The importance of snakes, and the Burmese python, as model organisms Sequencing the genome of the Burmese python (Python molurus bivittatus) as a model for studying extreme adaptations in snakes O P E N L E T T E R Open Access
2011

Todd A Castoe
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

Jason Ap
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

De Koning
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

Kathryn T Hall
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

Ken D Yokoyama
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

Wanjun Gu
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

Eric N Smith
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

Cédric Feschotte
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

Peter Uetz
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

David A Ray
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

Jason Dobry
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

Robert Bogden
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

Stephen P Mackessy
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

Anne M Bronikowski
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

Wesley C Warren
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

Stephen M Secor
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

David D Pollock firstname.lastname@example.com
Department of Biochemistry & Molecular Genetics
University of Colorado School of Medicine
12801 17th Ave80045AuroraCOUSA

The importance of snakes, and the Burmese python, as model organisms Sequencing the genome of the Burmese python (Python molurus bivittatus) as a model for studying extreme adaptations in snakes O P E N L E T T E R Open Access

Genome Biology
124062011*Correspondence: Full list of author information is available at the end of the article
The Consortium for Snake Genomics is in the process of sequencing the genome and creating transcriptomic resources for the Burmese python. Here, we describe how this will be done, what analyses this work will include, and provide a timeline.

The evolutionary origin of snakes involved extensive morphological and physiological adaptations that included the loss of limbs, lung reduction, and trunk and organ elongation. Most snakes also evolved a suite of radical adaptations to consume large prey relative to their body size, including the ability to endure extreme physiological and metabolic fluctuations [1,2] and produce diverse venom proteins [3,4]. These radical adaptations, centered around consuming large prey whole, have made snakes an interesting model for studying metabolic flux and organ physiology, regeneration, and regulation, with the most important example being the Burmese python. Within 2 to 3 days after feeding, the Burmese python (Python molurus bivittatus) can experience tremendous physiological changes, including: a 44-fold increase in metabolic rate (the highest among tetrapods); 35 to 100% increases in the mass of the heart, liver, pancreas, small intestine, and kidneys; 160-fold increase in plasma fatty acid and triglyceride content; and 5-fold increase in intestinal microvillus length [1,5]. After the completion of digestion, each of these phenotypes is reversed as digestive functions are downregulated and tissues undergo atrophy [6]. This extreme modulation of tissue morphology and function facilitates investigation into the signaling and cellular mechanisms that underlie regulation of organ performance and regeneration. These animals are also readily obtained from commercial breeders, non-aggressive, and easier and cheaper to care for than laboratory rats. The scientific potential of this system to reveal molecular mechanisms associated with these extreme reactions (and their reversal) is tremen dous, and can provide novel insight into vertebrate gene and systems function, novel strategies and drug targets for treating human diseases, and alternative disease models.

Snakes have also been used as model species for highprofile discoveries pertaining to vertebrate development, including the findings that vertebrate metamerism (somito genesis) can be controlled by changing the rate of somitogenesis [7], that the loss of limbs correlates with changes in expression of some regulatory genes [8] as well as Hox gene expression and gene structure [9], that particular developmental pathways are associated with tooth and fang development [10], and that limblessness in snakes may result from failure to activate core vertebrate signaling pathways during development and from changes in Hox gene expression [8,11]. Snakes are also important models for high-performance muscle physiology [12], genetic sex determination [13], evolution ary ecology [14,15], and molecular evolution and adaptation [16][17][18]. Enhanced snake genomic resources (eventually including comparative genomic data from multiple species) are expected to provide additional insight into how the unique structures and developmental processes of snakes evolved.

In addition to the python (which is non-venomous), venomous snake species are also important for biomedical research, as is developing a greater under standing of the genomic and adaptive contexts leading to the origin of venom genes. Worldwide, the World Health Sequencing the genome of the Burmese python (Python molurus bivittatus) as a model for studying extreme adaptations in snakes

O P E N L E T T E R Open Access

Organization estimates that there are about 2.5 million venomous snake bites per year (about 1,400 in the US), resulting in about 125,000 deaths [19]. As a consequence, the health relevance of snake venom research is extensive. Genes identified in snake venoms are related to genes used in normal housekeeping and digestive roles in other vertebrates [3,4], but the details of how these have been modified by evolution to become functionally diverse toxic venoms cannot readily be determined without good comparative information from the full complement of genes from both venomous and non-venomous snakes.

Phylogenetic position of snakes and the python

Among vertebrates, the snake lineage represents a speciose (about 3,100 species) and phenotypically diverse radiation. Because snakes represent such an ancient (about 150 million years old) lineage on the branch of the vertebrate tree of life ( Figure 1; squamate reptile divergence estimates based on [20]), understanding the content of snake genomes will contribute broadly to an under standing of vertebrate genomics. Together with the genome of the Anolis lizard, the availability of a snake genome (and eventually, multiple snake genomes) will contribute to better rooting of mammalian gene trees, and to more accurate reconstructions of amniote ances tral genome attributes. Below, we outline that in addition to the python genome, the genomes of the venomous king cobra and the non-venomous garter snake are also currently being sequenced. In the phylogenetic tree in Figure 1, we highlight that in addition to the major lineages being targeted by these three confirmed genome projects, there are two other major groups, blindsnakes and venomous vipers (for example, rattlesnakes), that are not yet explicitly targeted by ongoing genome sequencing projects (although multiple groups have cited these as potential targets). One purpose of the website that we have established [21] is to provide the community with updated information on targeting of species for genome sequencing.

Python genome project overview

A main goal of the python genome project is to provide key genomic resources to facilitate studies of how its extreme phenotypes are regulated and accomplished at the molecular level. Thus, a central component of the python genome project is to produce a draft python genome that contains genic and near-genic regions that are assembled and annotated. To provide a service to the broader research community, we have released a prepublication preliminary draft assembly of the python genome for conditional use. We are working under the Toronto Statement for prepublication release [22], and this letter provides the details of our plans and responsibilities, as outlined in the original paper describing this statement [22].

Properties of the python genome, and genomic resources currently available

Snake genomes are often smaller than mammalian genomes, ranging from about 1.3 Gbp to 3.8 Gbp, with an average of 2.08 Gbp [23]. There is no existing estimate for the genome of Python molurus, but the most recent estimate for the related species Python reticulatus is 1.44 Gbp; this suggests that the Burmese python genome is relatively small compared with most snakes. The karyotype of the Burmese python is known, and comprises 36 chromosomes (2n = 36), with 16 macrochromosomes and 20 microchromosomes [24]. All snakes are thought to have ZW genetic sex determination, with males being the homogametic sex (ZZ) and females heterogametic (ZW).

Since the early work of Olmo and colleagues [25,26] using DNA reassociation kinetics, it has been known that the genome of P. molurus had particularly low amounts of repetitive DNA compared with other snakes. This was recently confirmed with sequence-based evidence [27], using 454 sequencing of genomic shotgun libraries to randomly sample fractions of snake genomes, and using these fractions to estimate genomic repetitive element content and diversity ( Figure 2; data based on [27]). From these data, the python genome was estimated to be made up of 21% readily identifiable repetitive element sequence ( Figure 2), compared with more than double that (45%) in the venomous copperhead (a relative of the rattlesnake) with a similarly sized genome [27]. Despite the contrast in repetitive element abundance, both snakes contained a similarly broad diversity of transposable element types, which seems to be an emerging hallmark of squamate reptile (lizards and snakes) genomes [27][28][29]. Bov-B and CR1 LINE retroelements were among the most prominent transposable element types in the python genome ( Figure 2) [27], a characteristic in common with other snake genomes [27,29].

Burmese python genome draft version 1.0

We completed and publicly released an initial draft assem bly of the Burmese python genome (v1.0). This sequence was obtained from a single individual purchased from a commercial breeder, and did not originate from an inbred line (per se), and thus we expect moderate levels of heterozygosity.

This genome draft was built primarily from Illumina GAIIx sequencing of a short insert (325 bp) paired-end shotgun genomic library. Various amounts of sequence data were collected from this library using paired reads of three different lengths (114 bp: 15  17-fold coverage of the estimated 1.4 Gbp python genome, and is available from the NCBI accession AEQU000000000.1. This coverage is equivalent to about 35X 'virtual' or 'structural' coverage of the genome, which includes the gaps in the paired-end sequences.

Computational genome assembly was conducted using SOAP de novo v.1.04, with a k-mer size of 31. This assembly yielded 1.128 million contigs, with a mean length of 944 bp and an N50 length of 1,355 bp. Using paired-end sequence reads, contigs were assembled into 324,418 scaffolds that had a mean length of 1,397 bp and an N50 length of 2,186 bp. The total length of the scaffolded assembly was 1,177 Mbp. We note that the average contig and scaffold sizes in this draft are relatively small, in part because there are no sequences from longer mate-pair libraries or BAC references to increase structural coverage and improve assembly; such coverage will be added in future drafts.

Python BAC library resources

There is a high-quality high-density (about 5X coverage) BAC library available for the Burmese python, con structed using DNA from the same individual from which the draft genome was sequenced. This BAC library, along with mapping and sequencing services, is currently available commercially to the public from Amplicon Express [30].

Other resources

Limited transcriptomic resources have already been made available at the snake genomics website [21], and a  larger suite of transcriptomic resources will be made available with the release of the second assembly of the python genome (v2.0). There is also a preliminary set of repeat element consensus sequences, estimated from genomic sample sequencing of 454 genomic shotgun libraries [21,27].

Strategy for sequencing the python genome

Our strategy for improving the existing python genome is to add substantial additional sequence coverage from slightly longer insert (600 bp) paired-end Illumina sequen cing, together with 3-kb mate-pair paired-end sequence. We plan to have a total of 50X coverage of these mixed read types, predominantly from long (114 to 150 bp) Illumina GAIIx paired-end reads.

The second draft assembly will be updated with the new short and long insert paired-end sequence data. Genome assembly will involve four principal steps that progress from forming contigs from raw quality-filtered sequence reads, to connecting contigs into scaffolds using paired-end sequence data, to gap filling (using all reads) and error correction. The set of smaller contigs will serve as anchors for addition of longer range insert sizes to increase scaffold length.

We therefore expect that contig lengths will be sufficient for most gene predictions and post-assembly alignment-based analysis. We also expect that the attributes of the python genome, being smaller and also lower in repetitive content than mammalian genomes (or other snakes), for example [27], together with our use of relatively long sequence reads, will produce a reasonably good quality assembly with moderately long contigs and scaffolds.

We will assess the accuracy of the assembled python genome using several methods, including read chaff rate (proportion of reads not incorporated into the assembly), read depth of coverage, average quality values per contig, discordant read pairs, gene footprint coverage (as assessed by cDNA contigs) and comparative alignments to the most closely related species with a complete genome -the Anolis lizard (and eventually, other snake genome assemblies). We will also take advantage of mapped cDNA contigs from various python tissues to improve assembly contiguity and accuracy, further strengthen ing the genic component of this assembly.

Our internally contamination-screened genome assembly will be submitted to the whole genome shotgun division of GenBank for independent contamination analysis. The final assembly will be posted on the Ensembl [31], University of California Santa Cruz [32] and NCBI [33] genome browsers for public queries as soon as it is available and passes contamination analyses, and relevant announcements and links will be posted on the snake genomics website [21].

Description of sequencing project with anticipated milestones and timeline

We recently released a preliminary draft assembly of the python genome (v1.0) to the public, together with limited transcriptome data. This assembly includes primarily about 17X coverage from Illumina short-insert pairedend sequencing and is therefore expected to be relatively fragmentary. Our anticipated timeline includes the comple tion of data collection required for the updated assembly (v2.0) based on extended genome coverage (about 50X) from short and longer insert paired-end Illumina sequencing by the end of the summer of 2011. This will be accompanied by an extensive set of trans criptome data, from multiple organs, that will be incorporated into gene prediction annotations. Attainment of 50X genome coverage and completion of long mate-pair library sequencing will mark the end of the data collection phase and the start of assembly and analysis. The end of this phase will be marked clearly on the snake genomics website [21], as will milestones of data analysis and release. The maximum time between the end of data collection and submission of the genome paper will be 1 year. The Toronto Statement suggests that there be a 1-year period, after which global analyses and publication by the community would be unimpeded. We recognize the start of this 1-year period at approximately the time that this manuscript will be published, July 2011, and therefore this embargo period would end July 2012.

Biological questions and types of analyses to be addressed by the python genome project

Here we outline the major questions, types of analyses, and analytical goals that will be included in the core python genome marker paper. The Toronto Statement suggests this be done to identify these topics as being somewhat embargoed, and we also see this as providing expectations for the community regarding the types of analyses planned. Although vignettes of the topics below will, in most cases, appear in some form in the core python genome paper, a majority of these will also involve longer-term research (including other publications) by members of the working group. Ultimately, the goal of the Consortium for Snake Genomics is to make certain that research efforts are not duplicated, and also to put together clusters of researchers interested in similar questions. Thus, we continue to welcome additional members to join the Consortium for Snake Genomics, and because of this, the research scope of the group may continue to expand beyond even what we outline here because of the interests of new members.

The analytical goals of the python genome project focus on aspects of the extreme physiology and metabolism of pythons, and on making links between the extreme phenotypes and genotypes of the python and snakes in general. A main focus of analysis will include trans criptome data that describes the dynamics of gene expression that accompanies major physiological transitions brought about by feeding in the python. We will also be conducting genome-wide analysis of protein evolution to detect patterns of molecular evolution indicating positive selection that may relate to key adaptations of snakes, and the python specifically. In addition to focusing on all proteins in the genome, we intend to include detailed analysis of sets of genes involved in physiology, metabolism, heat sensing, vision, body elongation, limb loss, and the evolution of snake venoms. We anticipate analyzing how the protein families of interest identified above have differentially expanded or contracted in the snake and mammalian lineages.

We are also interested in analyses that focus on areas of the genome outside of the protein-coding regions. Complementing our analysis of protein-coding genes, we plan to use the python genome to investigate, essentially for the first time, unique properties of snake and reptilian gene and promoter architecture, and to make a first attempt to identify snake cis-regulatory elements and compare these to other species. Specifically, this analysis will include comparisons of nucleotide content and overrepresented motifs that occur in core upstream promoters of genes with well-predicted transcription starts. Our comparisons would highlight cis-regulatory structure in the python and anole lizard in relation to patterns in other vertebrates. We also are interested in studying the repetitive element landscape of the python genome, including identification of which types of transposable elements occur in the python genome and how these elements have expanded over evolutionary time, and how horizontal transfer may explain their origins in the python genome. Our genome analyses will additionally include identification of single nucleotide polymorphisms from genomic and transcriptomic data collected, and an effort to make available sets of sequences for use as molecular markers for snakes (for example, microsatellite primers and orthologous loci for use in phylogenetics and other applications). Lastly, we will be conducting a detailed analysis to identify genomic sequences that represent python sex chromosomes by using genomic sequences collected from multiple individuals from both sexes.

There are a number of potential research areas that would probably be productive to pursue but are outside of the scope of the current plans of the project -these topics are therefore potential research avenues that we encourage others to pursue. Because the python represents a relatively deep evolutionary lineage on the amniote vertebrate tree of life, using the python data together with other comparative data to estimate genomic characteristics of the ancestral amniote genome (or the ancestral squamate genome) would be fascinating, including estimation of ancestral gene family copy numbers, instances of differential expansion/contraction of gene families in mammals and squamate reptiles, evolution of long conserved non-coding sequences, and genomic features such as isochore structure. Analysis of genes and gene families involved in vertebrate hearing, locomotion, behavior, and coloration are other examples of projects outside of the scope of the current project.

Justification and strategies for expansion of snake genomics

Research incorporating snakes as model systems is becoming increasingly popular and diverse in its breadth of topics. The availability of the python genome and associated resources will provide a much-needed genetic and genomic reference infrastructure for further facili tating such research. In addition to the importance of the python as a model for research, different snake species have been used as model systems for different types of research. For example, research focusing on behavior, development, and evolutionary ecology has focused on smaller non-venomous species such as garter and corn snakes in the family Colubridae, whereas research related to snake venom and envenomation have centered on venomous species typically in the families Viperidae (for example, rattlesnakes, and adders) and Elapidae (for example, coral snakes, cobras, and mambas). In addition to these lineages that contain commonly used model research species, blindsnakes represent a lineage that diverged long ago from the rest of the snakes, and as such would be a major contribution for comparative and evolutionary analyses. In addition to the python, we are aware of two additional confirmed snake genome sequencing projects targeting the non-venomous garter snake [29], and the venomous king cobra (F Vonk, personal communication; Figure 1). We therefore expect that multiple snake genomes will be available to support diverse research projects in the near future, and the incorporation of additional lineages of snakes would further support their utility as research models.

Formation of the Consortium for Snake Genomics and a portal for snake genomic resources

To foster the growth of a productive and interactive community of researchers interested in snake genomics, and to also encourage the growth of snake genomic resources, we have established the Consortium for Snake Genomics (CSG) and a website to house related content [21]. A core concept guiding the establishment of the CSG is that through shared interest in developing resources for snake-related research, individual researchers would be able to benefit from the pooling of resources, research motivations, and expertise, while also avoiding redundant effort. Therefore, an integral part of this vision includes the recruitment of, and interaction among, a diverse working group of researchers interested in using snake genomic resources.

The CSG is also directly involved with the reptilian subset of the Genome10K project [34], with the intention of making certain that efforts to build resources for particular species are not duplicated, and that scientific arguments for the need for genomic resources of parti cular types, or for particular snake lineages, get translated into priorities for future sequencing initiatives, and that all this gets translated to the community through the snake genomics website [21]. At the website we have created pages with links to available snake genomic resources, and posted updates (news) on major projects, such as the status of various snake genomics sequencing projects and data releases; RSS feeds have been set up so that changes to the various pages can be updated through RSS readers automatically once subscribed to the feed. We have also set up an email list system so that interested researchers can request to receive occasional email updates related to snake genomics. Lastly, for researchers interested in becoming directly integrated into ongoing or future CSG projects, email contacts for the lead author are provided on the site.

.1 Gbp, 76 bp: 5.6 Gbp, and 36 bp: 2.9 Gbp), with the addition of a small amount (30 Mbp) of 454 shotgun library sequences. The v1.0 draft Burmese python genome, based on 23.7 Gbp of DNA sequence data, is equivalent to approximately

Figure 1 .
1Phylogenetic tree of major amniote vertebrate lineages. Approximate divergence times are indicated. The turtle lineage is not included, and the placement of that lineage on this tree is controversial.

Figure 2 .
2Repetitive elements in the Burmese python genome. The estimated proportion of the Burmese python genome sequence occupied by different repetitive elements (including the largest category, 'unannotated') is indicated. Results are based on genomic sample-sequencing using 454 genomic shotgun libraries, and identification of known and de novo repeat elements within these data was performed as reported in[27]. LINE, long interspersed element; LTR, long terminal repeat; SINE, short interspersed element.

Todd A Castoe 1 , AP Jason de Koning 1 , Kathryn T Hall 1 , Ken D Yokoyama 1 , Wanjun Gu 2 , Eric N Smith 3 , Cédric Feschotte 3 , Peter Uetz 4 , David A Ray 5 , Jason Dobry 6 , Robert Bogden 6 , Stephen P Mackessy 7 , Anne M Bronikowski 8 , Wesley C Warren 9 , Stephen M Secor 10 and David D Pollock 1 *
Castoe et al. Genome Biology 2011, 12:406 http://genomebiology.com/2011/12/7/406

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Prostatic stromal sarcoma with neuroectodermal differentiation

Hitoshi Yamazaki
Teppei Ohyama
Toshiki Tsuboi
Yoshinori Taoka
Dai Kohguchi
Hiroyoshi Iguchi
Teruaki Ao
Prostatic stromal sarcoma with neuroectodermal differentiation
C A S E R E P O R T Open AccessProstatic stromal sarcomaSTUMPImmunohistochemistryCD99CD56SynaptophysinNeuroectodermal differentiation
Prostatic stromal sarcoma is a fairly rare tumor that constitutes approximately 0.1-0.2% of all prostatic cancers. Detailed characteristics of the tumor are still unclear due to its rarity. We describe a case of prostatic stromal sarcoma in a 63 year-old man who suffered from urinary obstructive symptoms. Palliative transuterine resection was performed and the preliminary histopathological diagnosis was neuroendocrine carcinoma. After chemotherapy, total pelvic exenteration was performed. Histopathologically, the tumor was composed of monotonously proliferating small to medium-sized round cells, which existed in compact islands with loose or dense fibrovascular networks. Immunohistochemically, the tumor cells were widely positive for vimentin, CD56, CD99 and focally positive for synaptophysin, CD10, progesterone receptor, desmin and CD34, but negative for EMA, cytokeratin, estrogen receptor, S-100 and myoglobin. Most of the previously reported tumors exhibited positive stainability for CD10 and progesterone receptor. In addition to these markers, expressions of CD56, CD99 and synaptophysin were characteristically detected in our case. To the best of our knowledge, we present the first case of prostatic stromal sarcoma with characteristic immunohistochemical staining properties. Although the biological characteristics of this rare tumor have not yet been elucidated, these findings suggest prostatic stromal sarcoma can potentially show neuroectodermal differentiation. Virtual slide: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/ 7291874028051262

Background

Prostatic stromal sarcoma (PSS) is a fairly rare tumor, constituting approximately 0.1% of all prostatic cancers [1,2]. Prostatic sarcoma and related proliferative lesions, including prostatic phyllodes tumors, have been classified as prostatic stromal tumors of uncertain malignant potential (STUMP) and prostatic stromal sarcoma (PSS) based on cellularity, mitotic index, cellular atypia and necrosis (WHO 2004) [3]. Some STUMP cases were reported as malignant transformation into PSS [4]. There has not yet been a clear differentiation between PSS and STUMP due to the rarity of these tumors. In this article, prostatic stromal sarcoma has potentially neuroectodermal characteristics.

Case presentation

The patient was a 63 year-old man who presented to the urologist with a one week history of dysuria, pollakiuria and an unrelieved feeling after urination. He had a previous history of diabetes mellitus and asymptomatic multiple brain infarction. He habitually drank alcohol and smoked and his mother died of gastric cancer. Abdominal ultrasonography revealed 343 ml of residual urine volume. Computed tomography revealed a prostatic mass lesion which protruded into the bladder space ( Figure 1a). Gadrinium-enhanced T1 weighted magnetic resonance imaging also revealed the prostatic mass lesion had irregularly high signals (Figure 1b). Based on our clinical diagnosis of benign prostatic hypertrophy, palliative transurethral resection was performed. The specimen consisted of 20 grams of piecemealed prostate that was totally embedded in paraffin and histologically analyzed. Sections showed monotonously proliferating small to medium-sized round cells invading the edematous stroma with coarseness and fineness and sparse or dense patterns. The residual prostatic glands were identified. Immunohistochemically, the tumor cells were positive for vimentin, CD56, synaptophysin (focal) but negative for EMA, cytokeratin, S-100. The preliminary histopathological diagnosis to decide the strategy for further therapy was neuroendocrine carcinoma. He took one series of chemotherapy, a combination of cisplatin and irinotecan. Ten days after the last day of the chemotherapy, suprapubital radical cystprostatectomy with rethrotectomy was performed. During the operation, the urinary bladder could be detached from the rectum with difficulty. Finally, low anterior rectal resection was additionally performed, resulting in total pelvic exenteration. When the urethra was cut, a part of the tumor was pressed down from the urethral cavity. The gross features of the tumor were elastic soft and translucently whitish in color. After fixation in 10% formaldehyde the prostate weighed 125 g and contained an ill-defined gray whitish mass lesion, which focally exhibited necrosis ( Figure 2). The bladder and the retroperitoneal cavity were diffusely infiltrated by the whitish tumor, which was directly connected to the prostatic tumor. Although the rectum was attached to the tumor mass, the rectal parenchyma was free from tumor invasion. The bladder space was occupied by the tumor and narrowed, resulting in a slit-like space. Serial sections were analyzed and revealed a round cell sarcomatous tumor with occasional higher cellularity than that in the previous transuterine resection. Tumor necrosis was occasionally identified. The N/C ratio of the tumor cells was a relatively high grade. Mitotic activity was measured as 12/10 HPF in the higher cellularity area. The residual prostatic glands were compressed to the periphery (Figure 3a). No lymph nodal metastasis was observed. Immunohistochemically, the tumor cells were widely positive for vimentin, CD56, CD99 and focally positive for synaptophysin, CD10, progesterone receptor, desmin and CD34, but negative for EMA, cytokeratin, estrogen receptor, S-100, GFAP and myoglobin (Figure 3b,c,d). The Ki-67 index was about 70%. We finally diagnosed the tumor as prostatic stromal sarcoma. Sixteen months later, the patient is alive without local recurrence or distant metastasis.

Discussion

Prostatic stromal sarcoma is a rare tumor that constitutes approximately 0.1-0.2% of all prostatic cancers [1,2]. Prostatic mesenchymal tumors sometimes cannot be clearly classified as histological entities due to their rarity [5]. Except for prostatic mesenchymal tumors with specialized differentiation, a histologically characteristic classification involves prostatic stromal sarcoma and prostatic stromal proliferation of uncertain malignant potential (STUMP) [6][7][8]. These tumors have been described on the basis of the histological similarity to phyllodes tumors and have to be differentially diagnosed as other specialized types of tumor, such as GIST, leiomyosarcoma, rhabdomyosarcoma(RMS), and fibrosarcoma [3]. RMS is the most common sarcoma of the prostate, but is rarely reported in adult patients [9].  Prostatic stromal sarcoma and STUMP express female hormone receptors, especially progesterone receptor. These tumors seem to be derived from female hormonedependent stromal cells [10]. Depending on the cellularity, mitosis and necrosis, these two tumors are histologically evaluated. An acceptable histological grading of stromal sarcoma has not yet been proposed due to the rarity of the tumor. Hasegawa reported that the ki-67 index was related to the prognosis of the tumor [4].

Recent immunohistochemical analysis revealed that both prostatic stromal sarcoma and STUMP usually express CD10, CD34 and the progesterone receptor [3,7,8,[10][11][12]. This tumor sometimes positively reacts with smooth muscle actin. However, the detailed histopathological characteristics of the tumor cells have not yet been clearly elucidated. Kim reported a case of prostatic stromal sarcoma with rhabdoid features [13]. The tumor in our case was positive for CD56, CD99 (to our knowledge the second reported case) [14], synaptophysin and negative for EMA and cytokeratin. This is the first description of prostatic stromal sarcoma with immunohistochemically positive stainability for synaptophysin. There are other prostatic non-epithelial malignancies with potential neuroectodermal differentiation such as carcinosarcoma, ectomesenchymoma and primitive neuroectodermal tumor (PNET), which have to be distinguished from our case of prostatic stromal sarcoma. Patient age is a useful marker for differential diagnosis, although PNET and malignant ectomesenchymoma are rarely reported in young adult prostate cancer patients [15][16][17][18]. Most PNET cases reveal positive immunoreactivity for CD99, but are negative for progesterone receptor. Carcinosarcoma contains elements of epithelial malignancy which exhibit some types of cytokeratins.

PSS is suspiciously derived from mesenchymal pluripotent stem cells in the prostatic stroma. As few cases of STUMP change to PSS in the history, some genetic transformations are considered to be related to PSS [4,19]. Arva showed that some cases of prostatic squamous cell carcinoma were derived from hormonal or radiation-treated prostatic adenocarcinoma [20]. Unexpected irradiation may be one of the risk factors of PSS [21,22]. Babarović reported a case of high grade angiosarcoma arising in fibroadenoma and suggested the possibility that severe inflammatory reaction, for example silicon granuloma, may cause malignant transformation of stromal cells in the affected area [23,24]. These factors may give rise to an exuberant stromal response and cause some genetic events in the mesenchymal pluripotent stem cells. We could classify PSS as two categories, which are de novo tumorigenesis and malignant transformation step by step [20]. However, there are no authentic molecules which directly cause prostatic stromal tumorigenesis. Fibroblast growth factor 8 (FGF8), the eighth member of the fibroblast growth factor family contains alternatively spliced mRNA isoforms. Fibroblast growth factor 8b(FGF8b) is an androgen-induced growth factor with potent oncogenic activity [25][26][27]. Elo reported the results of prostate-targeted fibroblast growth factor 8b transgenic mice [28]. These mice showed progressive changes in prostatic stroma, as well as the prostatic epithelium. Furthermore, FGF8b is the predominant FGF8 spliceform necessary for proper posterior neural formation in Xenopus [29]. Amsterdam reported the relationship between fgf8 misregulation and neuronal tumors in Zebrafish [30]. This knowledge suggests FBF8b is related to potential neuroectodermal differentiation in prostatic stromal tumorigenesis. Some hormonal imbalances, including androgen imbalance, may cause prostatic stromal progression, which could lead to prostatic stromal neoplasia. We hereby point out that PSS may also possess neuroectodermal characteristics.

Conclusions

We describe a rare case of prostatic stromal sarcoma (PSS) presenting with characteristic immunohistochemical staining properties. To our knowledge, this case is the first case of PSS with positive stainability for synaptophysin, as well as CD99 and CD56.

Consent

Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of Diagnostic Pathology.

Figure 1
1Image analysis. a) Computed tomography shows a prostatic non-homogenous mass lesion irregularly protrudingd into the bladder space. b) Gadrinium-enhanced T1 weighted magnetic resonance imaging shows a heterogeneous hyperintense mass.

Figure 2
2Cut surface of the tumor. The prostate and the bladder space are widely occupied by the tumor tissue, which exhibited a whitish-yellow multinodular appearance with focal necrosis.

Figure 3
3Microscopic findings. a)The tumor was made up of sarcomatoid oval to spindle cells. (HE x 100) b) Immunostaining with CD56 showed positive results for tumor cells on the cell membrane (CD56 x 200). c) Immunostaining with synaptophysin focally showed positive results for the tumor cells in the cytoplasm (synaptophysin x 200). d) Immunostaining with CD99 showed positive results for tumor cells on the cell membrane (CD99 x 200).
http://www.diagnosticpathology.org/content/7/1/173
Competing interestsWe do not have any competing interests for our manuscript.Authors' contribution HY was responsible for data collection and drafted the manuscript. TO, TT, YT, DK and TA made contributions to acquisition of clinical data and performed surgical procedure. HI performed radiological interpretation and helped drafting the manuscript. All authors read and approved the final manuscript.Submit your next manuscript to BioMed
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Enhancing performance of ZnO dye-sensitized solar cells by incorporation of multiwalled carbon nanotubes

Wei-Chen Chang
Yao-Yi Cheng
Wan-Chin Yu
Yih-Chun Yao
Chia-Hua Lee
Hung-Han Ko
Enhancing performance of ZnO dye-sensitized solar cells by incorporation of multiwalled carbon nanotubes
N A N O E X P R E S SZnOnanoparticlemultiwalled carbon nanotubecomposite filmdye-sensitized solar cellsconversion efficiency
A low-temperature, direct blending procedure was used to prepare composite films consisting of zinc oxide [ZnO]   nanoparticles and multiwalled carbon nanotubes [MWNTs]. The mesoporous ZnO/MWNT films were fabricated into the working electrodes of dye-sensitized solar cells [DSSCs]. The pristine MWNTs were modified by an air oxidation or a mixed acid oxidation treatment before use. The mixed acid treatment resulted in the disentanglement of MWNTs and facilitated the dispersion of MWNTs in the ZnO matrix. The effects of surface property and loading of MWNTs on DSSC performance were investigated. The performance of DSSCs was found to depend greatly on the type and the amount of MWNTs incorporated. At a loading of 0.01 wt%, the acid-treated MWNTs were able to increase the power conversion efficiency of fabricated cells from 2.11% (without MWNTs) to 2.70%.

Introduction

Dye-sensitized solar cells [DSSCs] are considered as promising third-generation solar energy devices because of their low fabrication cost, compatibility with flexible substrates, and practicable high conversion efficiency [1,2]. The maximum conversion efficiencies attained by DSSCs so far (approximately 11%), although considerably lower than those of silicon solar cells, may meet the requirements of many practical applications [1,3]. Further improvement in the conversion efficiency of DSSCs is possible, as theoretical prediction of the maximum conversion efficiency of DSSCs is approximately 20% [4].

A DSSC is a photoelectrochemical system in which a porous nanostructured oxide film with adsorbed dye molecules acts as the photoanode and plays a significant role in converting photons into electrical energy. It has been shown that the performance of DSSCs is closely related to the structure of the photoelectrode film [5]. A rational design of the photoelectrode film structure may result in better light harvesting and electron transport. Zinc oxide [ZnO] has been used widely for the fabrication DSSC photoanodes. ZnO is regarded as an attractive alternative to titanium dioxide [TiO 2 ] because it has a similar band gap level to that of TiO 2 while possesses a higher electron mobility and more flexibility in synthesis and morphologies [6,7]. Among the nanostructures investigated for the fabrication of DSSC photoelectrode films, nanoparticles are most widely used. This is likely due to a high specific surface area provided by nanoparticle-based films. Another reason is probably the ease of preparation of nanoparticles through simple chemical solution methods and the ease of film formation through the doctor-blade method.

Nanoparticle-based films can provide a large surface area, but the existence of numerous boundaries in the nanoparticle network may hinder the transport of electrons in photoelectrode and thus limit energy conversion efficiency of DSSCs. Incorporating one-dimensional [1-D] nanostructures into nanoparticulate films may overcome the problem by providing direct pathways for electron transport [8]. Carbon nanotubes [CNTs], a type of 1-D nanostructure, possess several unique properties including hollow and layered structures, a high aspect ratio, excellent electrical and thermal conductivity, high mechanical strength, and a large specific surface area [9]. Incorporating CNTs into ZnO electrodes should provide highly conductive paths to the ZnO nanostructures, thereby promoting faster transport of photoinduced electrons in DSSCs and higher current. The combination of CNTs with ZnO nanoparticles is thus a promising approach to boost conversion efficiency of DSSCs. In fact, this approach has been used to improve the performance of TiO 2 -based DSSCs [10][11][12]. However, the effect of CNTs on ZnO nanoparticle-based DSSCs has not been reported before. The major barrier for the application of CNTs in DSSCs is the insolubility of CNTs in most solvents. To obtain homogeneous dispersion of CNTs, CNTs need to be pre-treated before mixing them with nanoparticles. The air oxidation treatment has been found to completely remove amorphous carbon and metal oxide impurities from CNTs [13]. The mixed acid treatment not only effectively purifies CNTs but also leads to the formation of oxygen-containing groups, mainly carboxylic, on the graphitic surface [14,15]. The carboxylic groups thus formed facilitate the exfoliation of CNT bundles and therefore the dispersion of CNTs. The oxygen-containing groups should also improve the interfacial bonding between CNTs and ZnO nanoparticles.

In this study, DSSC photoanodes were fabricated using nanostructured films based on commercial ZnO nanoparticles. To study the effects of incorporating CNTs on device performance, two different types of multiwalled carbon nanotubes [MWNTs], i.e., oxidized MWNT [O-MWNT] (or oxidized in air) and acid-MWNT (mixed acid treated), were prepared and blended with ZnO nanoparticles at various levels. The effects of air oxidation and mixed acid treatments on the morphologies of MWNT were studied using transmission electron microscopy [TEM], and the morphologies of the prepared ZnO/MWNT composite films were investigated by using field emission scanning electron microscopy [FE-SEM]. The energy conversion efficiency [h] and the electrochemical impedance of the fabricated DSSCs were also determined.

Experimental methods

Pristine MWNTs that were 10 to 30 nm in diameter and 5 to 15 mm in length were purchased from Nanotech Port (Genesis Nanotech Corporation, Tainan, Taiwan, Republic of China). These MWNTs were produced via a chemical vapor deposition method. To obtain the O-MWNT, the pristine unmodified-MWNT [U-MWNT] was purified by thermal treatment in air at 550°C for 45 min to remove amorphous carbon and residual metal catalysts. The carboxylic group-modified MWNT [acid-MWNT] was prepared by treating the O-MWNT with a sulfuric acid/nitric acid mixture (3:1, v/ v) at 50°C for 2 h under ultrasonication. After the mixed acid treatment, the acid-MWNT was collected by filtration and then washed with distilled water and methanol [16].

Commercial ZnO nanoparticles (UniRegion Bio-Tech, Taiwan, Republic of China) that were about 20 nm in size were dispersed in equal proportion of distilled water and tert-butanol to form ZnO pastes. The MWNTs were added at this step when ZnO/MWNT composite films were to be made. The nanoporous films were prepared by applying the pastes onto commercial fluorine-doped tin oxide [FTO] substrates (Nippon Sheet Glass, 8 to 10 Ω/□, 3 mm thick) (Nippon Sheet Glass Company, Tokyo, Japan) by the doctor-blade method using adhesive tapes as a frame and spacer. The active electrode area was 0.25 cm 2 , and the films had a thickness of 8 μm. The resulting films were then gradually heated and annealed at 150°C for 1 h to remove organic materials in the paste and to increase crystallinity of ZnO. After being cooled to room temperature, the porous films were sensitized by immersing them into a dye solution that contained 0.5 mM of cis-bis (isothiocyanato) bis-(2,2'-bipyridyl-4,4'-dicarboxylato)ruthenium(II)bis-tetrabutylammonium (Solaronix, N719) (Solaronix SA, Aubonne, Switzerland) in a mixed solvent consisting of equal parts of acetonitrile and tertbutanol. The dye-adsorption time lasted for 2 h. The dye-loaded electrodes were then rinsed with acetonitrile and dried in the air. The counter electrode was made of FTO glass, onto which a nanocrystalline platinum [Pt] catalyst was deposited by decomposition of H 2 PtCl 6 at 400°C for 20 min. The ZnO photoanode and the counter electrode were sealed together with a 60 μm-thick hot-melting spacer (DuPont, Surlyn, Shanghai, People's Republic of China), and the inner space was filled through a hole with a volatile electrolyte composed of 0.1 M lithium iodide, 0.6 M 1, 2-dimethyl-3-propylimidazolium iodide (PMII, Merk Ltd., Taiwan, Republic of China) 0.05 M I 2 (Sigma-Aldrich China, Inc., Shanghai, People's Republic of China), and 0.5 M tert-butylpyridine (Sigma-Aldrich China, Inc., Shanghai, People's Republic of China) in acetonitrile.

The surface morphologies and dimensions of the ZnO/MWNT composite films were characterized using a FEI Nova230 FE-SEM (FEI Inc., Hillsboro, Oregon). TEM images of MWNTs were taken by employing a JEOL-JEM-1230 transmission electron microscope (Jeol (Beijing) Co., Ltd., Beijing, China). X-ray diffraction [XRD] patterns were obtained by using a diffractometer (PANalytical X'Pert PRO) (Spectris Instrumentation and Systems Shanghai Ltd., Shanghai, People's Republic of China) with Cu Kα radiation. The thickness of the ZnO nanocrystalline layer was measured by using a microfigure measuring instrument (Surfcorder ET3000, Kosaka Laboratory, Kosaka, Japan). The h was measured under a white light source (YSS-100A, Yamashita Denso Company, Tokyo, Japan), which gave an irradiance of 100 mW cm -2 at the equivalent of air mass [AM] 1.5 on the surface of the solar cell. The irradiance of simulated light was calibrated using a silicon photodiode (BS-520, Bunko Keiki Co., Ltd, Tokyo, Japan). The evolution of the electron transport process in the cell was investigated using electrochemical impedance spectroscopy [EIS]. Impedance spectroscopy was performed using an electrochemical analyzer (Autolab PGSTAT30) (Eco-Chemie, Utrecht, The Netherlands). The impedance measurements were carried out by applying a direct current bias at open circuit voltage [V OC ] and an alternating current voltage with amplitude of 10 mV in a frequency range from 10 -2 to 10 5 Hz under AM 1.5 illumination.

Results and discussion

The TEM images of the pristine U-MWNT and the modified MWNTs (O-MWNT and acid-MWNT) are shown in Figure 1. As shown in Figure 1a, U-MWNTs exhibited highly curved, aggregated random-coil feature, which can be attributed to the intrinsic van der Waals attractions between the individual nanotubes. The black spot on the tip of U-MWNT (Figure 1b) was not observed on O-MWNT (Figure 1d), meaning that most of the impurities had been removed by air oxidation treatment. The air oxidation treatment purified the pristine MWNTs, but did not change significantly the aspect ratio or the aggregated random-coil nature of U-MWNTs, as demonstrated by Figure 1a, c. Figure 1e, f shows that the mixed acid treatment greatly modified the structure of MWNTs and opened the tip of acid-MWNTs. Figure 1e also shows that acid-MWNTs were no longer entangled together, which could be attributed to the carboxyl layer formed on the surface of acid-MWNTs. According to the Fourier transform infrared spectroscopy spectra reported earlier in the literature, the mixed acid treatment generates a carboxyl layer on the surface of CNTs [16]. However, the mix acid treatment also shortened MWNTs and generated significantly more defect sites on acid-MWNTs.

The FE-SEM micrograph in Figure Figure 2c. It is well known that the surface of ZnO particles possesses a lot of hydroxyl groups, which can undergo interaction with the carboxylic groups on acid-MWNT surface. This should be the reason why better dispersion was achieved with acid-MWNTs. The same principle has been applied in a previous study to reduce microcrack formation on electrophoretically deposited TiO 2 film through the addition of acid-treated MWNTs [17]. Like ZnO, the surface of TiO 2 particle also possesses hydroxyl groups. However, the mechanical mixing method used in this study was probably not sufficient to form uniformly dispersed ZnO/MWNT paste, so acid-MWNTs were not uniformly distributed in the resulting composite films.

The XRD spectra of the pure ZnO film and two ZnO/ MWNT composite films are shown in  It has been reported that the incorporation of a small amount of CNTs into the TiO 2 films can enhance the collection and transport of electrons [11,12]. The DSSC containing 0.01 wt% of acid-MWNTs yielded the highest h. The peak h of 2.70% represents a 30% increase compared to that of the pure ZnO-based cell. The increase in h by the incorporation of 0.01 wt% of acid-MWNTs was attributed to a strong enhancement in J SC coupled with a slight decrease in V OC . The improvement of h for the DSSC modified by   0.01 wt% of acid-MWNTs was probably due to the decrease in the charge transport resistance at the ZnO/ dye/electrolyte interface, a larger number of injected electrons, and more efficient electron transport effected by acid-MWNTs (see Figure 5). Lower h resulted when O-MWNT or a higher percentage of acid-MWNT was used. The decline in h was most significant in DSSC containing 0.1 wt% of O-MWNT, where the aggregation of MWNT was likely to be most severe. The degradation of DSSC performance was probably caused by a loss in optical transparency at higher MWNT loadings [10,11].

To further understand the effect of MWNTs on cell performance, some of the solar cells were analyzed by EIS, and the experimental impedance data were fitted to an equivalent circuit. The experimental impedance data, obtained from DSSCs made with 8 μm-thick films, are given by the Nyquist plots in Figure 6a. The Nyquist plots in Figure 6a are the impedance spectra of the pure ZnO-based cell and the ones containing 0.01 wt% acid-MWNTs and 0.01 wt% O-MWNTs. The corresponding J SC , V OC , fill factor [FF], and conversion efficiencies of these cells are summarized in Table 1. Generally, the impedance spectra of DSSCs exhibit three semicircles. The semicircle in the high frequency range corresponds to charge transfer behavior at the Pt/electrolyte (R Pt and C Pt ), the FTO/electrolyte (R FTO and C FTO ) and the FTO/ZnO (R FZ and C FZ ) interfaces. The semicircle in the middle frequency range is assigned to the electron transfer at the ZnO/dye/electrolyte interfaces, which is related to electron-transport resistance in the ZnO network [R w ], charge-transfer resistance related to recombination of electrons at the ZnO/electrolyte interface [R k ], and the chemical capacitance of the ZnO electrode [C μ ]. The semicircle in the low frequency range reflects the Warburg diffusion process of I -/I 3 in the electrolyte (Z N ) [19][20][21]. To extract DSSC parameters related to electron transport and recombination, the experimental impedance data was fitted to an equivalent circuit based on the diffusion-recombination model [19,20], as shown in Figure 6b. The results from fitting the Nyquist plot data to the equivalent circuit are summarized in Table  2. As shown, the incorporation of MWNTs lowered the R w . The lowest R w occurred when the photoelectrode was modified with 0.1 wt% of O-MWNTs. This was likely due to the high electrical conductivity of O-MWNTs. It should be noted that acid-MWNTs have considerably lower electrical conductivity than  O-MWNTs [22], because mixed acid treatment leads to pronounced structural damages [15,22]. On the other hand, the incorporation of MWNTs also led to lower R k , which is related to the charge recombination between injected electrons and electron acceptors in the redox electrolyte at the ZnO/electrolyte interface, meaning recombination loss was more significant when photoelectrode was modified with MWNTs. This recombination loss was most severe with the O-MWNT-modified device, whose R k value was about half that of the pure ZnO-based cell. This was likely due to the severe aggregation of O-MWNT in the ZnO matrix. As shown in Figure 2c, O-MWNT formed bundles in the ZnO matrix. Therefore, only the outer layer of O-MWNTs was in contact with ZnO nanoparticles. The interior of the O-MWNT bundles was most likely exposed directly to electrolyte, resulting in the highest recombination loss. Despite their lower R k values, the MWNT-modified devices still had higher ratio of R k to R w [R k /R w ] values than the pure ZnO device, suggesting that a larger number of electrons were injected in the MWNT-containing electrodes. This result could explain the higher J SC observed in MWNT-containing cells [20,23]. The reciprocal of the characteristic frequency (1/ω max ) represents the mean electron lifetime [τ eff ]. The ω max values were found to be 97.09, 145.98, and 125.01 Hz for the ZnO, ZnO/O-MWNT, and ZnO/acid-MWNT devices, respectively. The τ eff was longer in the pure ZnO-based device compared to the MWNT-containing devices ( Table 2). This indicates that the incorporation of MWNTs increased the recombination reaction. This is consistent with the low R k values for MWNT containing cells. The effective electron diffusion coefficient in the photoanode [D eff ] is calculated by using the relation: D eff = (R k /R w )(L 2 /τ eff ), where L is the thickness of the ZnO film (8 μm). As shown in Table 2 the D eff for the MWNT-containing cells were much larger than that of the pure ZnO-based cell. The higher D eff for MWNT-modified DSSCs could be explained by more injected electrons and the induced faster transport of electrons. The effective electron diffusion length [L eff ], calculated by the relation: L eff = (D eff × τ eff ) 1/2 , expresses the competition between the collection and the recombination of electrons. The L eff values were estimated to be 35.4 μm for the ZnO/O-MWNT device and 27.1 μm for the ZnO/acid-MWNT device. The L eff in the MWNT-modified DSSCs was higher than that in the pure ZnO-based cells (22.2 μm). The higher value of L eff was consistent with the higher J SC values of the MWNT-modified cells ( Table 1).

As shown in Table 1 the ZnO/O-MWNT device had the highest J SC value, but its overall h was lower than that of the ZnO/acid-MWNT device. This was due to the low V OC value of the ZnO/O-MWNT device, which was likely resulted from the severe aggregation of O-MWNTs in the ZnO matrix. Because O-MWNTs formed bundles (Figure 2c), only the outer surface of the bundle was in contact with ZnO nanoparticles. The interior of the O-MWNT bundle was most likely exposed directly to electrolyte, resulting in the highest recombination loss (lowest R k as shown in Table 2) and consequently the lowest V OC . Therefore, despite its highest J SC , the ZnO/O-MWNT device had lower h than the ZnO/acid-MWNT cell.  The thickness of the ZnO or ZnO/MWNT film was 8 μm.

Conclusions

DSSCs based on ZnO/MWNT composite films were fabricated using a low-temperature, direct blending procedure and compared with the pure ZnO nanoparticlebased cells. The cell performance was found to depend on the loading and surface modification of MWNTs. At a loading of 0.01 wt%, both O-MWNT and acid-MWNT could enhance the h of DSSCs, which was attributed to higher J SC values. However, the acid-MWNT modified cell had higher h. This is because the acid-MWNTs were disentangled and better dispersed in the ZnO matrix, leading to less recombination loss as compared to the O-MWNTs containing cells. The solar cell containing 0.01 wt% of acid-MWNT yielded the highest h, which was characterized by the following parameters: J SC = 5.68 mA/cm 2 , V OC = 0.66 V, FF = 0.72, and h = 2.70%.

2a reveals the surface morphology of the ZnO/acid-MWNT film with exfoliated MWNTs visible on the surface. The cross-sectional view of the ZnO/acid-MWNT film, given in Figure 2b, shows that acid-MWNTs were embedded in the ZnO nanoparticle network. Although the acid-MWNTs were not uniformly distributed, they did not form bundles like O-MWNTs, as shown in

Figure 3 .
3All three films exhibited peaks at 2θ = 31.7°, 34.5°, 36.3°, 47.4°, 56.6°, 62.7°, and 68.2°, which correspond to the (100), (002), (101), (102), (110), (103), and (201) planes of ZnO (Joint Committee on Powder Diffraction Standards 36-1451), respectively[18]. However, the characteristic peak of MWNT (2θ = 26.3°) was not observed in the XRD spectra. The amount of MWNT in the composite films might be too small to generate visible peaks for MWNTs.Comparison of the J-V characteristics of pure ZnO film-based and ZnO/MWNT composite film-based DSSCs is presented inFigure 4. The photoelectric conversion performances of the ZnO/MWNT film-based DSSCs depended strongly on the type and loading of MWNTs in the electrodes. The value of short-circuit

Figure 1
1TEM images of MWNTs. (a) and (b) U-MWNTs; (c) and (d) O-MWNTs; (e) and (f) acid-MWNTs. [J SC ] was greatly increased when 0.01 wt% of O-MWNTs or acid-MWNTs were incorporated in the electrode.

Figure 2
2FE-SEM micrographs of ZnO/MWNT composite film containing 0.01 wt% of MWNTs. (a) top view and (b) crosssectional view of ZnO/acid-MWNT film; (c) cross-sectional view of ZnO/O-MWNT film.

Figure 3
3XRD spectra. (a) MWNT; (b) FTO glass substrate; (c) ZnO; (d) ZnO/O-MWNT film; (e) ZnO/acid-MWNT film. (M, MWNT; S, FTO substrate (SnO 2 ); Z, ZnO).

Figure 4 J
4-V characteristics of fabricated DSSCs.

Figure 5
5Proposed mechanism for enhanced electron transport in ZnO/MWNT composite electrodes. CB, conduction band; VB, valence band.

Figure 6
6Equivalent circuit and Nyquist plots. (a) Equivalent circuit for the simulation of DSSC impedance spectra. (b) Nyquist plots of fabricated DSSCs determined under an AM 1.5 G solar illumination of 100 mW cm -2 . MWNT loading was 0.01 wt%, and the film thickness was 8 μm.

acid-MWNT: group-modified MWNT; CNT: carbon nanotube; DSSC: dyesensitized solar cells; MWNTs: multiwalled carbon nanotubes; EIS: electrochemical impedance spectroscopy; FE-SEM: field emission scanning electron microscopy; FF: fill factor; FTO: fluorine-doped tin oxide; J SC : shortcircuit photocurrent density; O-MWNT: oxidized MWNT; TEM: transmission electron microscopy; U-MWNT: unmodified-MWNT; V OC : open circuit voltage; XRD: X-ray diffraction.

Table 1
1Effects of incorporating MWNTs on J-V characteristics of DSSCsThe thickness of the ZnO or ZnO/MWNT film was 8 μm.Electrode composition
Short-circuit photocurrent
density (mA/cm 2 )

Open circuit voltage (volt)
Fill factor
Energy conversion
efficiency (percent)

ZnO
4.54
0.68
0.68
2.11

ZnO/0.01 wt% O-MWNT
5.90
0.60
0.66
2.33
ZnO/0.01 wt% acid-MWNT
5.68
0.66
0.72
2.70

Table 2
2Effects of incorporating MWNTs on the electron transport properties of fabricated cellsElectrode
composition

Charge-transfer resistance
at the ZnO/electrolyte
interface [R k ] (ohm)

Electron transport
resistance in the ZnO
network [R w ] (ohm)

Ratio
of R k
to R w

Mean
electron
lifetime
(ms)

Electron diffusion
coefficient in the
photoanode
(10 -4 cm 2 s -1 ×)

Effective electron
diffusion length
(micrometer)

ZnO
50.17
6.49
7.73
10.30
4.80
22.2

ZnO/0.01 wt%
O-MWNT

27.15
1.39
19.53
6.85
18.25
35.4

ZnO/0.01 wt%
acid-MWNT

41.56
3.62
11.48
8.00
9.18
27.1

AcknowledgementsThe authors acknowledge the financial support from Bureau of Energy, Ministry of Economic Affairs, Taiwan (project no. A455DR2110). The authors also thank Prof Chung-Wen Lan at the Department of Chemical Engineering, National Taiwan University for the instrument support.Authors' contributions WCY proposed the idea, helped in the manuscript preparation, and oversaw the study. WCC designed and carried out the experiment, analyzed the data, and prepared the manuscript. YYC helped define the research theme and experimental design. YCY carried out the fabrication of ZnO/MWNT DSSCs. CHL provided a helpful discussion and helped in the manuscript preparation. HHK contributed to the preparation of MWNT. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
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