glucose levels, oxidative stress, and inflammatory response.Diabetes was induced by administration of alloxan monohydrate. Mice were divided into 4 groups; CON (non-diabetic control mice fed AIN 93 G purified rodent diet), DM (diabetic mice fed AIN 93 G purified rodent diet), VCE (diabetic mice fed 0.5% vitamin C and 0.5% vitamin E supplemented diet), and Comb . After 10 days of dietary antioxidant supplementation, cutaneous full-thickness excisional wounds were performed, and the rate of wound closure was examined. TBARS as lipid peroxidation products and vitamin E levels were measured in the liver. Expression levels of oxidative stress and inflammatory response related proteins were measured in the cutaneous wound site.Dietary antioxidant supplementation improved blood glucose levels and wound closure rate and increased liver vitamin E, but not liver TBARS levels in the diabetic mice as compared to those of the CON. In addition, dietary antioxidant supplementation modulated the expression levels of pIκBα, HO-1, CuZnSOD, iNOS and COX-2 proteins in the diabetic mice.These findings demonstrated that delayed wound healing is associated with an inflammatory response induced by hyperglycaemia, and suggests that dietary antioxidant supplementation may have beneficial effects on wound healing through selective modulation of blood glucose levels, oxidative stress, and inflammatory response. Diabetes mellitus (DM) is a disease in which injury of peripheral tissue is induced by oxidative stress caused by chronic hyperglycaemia. The number of DM patients is now approximately 250 million people and is expected to reach 400 million by 2025 . It is kDuring the inflammatory stage, the first stage in the wound healing process, neutrophils and macrophages infiltrate the wound site and phagocytose infectious agents and fragments of tissue degradation release proteases and various reactive oxygen species (ROS) into the wound environment . They alDiabetic patients have impaired wound healing, which is considered as chronic and delayed healing if it continues beyond 8 weeks . They arFemale ICR mice (5.5 weeks old) were purchased from the Central Lab. . The mice were individually housed in polycarbonate shoebox type cages with wire tops in a room maintained at 22 ± 1°C and 50 ± 1% humidity on a 12-h light/dark cycle, with free access to water and chow diet for 1 week. Diabetes was induced by alloxan monohydrate  in 0.9% NaCl. Non-diabetic control mice were injected with saline. After 5 days, the induction of diabetes was confirmed by measuring fasting blood glucose levels and monitoring for 10 days. Mice with a fasting blood glucose level ≥ 250 mg/dl were used for this study. Glucose levels were measured by a one-touch blood glucose meter  from the tail vein at the same time to minimize the effect of diurnal fluctuation. All mice were used in accordance with animal protocols approved by the Kyung Hee University Institutional Animal Care and Use Committee.Mice were divided into 4 groups that were fed different antioxidant supplementation; i) Group 1 (CON): non-diabetic control mice were fed AIN 93 G Rodent purified diet; ii) Group 2 (DM): diabetic mice were fed AIN 93 G Rodent purified diet; iii) Group 3 (VCE): diabetic mice were fed 0.5% vitamin C  and 0.5% vitamin E ((+/-) α-tocopherol, synthetic, > = 96%; Sigma-Aldrich); and iv) Group 4 (Comb): diabetic mice were fed 0.5% vitamin C, 0.5% vitamin E, and 2.5% NAC  for 10 days.Blood, wounds, and livers at 0 h , 24 h, 48 h, and 72 h, after wounding, were collected. Blood samples were taken using a 1 cc syringe with heparin and centrifuged at 3000 rpm for 10 min at 4°C. All samples were kept at -80°C until processed.The liver thiobarbituric acid reacting substances (TBARS) level was measured as an index of lipid peroxidation . BrieflyThe level of vitamin E in the liver was measured using a high-performance liquid chromatography (HPLC) system as previously described . The mouFull thickness excisional wounds were made on the back of the mouse. The mice were, briefly, anesthetized with isoflurane  and the backs of all mice were shaved using a hair clipper and then sterilized by 70% (v/v) ethanol. The wound biopsy model used in this experiment has been previously described . The fulWounds in each individual mouse were photographed digitally every day, beginning on the day of wounding (d 0) with a standard dot equivalent to the initial wound area placed beside the wound. The quantification of wound closure used in this experiment was previously described . Wound cSkin was homogenized in a lysis buffer  with a protease inhibitor  and centrifuged at 16,000 rpm for 30 minutes at 4°C. The sample (60 μg protein) was separated on 10% SDS PAGE gels and then transferred to the PVDF membrane. After blocking for 1 hour in 5% skim milk, the membranes were incubated with specific monoclonal and polyclonal antibodies against pIκBα , HO-1 , CuZnSOD , iNOS , COX-2  and β-actin  overnight at 4°C, and then incubated with a secondary antibody for 1 hour at room temperature. The blots were detected by enhanced chemiluminescence and measured by the Image J program. β-actin was used for normalization of the target protein expression in each sample.P < .05.All values are expressed as means ± SEM. Data were analyzed by 1-way ANOVA using SPSS (version 12) statistical analysis program, and then differences among means were analyzed using Duncan's test. The relationships between relative wound size and blood glucose levels were evaluated by Pearson's correlation coefficients. For all tests, differences were considered significant at The body weights of all diabetic mice were similar to those of the CON group regardless of antioxidant supplementation (data not shown).Blood glucose levels of all diabetic mice were significantly increased after alloxan injection. Although blood glucose levels of the VCE and the Comb groups did not go back to the steady levels, there was a significant reduction as compared to those of the DM group Figure .Liver TBARS levels of the DM group were much higher than the CON group but they were not significantly reduced in the VCE and the Comb groups Figure .Vitamin E levels of the DM group were not significantly different from those of the CON group. However, the levels of the vitamin E in the VCE and the Comb groups, supplemented with dietary antioxidants, including vitamin E, were significantly increased 13 folds) as compared to those of the DM group  on days 6-8 after wounding  in each diet treatment group was positively correlated to the blood glucose levels on day 5 (starting day of antioxidant supplementation) up to day 24 (day 8 after wounding) after alloxan injection Table .The protein levels of pIκBα showed a similar result to those of COX-2 at 0 h. However, there were no significant differences among all groups at 24 h, 48 h and 72 h Figure . The expAs shown in Figure The inflammatory stage is the most important in the wound healing process because the proper healing process requires recruitment of inflammatory cells and secretion of various mediators. However, an increase of oxidative stress, caused by wound occurrence with continuous hyperglycaemia in diabetes, prolongs inflammatory response and causes an ulcer by delaying a shift to the next steps. Therefore, controlling oxidative stress and inflammatory response through appropriate regulation of blood glucose levels is essential to accelerating the wound closure rate. We hypothesized that the regulation of blood glucose levels through dietary antioxidant supplementation reduces oxidative stress in the inflammatory stage and improves the early wound closure rate during cutaneous wound healing.In previous studies, dietary antioxidant supplementation itself did not affect blood glucose levels , but thiet al. [et al. [Oxidative stress is increased by enhancing the rate of ROS production and declining antioxidant defense in diabetes. Therefore, it makes it easier to cause oxidative damage in various tissues in diabetes. In this study, we confirmed that liver TBARS levels, the end product of lipid peroxidation, were increased in diabetes, but they were not significantly decreased by dietary antioxidant supplementation. This result stands in contrast with earlier works by Kim et al.  and Odet [et al. , showing [et al. . The resThe present study showed correlations between wound size and blood glucose levels after antioxidant supplementation. Blood glucose levels from the start day of diet treatment during the inflammatory stage affected relative wound size on days 6 to 8 after wounding. We suggested that it is possible to accelerate the wound closure rate by regulating blood glucose levels through dietary antioxidant supplementation, which attributed to lower oxidative stress, confirmed by decreased TBARS, HO-1 and CuZnSOD levels during the early inflammatory stage.To examine the change of the inflammatory response by dietary antioxidant supplementation at molecular levels that modify the rate of the wound closure, we investigated the expressions of oxidative stress and inflammatory response related proteins. NFκB is composed of a family of inducible transcription factors that serve as essential regulators of the host immune and inflammatory response . NFκB prPrevious studies showed that SOD levels were decreased in diabetes by a deteriorated antioxidant defense system . On the The levels of iNOS, one of the most important factors playing a key role in the wound healing process, increased in the early inflammatory stage after wounding and subsequently declined until the wound was healed. In this study, iNOS protein levels significantly increased at a basal level and decreased at 24 h after wounding, but again rose at 48 h and 72 h in the DM group. The results suggest that reduced iNOS protein levels at 24 h in the DM group were up-regulated due to increased oxidative stress caused by hyperglycaemia. Furthermore, increased iNOS protein levels, maintained until 72 h in the DM group, may decrease collagen synthesis, which plays an essential role in the proliferation and the remodeling stages next to the inflammatory stage, thus delaying the entire wound healing process. The expression levels of iNOS in the Comb group decreased gradually from 48 h, which paralleled the accelerated rate of wound closure on day 2. In particular, a significant decrease of the iNOS levels in the VCE and the Comb groups at 72 h, as compared to those of the DM group, influenced the improved wound closure rate, which showed a similar rate as the controls on day 3. Oxidative stress due to hyperglycaemia caused by diabetes increases secretion of various cytokines, which induce COX-2, a modulator of the inflammatory response in the biosynthesis of prostaglandins. Over-expression of the COX-2 protein decreases collagen synthesis and retards proliferation, thus delaying the entire cutaneous wound healing . We founTaken together, the results in this study suggest that dietary antioxidant supplementation creates synergetic effects that improve the condition of diabetes through the regulation of blood glucose levels and effects that accelerate the early inflammatory responses during cutaneous wound healing. Reduced iNOS and COX-2 expression levels may prevent prolonged inflammatory response by decreasing oxidative stress in dietary antioxidant supplementation, a fact that is confirmed by oxidative stress associated markers, namely, HO-1 and CuZnSOD.The current study demonstrated that the regulation of selective inflammatory mediators by dietary antioxidant cocktail supplementation selectively normalizes the early wound healing process in diabetes. Based on our results, antioxidant cocktail supplementation, including vit C, E and NAC, could be effectively used for diabetic wound healing therapy. However, our research was limited to animal studies; thus, it is important to determine the precise mechanisms in delayed wound healing for application to a human study. Furthermore, it is necessary to conduct a thorough investigation of each stage of the very intricate wound healing process that occurs in diabetes. Finally, further research should be conducted to verify a safe concentration of each antioxidant for clinical usage in diabetic patients.The authors declare that they have no competing interests.The author's responsibilities were as follows: NYP. performed the experiments and analysis and participated to data interpretation and manuscript writing. YL. contributed to study design, data interpretation and manuscript writing. All authors read and approved the final manuscript.


Particle lithography offers generic capabilities for the high-throughput fabrication of nanopatterns from organosilane self-assembled monolayers, which offers the opportunity to study surface-based chemical reactions at the molecular level. Nanopatterns of octadecyltrichlorosilane (OTS) were prepared on surfaces of Si(111) using designed protocols of particle lithography combined with either vapor deposition, immersion, or contact printing. Changing the physical approaches for applying molecules to masked surfaces produced OTS nanostructures with different shapes and heights. Ring nanostructures, nanodots and uncovered pores of OTS were prepared using three protocols, with OTS surface coverage ranging from 10% to 85%. Thickness measurements from AFM cursor profiles were used to evaluate the orientation and density of the OTS nanostructures. Differences in the thickness and morphology of the OTS nanostructures are disclosed based on atomic force microscopy (AFM) images. Images of OTS nanostructures prepared on Si(111) that were generated by the different approaches provide insight into the self-assembly mechanism of OTS, and particularly into the role of water and solvents in hydrolysis and silanation. Self-assembled monolayers (SAMs) of organosilanes have become important as surface resists and functional coatings for micro- and nanopatterning applications –9. The s9 nanostructures per square centimeter. Different approaches with particle lithography have been successful for producing periodic, 2D arrays of nanostructures of different materials and molecular films, including metals [For methods of particle lithography, a surface mask of polystyrene latex or silica mesospheres is used to direct the deposition of organic thin films and nanomaterials. The surface density of nanostructures can be designed by selecting the diameter of mesospheres, for high-throughput patterning on the order of 10g metals –15, nanog metals –19, protg metals –22, polyg metals –26 and Sg metals –31. A sig metals , mica [3g metals –35, quarg metals –37, indig metals , or silig metals ,32,39–42g metals –44. ThisThe morphology of SAMs or nanostructures of OTS reflects a balance of the interactions that occur between the silane precursor and the silanol groups, interactions between the end groups, interactions between the alkyl chains of the silane molecules, and the nature of the substrates –46. Thes11Molecular-level differences in the thickness and morphology of OTS nanostructures prepared by different lithography procedures can be investigated by performing atomic force microscopy (AFM) studies –53. PartA comparison of the geometries and thicknesses of the nanostructures produced by particle lithography was used to systematically investigate parameters for surface self-assembly of octadecyltrichlorosilane (OTS). Three methods of particle lithography for preparing organosilane nanostructures are compared, as shown in 2) in 2 frame of 2 close-up view, 29 patterns are packed closely together , as shown in the contact-mode AFM images in together . This sc11, as shWhen the latex masks were dried, a water meniscus persisted at the base of each latex sphere on the surface, and this defined the reaction sites for hydrolysis and condensation of the organosilanes . For the2 images of 9 nanostructures/cm2. This value is comparable to the pattern density for To produce monolayer nanostructures of OTS, particle lithography with contact printing and immersion were evaluated to optimize the deposition conditions for achieving a densely packed SAM. Images of a nanostructured film of OTS prepared by using particle lithography combined with contact printing are shown in Multiple replicate samples were prepared using contact printing, for different size masks, showing that the heights were consistent with the example of 9 nanostructures/cm2, showing ~120 nanopatterns within the 2.5 × 2.5 µm2 frame shown in A completely different morphology other than rings or nanopores was observed for OTS nanostructures produced by the immersion of particle masks. Dot-shaped nanostructures were produced by using latex-particle lithography with immersion, as shown in Immersion of a masked substrate in a solvent is the most common approach for preparing films of OTS, and has produced the most consistent thickness of a monolayer. However, immersion in solvents causes rapid detachment of the latex masks. To enable an immersion process for particle lithography, a brief heating step was developed to solder the latex beads to the substrate (75 °C for 30 min). Latex deforms when heated, leaving less surface area available for OTS deposition . After t2) and high-magnification images (2).The topography frames reveal periodic patterns within a monolayer film of OTS, with exquisitely small holes at the locations where silica mesospheres (250 nm diameter) were displaced. There are 38 nanopores in the zoom-in views of 9 patterns/cm2. The depth of the OTS film was measured to be 2.0 ± 0.2 nm . Organosilane thin films were characterized using models 5420 and 5500 scanning probe microscopes operated in contact or tapping-mode AFM. . Lateral force images were acquired for either the trace or retrace views corresponding to the scan direction of the selected topography frames. The color scales of lateral-force images indicate differences in tip–surface interactions, but were not normalized for the comparison of friction changes between different tips or experiments. The tips were silicon nitride probes. Tips used with tapping-mode AFM were rectangular shaped ultrasharp silicon tips that have an aluminium reflex coating, with a spring constant of 48 N/m . For contact-mode images, V-shaped tips  with an average force constant of 0.5 N/m were used. Data files were processed by using Gwyddion open-source software, which is freely available on the internet and supported by the Czech Metrology Institute [nstitute . Estimatnstitute .Preparation of latex-particle masks. Polished silicon wafers doped with boron  were used as substrates. Pieces of Si(111) were cleaned by immersion in a 3:1 (v/v) piranha solution for 1 h. Piranha solution consists of sulfuric acid and hydrogen peroxide, which is highly corrosive, and should be handled carefully. After acid cleaning, the substrates were rinsed with copious amounts of deionized water and dried in air. Size-sorted, monodisperse polystyrene latex mesospheres (200 nm diameter) were used as surface masks for patterning . Aqueous solutions of latex were cleaned by centrifugation to remove surfactants or contaminants. Approximately 300 µL of the latex solution was placed into a microcentrifuge tube and centrifuged at 15,000 rpm for 15 min. A solid pellet was formed, and the supernatant was removed and replaced with deionized water. The latex pellet was resuspended with 300 µL of deionized water by vortex mixing to prepare a 1% w/v solution. The washing process was repeated twice. A drop (10–15 µL) of the cleaned mesospheres was deposited onto clean Si(111) substrates and dried under ambient conditions  for at least one hour, in order to form surface masks for nanolithography.Particle lithography combined with vapor deposition. The masked substrates were placed into sealed glass vessels for vapor deposition of organosilane. The samples were placed on a raised platform in a jar containing 300 µL of neat octadecyltrichlorosilane . A vapor was generated by heating the vessel in an oven at 70 °C. After at least 6 h, the samples were removed and rinsed with ethanol and water to remove the latex masks.Particle lithography with contact printing. For contact printing, an inked block of polydimethylsiloxane (PDMS)  was used to transfer OTS to the substrate through a physical mask of latex spheres. A drop (10–12 µL) of an OTS solution in bicyclohexyl was deposited onto a clean, dry block of PDMS (2 × 2 cm2). A 30 µL volume of a 40% v/v solution of OTS in bicyclohexyl was deposited and spread evenly over the PDMS block, then quickly dried in a stream of ultra-high-purity argon. The PDMS block coated with OTS was placed on top of the masked substrate. The film of OTS was transferred from the PDMS block through the latex mask to the substrate by permeation. The areas of the Si(111) surface located directly underneath the latex particles were protected from silane deposition. After 1 h of physical contact, the PDMS block was removed. The sample was rinsed with copious amounts of deionized water. In the final step, the mask of latex particles was cleanly removed by sonication and rinsing with ethanol and deionized water. After removal of the mask, a nanostructured film of OTS was generated on the surface.Particle lithography with immersion. For the immersion strategy of particle lithography, the masked substrates of latex were heated for 30 min at 75 °C in order to anneal the beads to the surface. Masked substrates of colloidal silica mesospheres were heated for 12 h at 140 °C. After heating, the samples were cooled for at least 20 min under ambient conditions. The mesosphere-coated substrates were then immersed into a 0.1% solution of OTS in bicyclohexyl or anhydrous toluene for 1 h. Next, the samples were removed and rinsed with ethanol and deionized water, and sonication was used to remove the latex masks.