Engineered multiple translation initiation sites: a novel tool to enhance protein production in Bacillus licheniformis and other industrially relevant bacteria.

Gram-positive bacteria are a nascent platform for synthetic biology and metabolic engineering that can provide new opportunities for the production of biomolecules. However, the lack of standardized methods and genetic parts is a major obstacle towards attaining the acceptance and widespread use of Gram-positive bacterial chassis for industrial bioproduction. In this study, we have engineered a novel mRNA leader sequence containing more than one ribosomal binding site (RBS) which could initiate translation from multiple sites, vastly enhancing the translation efficiency of the Gram-positive industrial strain Bacillus licheniformis. This is the first report elucidating the impact of more than one RBS to initiate translation and enhance protein output in B. licheniformis. We also explored the application of more than one RBS for both intracellular and extracellular protein production in B. licheniformis to demonstrate its efficiency, consistency and potential for biotechnological applications. Moreover, we applied these concepts for use in other industrially relevant Gram-positive bacteria, such as Bacillus subtilis and Corynebacterium glutamicum. In all, a highly efficient and robust broad-host expression element has been designed to strengthen and fine-tune the protein outputs for the use of bioproduction in microbial cell factories.

ZSWIM1 Promotes the Proliferation and Metastasis of Lung Adenocarcinoma Cells through the STK38/MEKK2/ERK1/2 Axis.

Investigating the functions of the proteins with no or less functional annotations is an important goal of the HPP (Human Proteome Project) Grand Project. In this study, we investigated the function of such a protein, ZSWIM1 (C20orf162), its gene located on chromosome 20. Its expression is upregulated in lung adenocarcinoma compared with the adjacent normal tissues and negatively correlated with the overall survival. Overexpressing ZSWIM1 markedly promotes the proliferation, migration, invasion as well as epithelial-to-mesenchymal transition in lung adenocarcinoma cells, while knocking down ZSWIM1 functions oppositely. The interactome of ZSWIM1 was identified by immunoprecipitation-mass spectrometry, and we verified the interaction of ZSWIM1 with the potential partner, STK38. ZSWIM1 antagonized the function of STK38. Mechanically, ZSWIM1 promoted the activation of MEKK2/ERK1/2 pathway through interacting with STK38, leading to the release of MEKK2. Taken together, ZSWIM1 can be annotated as an oncogene in lung adenocarcinoma, and the STK38/MEKK2/ERK1/2 axis mediates its promoting role in lung adenocarcinoma.

TranslatomeDB: a comprehensive database and cloud-based analysis platform for translatome sequencing data.

Translation is a key regulatory step, linking transcriptome and proteome. Two major methods of translatome investigations are RNC-seq (sequencing of translating mRNA) and Ribo-seq (ribosome profiling). To facilitate the investigation of translation, we built a comprehensive database TranslatomeDB (http://www.translatomedb.net/) which provides collection and integrated analysis of published and user-generated translatome sequencing data. The current version includes 2453 Ribo-seq, 10 RNC-seq and their 1394 corresponding mRNA-seq datasets in 13 species. The database emphasizes the analysis functions in addition to the dataset collections. Differential gene expression (DGE) analysis can be performed between any two datasets of same species and type, both on transcriptome and translatome levels. The translation indices translation ratios, elongation velocity index and translational efficiency can be calculated to quantitatively evaluate translational initiation efficiency and elongation velocity, respectively. All datasets were analyzed using a unified, robust, accurate and experimentally-verifiable pipeline based on the FANSe3 mapping algorithm and edgeR for DGE analyzes. TranslatomeDB also allows users to upload their own datasets and utilize the identical unified pipeline to analyze their data. We believe that our TranslatomeDB is a comprehensive platform and knowledgebase on translatome and proteome research, releasing the biologists from complex searching, analyzing and comparing huge sequencing data without needing local computational power.

Genome-Wide and Experimental Resolution of Relative Translation Elongation Speed at Individual Gene Level in Human Cells.

In the process of translation, ribosomes first assemble on mRNAs (translation initiation) and then translate along the mRNA (elongation) to synthesize proteins. Elongation pausing is deemed highly relevant to co-translational folding of nascent peptides and the functionality of protein products, which positioned the evaluation of elongation speed as one of the central questions in the field of translational control. By integrating three types of RNA-seq methods, we experimentally and computationally resolved elongation speed, with our proposed elongation velocity index (EVI), a relative measure at individual gene level and under physiological condition in human cells. We successfully distinguished slow-translating genes from the background translatome. We demonstrated that low-EVI genes encoded more stable proteins. We further identified cell-specific slow-translating codons, which might serve as a causal factor of elongation deceleration. As an example for the biological relevance, we showed that the relatively slow-translating genes tended to be associated with the maintenance of malignant phenotypes per pathway analyses. In conclusion, EVI opens a new view to understand why human cells tend to avoid simultaneously speeding up translation initiation and decelerating elongation, and the possible cancer relevance of translating low-EVI genes to gain better protein quality.

Steady-state structural fluctuation is a predictor of the necessity of pausing-mediated co-translational folding for small proteins.

Translational pausing coordinates protein synthesis and co-translational folding. It is a common factor that facilitates the correct folding of large, multi-domain proteins. For small proteins, pausing sites rarely occurs in the gene body, and the 3'-end pausing sites are only essential for the folding of a fraction of proteins. The determinant of the necessity of the pausings remains obscure. In this study, we demonstrated that the steady-state structural fluctuation is a predictor of the necessity of pausing-mediated co-translational folding for small proteins. Validated by experiments with 5 model proteins, we found that the rigid protein structures do not, while the flexible structures do need 3'-end pausings to fold correctly. Therefore, rational optimization of translational pausing can improve soluble expression of small proteins with flexible structures, but not the rigid ones. The rigidity of the structure can be quantitatively estimated in silico using molecular dynamic simulation. Nevertheless, we also found that the translational pausing optimization increases the fitness of the expression host, and thus benefits the recombinant protein production, independent from the soluble expression. These results shed light on the structural basis of the translational pausing and provided a practical tool for industrial protein fermentation.

Differential protein analysis of serum exosomes post-intravenous immunoglobulin therapy in patients with Kawasaki disease.

BACKGROUND: Kawasaki disease, which is characterised by systemic vasculitides accompanied by acute fever, is regularly treated by intravenous immunoglobulin to avoid lesion formation in the coronary artery; however, the mechanism of intravenous immunoglobulin therapy is unclear. Hence, we aimed to analyse the global expression profile of serum exosomal proteins before and after administering intravenous immunoglobulin. METHODS: Two-dimensional electrophoresis coupled with mass spectrometry analysis was used to identify the differentially expressed proteome of serum exosomes in patients with Kawasaki disease before and after intravenous immunoglobulin therapy. RESULTS: Our analysis revealed 69 differential protein spots in the Kawasaki disease group with changes larger than 1.5-fold and 59 differential ones in patients after intravenous immunoglobulin therapy compared with the control group. Gene ontology analysis revealed that the acute-phase response disappeared, the functions of the complement system and innate immune response were enhanced, and the antibacterial humoral response pathway of corticosteroids and cardioprotection emerged after administration of intravenous immunoglobulin. Further, we showed that complement C3 and apolipoprotein A-IV levels increased before and decreased after intravenous immunoglobulin therapy and that the insulin-like growth factor-binding protein complex acid labile subunit displayed reverse alteration before and after intravenous immunoglobulin therapy. These observations might be potential indicators of intravenous immunoglobulin function. CONCLUSIONS: Our results show the differential proteomic profile of serum exosomes of patients with Kawasaki disease before and after intravenous immunoglobulin therapy, such as complement C3, apolipoprotein A-IV, and insulin-like growth factor-binding protein complex acid labile subunit. These results may be useful in the identification of markers for monitoring intravenous immunoglobulin therapy in patients with Kawasaki disease.

Rapid magnetic solid-phase extraction of Congo Red and Basic Red 2 from aqueous solution by ZIF-8@CoFe2 O4 hybrid composites.

Core-shell metal-organic framework materials have attracted considerable attention mainly due to their enhanced or new physicochemical properties compared with their single-component counterparts. In this work, a core-shell heterostructure of CoFe2 O4 -Zeolitic Imidazolate Framework-8 (ZIF-8@CoFe2 O4 ) is successfully fabricated and used as an solid-phase extraction adsorbent to efficiently extract Congo Red and Basic Red 2 dyes from contaminated aqueous solution. Vibrating sample magnetometry indicates that the saturated magnetization of ZIF-8@CoFe2 O4 is 3.3 emu/g, which is large enough for magnetic separation. The obtained hybrid magnetic metal-organic framework based material ZIF-8@CoFe2 O4 can remove the investigated dyes very fast within 1 min of the contact time. The adsorbent ZIF-8@CoFe2 O4 also shows a good reusability. After regeneration, the adsorbent can still exhibit high removal efficiency (∼97%) toward Congo Red for five cycles of desorption-adsorption. This work reveals the great potential of core-shell ZIF-8@CoFe2 O4 sorbents for the fast separation and preconcentration of organic pollutants in aqueous solution before high-performance liquid chromatography analysis.

Translating mRNAs strongly correlate to proteins in a multivariate manner and their translation ratios are phenotype specific.

As a well-known phenomenon, total mRNAs poorly correlate to proteins in their abundances as reported. Recent findings calculated with bivariate models suggested even poorer such correlation, whereas focusing on the translating mRNAs (ribosome nascent-chain complex-bound mRNAs, RNC-mRNAs) subset. In this study, we analysed the relative abundances of mRNAs, RNC-mRNAs and proteins on genome-wide scale, comparing human lung cancer A549 and H1299 cells with normal human bronchial epithelial (HBE) cells, respectively. As discovered, a strong correlation between RNC-mRNAs and proteins in their relative abundances could be established through a multivariate linear model by integrating the mRNA length as a key factor. The R(2) reached 0.94 and 0.97 in A549 versus HBE and H1299 versus HBE comparisons, respectively. This correlation highlighted that the mRNA length significantly contributes to the translational modulation, especially to the translational initiation, favoured by its correlation with the mRNA translation ratio (TR) as observed. We found TR is highly phenotype specific, which was substantiated by both pathway analysis and biased TRs of the splice variants of BDP1 gene, which is a key transcription factor of transfer RNAs. These findings revealed, for the first time, the intrinsic and genome-wide translation modulations at translatomic level in human cells at steady-state, which are tightly correlated to the protein abundance and functionally relevant to cellular phenotypes.

Experimental assessment of robust reference genes for qRT-PCR in lung cancer studies.

Stable internal reference genes are crucial for quantitative real-time PCR (qRT-PCR) analyses in lung cancer studies. Widely used reference genes are mostly chosen by intuition or from pan-cancer transcriptome data and lack experimental validation by qRT-PCR in the context of lung cancer. This study evaluated the stability of candidate reference genes in lung cancer cell lines under normal homeostasis, hypoxia, and serum deprivation to screen for robust reference genes for qRT-PCR in lung cancer studies. The stability of reference gene combinations was also assessed. We found that most of the stably expressed genes from pan-cancer transcriptome analyses were not sufficiently stable under some of the tested conditions. CIAO1, CNOT4, and SNW1 were found to be the most stable reference genes under various conditions. Greater stability was achieved by combining more reference genes. We further used the hypoxia biomarker hypoxia-inducible factor (HIF)-2α to demonstrate that choosing inappropriate reference genes can lead to incorrect qRT-PCR results. We also found that the stable reference genes were irrelevant to malignancy, which may explain their stability under various conditions that cancer cells often encounter. This study provides a list of validated and stable qRT-PCR reference genes and reference gene combinations for lung cancer that may standardize qRT-PCR experiments in future lung cancer studies.

A multi-omics dataset of human transcriptome and proteome stable reference.

The development of high-throughput omics technology has greatly promoted the development of biomedicine. However, the poor reproducibility of omics techniques limits their application. It is necessary to use standard reference materials of complex RNAs or proteins to test and calibrate the accuracy and reproducibility of omics workflows. The transcriptome and proteome of most cell lines shift during culturing, which limits their applicability as standard samples. In this study, we demonstrated that the human hepatocellular cell line MHCC97H has a very stable transcriptome (r = 0.983~0.997) and proteome (r = 0.966~0.988 for data-dependent acquisition, r = 0.970~0.994 for data-independent acquisition) after 9 subculturing generations, which allows this steady standard sample to be consistently produced on an industrial scale in long term. Moreover, this stability was maintained across labs and platforms. In sum, our study provides omics standard reference material and reference datasets for transcriptomic and proteomics research. This helps to further standardize the workflow and data quality of omics techniques and thus promotes the application of omics technology in precision medicine.

Towards an accurate and robust analysis pipeline for somatic mutation calling.

Accurate and robust somatic mutation detection is essential for cancer treatment, diagnostics and research. Various analysis pipelines give different results and thus should be systematically evaluated. In this study, we benchmarked 5 commonly-used somatic mutation calling pipelines (VarScan, VarDictJava, Mutect2, Strelka2 and FANSe) for their precision, recall and speed, using standard benchmarking datasets based on a series of real-world whole-exome sequencing datasets. All the 5 pipelines showed very high precision in all cases, and high recall rate in mutation rates higher than 10%. However, for the low frequency mutations, these pipelines showed large difference. FANSe showed the highest accuracy (especially the sensitivity) in all cases, and VarScan and VarDictJava outperformed Mutect2 and Strelka2 in low frequency mutations at all sequencing depths. The flaws in filter was the major cause of the low sensitivity of the four pipelines other than FANSe. Concerning the speed, FANSe pipeline was 8.8∼19x faster than the other pipelines. Our benchmarking results demonstrated performance of the somatic calling pipelines and provided a reference for a proper choice of such pipelines in cancer applications.

Efficient Detection of the Alternative Spliced Human Proteome Using Translatome Sequencing.

Alternative splicing (AS) isoforms create numerous proteoforms, expanding the complexity of the genome. Highly similar sequences, incomplete reference databases and the insufficient sequence coverage of mass spectrometry limit the identification of AS proteoforms. Here, we demonstrated full-length translating mRNAs (ribosome nascent-chain complex-bound mRNAs, RNC-mRNAs) sequencing (RNC-seq) strategy to sequence the entire translating mRNA using next-generation sequencing, including short-read and long-read technologies, to construct a protein database containing all translating AS isoforms. Taking the advantage of read length, short-read RNC-seq identified up to 15,289 genes and 15,906 AS isoforms in a single human cell line, much more than the Ribo-seq. The single-molecule long-read RNC-seq supplemented 4,429 annotated AS isoforms that were not identified by short-read datasets, and 4,525 novel AS isoforms that were not included in the public databases. Using such RNC-seq-guided database, we identified 6,766 annotated protein isoforms and 50 novel protein isoforms in mass spectrometry datasets. These results demonstrated the potential of full-length RNC-seq in investigating the proteome of AS isoforms.

Identifying a 6-Gene Prognostic Signature for Lung Adenocarcinoma Based on Copy Number Variation and Gene Expression Data.

The occurrence of lung adenocarcinoma (LUAD) is a complicated process, involving the genetic and epigenetic changes of proto-oncogenes and oncogenes. The objective of this study was to establish new predictive signatures of lung adenocarcinoma based on copy number variations (CNVs) and gene expression data. Next-generation sequencing was implemented to obtain gene expression and CNV information. According to univariate, multivariate survival Cox regression analysis, and LASSO analysis, the expression profiles of lung adenocarcinoma patients were screened and a risk score formula was established and experimentally validated in a local cohort. The model was evaluated by three independent cohorts (TCGA-LUAD, GSE31210, and GSE30219), and then validated by clinical samples from LUAD patients. A total of 844 CNV-related differentially expressed genes (CNV-related DEGs) were identified. These genes are significantly associated with the imbalance of various oxidative stress pathways. A CNV-associated-six gene signature was dramatically linked to overall survival in lung adenocarcinoma samples from both training and validation groups. Functional enrichment analysis further revealed involvement of genes in p53 signaling pathway and cell cycle as well as the mismatch repair pathway. Risk score is an independent marker considering clinical parameters and had better prediction in clinical subpopulation. The same signature also classified tumor tissues of clinical patients with CNV detected from their corresponding nontumorous tissues with an accuracy of 0.92. In conclusion, we identified a new class of 6 CNV-related gene markers that may act as efficient prognostic predictors of lung adenocarcinoma, thus contributing to individualized treatment decisions in patients.

The Ultrafast and Accurate Mapping Algorithm FANSe3: Mapping a Human Whole-Genome Sequencing Dataset Within 30 Minutes.

Aligning billions of reads generated by the next-generation sequencing (NGS) to reference sequences, termed "mapping", is the time-consuming and computationally-intensive process in most NGS applications. A Fast, accurate and robust mapping algorithm is highly needed. Therefore, we developed the FANSe3 mapping algorithm, which can map a 30 × human whole-genome sequencing (WGS) dataset within 30 min, a 50 × human whole exome sequencing (WES) dataset within 30 s, and a typical mRNA-seq dataset within seconds in a single-server node without the need for any hardware acceleration feature. Like its predecessor FANSe2, the error rate of FANSe3 can be kept as low as 10-9 in most cases, this is more robust than the Burrows-Wheeler transform-based algorithms. Error allowance hardly affected the identification of a driver somatic mutation in clinically relevant WGS data and provided robust gene expression profiles regardless of the parameter settings and sequencer used. The novel algorithm, designed for high-performance cloud-computing after infrastructures, will break the bottleneck of speed and accuracy in NGS data analysis and promote NGS applications in various fields. The FANSe3 algorithm can be downloaded from the website: http://www.chi-biotech.com/fanse3/.

Enhancing co-translational folding of heterologous protein by deleting non-essential ribosomal proteins in Pichia pastoris.

BACKGROUND: Translational regulation played an important role in the correct folding of heterologous proteins to form bioactive conformations during biogenesis. Translational pausing coordinates protein translation and co-translational folding. Decelerating translation elongation speed has been shown to improve the soluble protein yield when expressing heterologous proteins in industrial expression hosts. However, rational redesign of translational pausing via synonymous mutations may not be feasible in many cases. Our goal was to develop a general and convenient strategy to improve heterologous protein synthesis in Pichia pastoris without mutating the expressed genes. RESULTS: Here, a large-scale deletion library of ribosomal protein (RP) genes was constructed for heterologous protein expression in Pichia pastoris, and 59% (16/27) RP deletants have significantly increased heterologous protein yield. This is due to the delay of 60S subunit assembly by deleting non-essential ribosomal protein genes or 60S subunit processing factors, thus globally decreased the translation elongation speed and improved the co-translational folding, without perturbing the relative transcription level and translation initiation. CONCLUSION: Global decrease in the translation elongation speed by RP deletion enhanced co-translational folding efficiency of nascent chains and decreased protein aggregates to improve heterologous protein yield. A potential expression platform for efficient pharmaceutical proteins and industrial enzymes production was provided without synonymous mutation.

Low-cost, Low-bias and Low-input RNA-seq with High Experimental Verifiability based on Semiconductor Sequencing.

Low-input RNA-seq is powerful to represent the gene expression profiles with limited number of cells, especially when single-cell variations are not the aim. However, pre-amplification-based and molecule index-based library construction methods boost bias or require higher throughput. Here we demonstrate a simple, low-cost, low-bias and low-input RNA-seq with ion torrent semiconductor sequencing (LIEA RNA-seq). We also developed highly accurate and error-tolerant spliced mapping algorithm FANSe2splice to accurately map the single-ended reads to the reference genome with better experimental verifiability than the previous spliced mappers. Combining the experimental and computational advancements, our solution is comparable with the bulk mRNA-seq in quantification, reliably detects splice junctions and minimizes the bias with much less mappable reads.

New Early Cretaceous palaeomagnetic and geochronological results from the far western Lhasa terrane: Contributions to the Lhasa-Qiangtang collision.

To better constrain the Lhasa-Qiangtang collision, a combined palaeomagnetic and geochronological study of the far western Lhasa terrane was conducted on the Duoai Formation lava flows (~113-116 Ma), as well as on the Early Cretaceous Jiega Formation limestone. Following detailed rock magnetic, petrographical, and palaeomagnetic experiments, characteristic remanent magnetisation directions were successfully isolated from most samples using principal component analysis. The tilt-corrected direction groups yielded a palaeopole at 69.1°N, 319.8°E with A95 = 4.8° (N = 19). A primary origin for the magnetisation is consistent with positive fold tests. Our results from the Early Cretaceous units, combined with published palaeomagnetic data obtained from Cretaceous strata from the Lhasa and western Qiangtang terranes, show that these two terranes had already collided by the Early Cretaceous, the Lhasa terrane had a relatively east-west alignment, and it remained at a relatively stable palaeolatitude during the entire Cretaceous. Comparing the Cretaceous palaeolatitude calculated for the western Lhasa terrane with those from Eurasia and Mongolia suggests a latitudinal convergence of ~1400 ± 290 km and ~1800 ± 300 km, respectively, since the Early Cretaceous.

Early Cretaceous paleomagnetic and geochronologic results from the Tethyan Himalaya: Insights into the Neotethyan paleogeography and the India-Asia collision.

To better understand the Neotethyan paleogeography, a paleomagnetic and geochronological study has been performed on the Early Cretaceous Sangxiu Formation lava flows, which were dated from ~135.1 Ma to ~124.4 Ma, in the Tethyan Himalaya. The tilt-corrected site-mean characteristic remanent magnetization (ChRM) direction for 26 sites is Ds = 296.1°, Is = -65.7°, ks = 51.7, α95 = 4.0°, corresponding to a paleopole at 5.9°S, 308.0°E with A95 = 6.1°. Positive fold and reversal tests prove that the ChRM directions are prefolding primary magnetizations. These results, together with reliable Cretaceous-Paleocene paleomagnetic data observed from the Tethyan Himalaya and the Lhasa terrane, as well as the paleolatitude evolution indicated by the apparent polar wander paths (APWPs) of India, reveal that the Tethyan Himalaya was a part of Greater India during the Early Cretaceous (135.1-124.4 Ma) when the Neotethyan Ocean was up to ~6900 km, it rifted from India sometime after ~130 Ma, and that the India-Asia collision should be a dual-collision process including the first Tethyan Himalaya-Lhasa terrane collision at ~54.9 Ma and the final India-Tethyan Himalaya collision at ~36.7 Ma.

Rational design of translational pausing without altering the amino acid sequence dramatically promotes soluble protein expression: a strategic demonstration.

The production of many pharmaceutical and industrial proteins in prokaryotic hosts is hindered by the insolubility of industrial expression products resulting from misfolding. Even with a correct primary sequence, an improper translation elongation rate in a heterologous expression system is an important cause of misfolding. In silico analysis revealed that most of the endogenous Escherichia coli genes display translational pausing sites that promote correct folding, and almost 1/5 genes have pausing sites at the 3'-termini of their coding sequence. Therefore, we established a novel strategy to efficiently promote the expression of soluble and active proteins without altering the amino acid sequence or expression conditions. This strategy uses the rational design of translational pausing based on structural information solely through synonymous substitutions, i.e. no change on the amino acids sequence. We demonstrated this strategy on a promising antiviral candidate, Cyanovirin-N (CVN), which could not be efficiently expressed in any previously reported system. By introducing silent mutations, we increased the soluble expression level in E. coli by 2000-fold without altering the CVN protein sequence, and the specific activity was slightly higher for the optimized CVN than for the wild-type variant. This strategy introduces new possibilities for the production of bioactive recombinant proteins.

FANSe2: a robust and cost-efficient alignment tool for quantitative next-generation sequencing applications.

Correct and bias-free interpretation of the deep sequencing data is inevitably dependent on the complete mapping of all mappable reads to the reference sequence, especially for quantitative RNA-seq applications. Seed-based algorithms are generally slow but robust, while Burrows-Wheeler Transform (BWT) based algorithms are fast but less robust. To have both advantages, we developed an algorithm FANSe2 with iterative mapping strategy based on the statistics of real-world sequencing error distribution to substantially accelerate the mapping without compromising the accuracy. Its sensitivity and accuracy are higher than the BWT-based algorithms in the tests using both prokaryotic and eukaryotic sequencing datasets. The gene identification results of FANSe2 is experimentally validated, while the previous algorithms have false positives and false negatives. FANSe2 showed remarkably better consistency to the microarray than most other algorithms in terms of gene expression quantifications. We implemented a scalable and almost maintenance-free parallelization method that can utilize the computational power of multiple office computers, a novel feature not present in any other mainstream algorithm. With three normal office computers, we demonstrated that FANSe2 mapped an RNA-seq dataset generated from an entire Illunima HiSeq 2000 flowcell (8 lanes, 608 M reads) to masked human genome within 4.1 hours with higher sensitivity than Bowtie/Bowtie2. FANSe2 thus provides robust accuracy, full indel sensitivity, fast speed, versatile compatibility and economical computational utilization, making it a useful and practical tool for deep sequencing applications. FANSe2 is freely available at http://bioinformatics.jnu.edu.cn/software/fanse2/.