{ "cells": [ { "cell_type": "code", "execution_count": 1, "id": "35f9681a", "metadata": { "execution": { "iopub.execute_input": "2025-03-25T06:47:02.803166Z", "iopub.status.busy": "2025-03-25T06:47:02.802919Z", "iopub.status.idle": "2025-03-25T06:47:02.968091Z", "shell.execute_reply": "2025-03-25T06:47:02.967707Z" } }, "outputs": [], "source": [ "import sys\n", "import os\n", "sys.path.append(os.path.abspath(os.path.join(os.getcwd(), '../..')))\n", "\n", "# Path Configuration\n", "from tools.preprocess import *\n", "\n", "# Processing context\n", "trait = \"Atrial_Fibrillation\"\n", "cohort = \"GSE143924\"\n", "\n", "# Input paths\n", "in_trait_dir = \"../../input/GEO/Atrial_Fibrillation\"\n", "in_cohort_dir = \"../../input/GEO/Atrial_Fibrillation/GSE143924\"\n", "\n", "# Output paths\n", "out_data_file = \"../../output/preprocess/Atrial_Fibrillation/GSE143924.csv\"\n", "out_gene_data_file = \"../../output/preprocess/Atrial_Fibrillation/gene_data/GSE143924.csv\"\n", "out_clinical_data_file = \"../../output/preprocess/Atrial_Fibrillation/clinical_data/GSE143924.csv\"\n", "json_path = \"../../output/preprocess/Atrial_Fibrillation/cohort_info.json\"\n" ] }, { "cell_type": "markdown", "id": "7772aced", "metadata": {}, "source": [ "### Step 1: Initial Data Loading" ] }, { "cell_type": "code", "execution_count": 2, "id": "8c22bb2e", "metadata": { "execution": { "iopub.execute_input": "2025-03-25T06:47:02.969520Z", "iopub.status.busy": "2025-03-25T06:47:02.969373Z", "iopub.status.idle": "2025-03-25T06:47:03.016747Z", "shell.execute_reply": "2025-03-25T06:47:03.016422Z" } }, "outputs": [ { "name": "stdout", "output_type": "stream", "text": [ "Background Information:\n", "!Series_title\t\"Transcriptome analysis from human epicardial adipose tissue biopsies analyzed according to postoperative atrial fibrillation occurrence after cardiac surgery\"\n", "!Series_summary\t\"Introduction: Post-operative atrial fibrillation (POAF) is a frequent complication after cardiac surgery, but its pathophysiology remains incompletely understood. Considering that epicardial adipose tissue (EAT) is in close vicinity with the atrial myocardium, we hypothesized that a specific pre-operative EAT phenotype would be associated to POAF onset following surgery. Methods: Patients undergoing cardiac surgery prospectively enrolled in the POMI-AF cohort between February 2016 and June 2017 were studied. EAT samples were collected at the beginning of surgery. Whole-tissue gene expression patterns and the stromal and vascular fraction (SVF) cellular composition were explored. Patients were followed after surgery by continuous ECG to detect POAF onset. Results: Among the 60 patients included in the cohort, 15 POAF and 15 non-POAF patients were matched based on pre-operative characteristics. Gene set enrichment analysis of transcriptomic data from pre-operative EAT samples revealed 40 enriched biological processes in POAF vs non-POAF patients. Most of these processes were related to cellular immune response. Leukocytes (63±15% of total cells), and more specifically lymphocytes (56±13% of total CD45+ cells), represented the major cell subset in the preoperative EAT SVF, with no quantitative differences between POAF and SR patients (76 [52; 84]% vs 56 [50; 64]%, p=0.22). However, POAF patients presented a significantly higher cytotoxic CD8+/helper CD4+ T lymphocyte ratio than SR patients (respectively, 0.69[0.55; 1.19] vs 0.50 [0.31; 0.54], p=0.03) suggesting a cytotoxic shift prior to surgery. Conclusion: Epicardial fat from patients who develop POAF displays a specific pre-operative transcriptome signature characteristic of cellular immune response and cytotoxic lymphocyte enrichment.\"\n", "!Series_overall_design\t\"30 matched samples analyzed (15 POAF vs 15 SR patients)\"\n", "Sample Characteristics Dictionary:\n", "{0: ['tissue: epicardial adipose tissue'], 1: ['patient diagnosis: sinus rhythm after surgery', 'patient diagnosis: postoperative atrial fibrillation after surgery (POAF)']}\n" ] } ], "source": [ "from tools.preprocess import *\n", "# 1. Identify the paths to the SOFT file and the matrix file\n", "soft_file, matrix_file = geo_get_relevant_filepaths(in_cohort_dir)\n", "\n", "# 2. Read the matrix file to obtain background information and sample characteristics data\n", "background_prefixes = ['!Series_title', '!Series_summary', '!Series_overall_design']\n", "clinical_prefixes = ['!Sample_geo_accession', '!Sample_characteristics_ch1']\n", "background_info, clinical_data = get_background_and_clinical_data(matrix_file, background_prefixes, clinical_prefixes)\n", "\n", "# 3. Obtain the sample characteristics dictionary from the clinical dataframe\n", "sample_characteristics_dict = get_unique_values_by_row(clinical_data)\n", "\n", "# 4. Explicitly print out all the background information and the sample characteristics dictionary\n", "print(\"Background Information:\")\n", "print(background_info)\n", "print(\"Sample Characteristics Dictionary:\")\n", "print(sample_characteristics_dict)\n" ] }, { "cell_type": "markdown", "id": "9963ba7d", "metadata": {}, "source": [ "### Step 2: Dataset Analysis and Clinical Feature Extraction" ] }, { "cell_type": "code", "execution_count": 3, "id": "10b2caf3", "metadata": { "execution": { "iopub.execute_input": "2025-03-25T06:47:03.017859Z", "iopub.status.busy": "2025-03-25T06:47:03.017749Z", "iopub.status.idle": "2025-03-25T06:47:03.024614Z", "shell.execute_reply": "2025-03-25T06:47:03.024290Z" } }, "outputs": [ { "name": "stdout", "output_type": "stream", "text": [ "Preview of selected clinical features:\n", "{'characteristic_1': [0.0], 'characteristic_2': [1.0]}\n", "Clinical data saved to ../../output/preprocess/Atrial_Fibrillation/clinical_data/GSE143924.csv\n" ] } ], "source": [ "# Define variables and conversion functions for dataset analysis\n", "\n", "# 1. Gene Expression Data Availability\n", "# Based on the description, this dataset contains transcriptome analysis data\n", "is_gene_available = True\n", "\n", "# 2. Variable Availability and Data Type Conversion\n", "# 2.1 Data Availability\n", "# For trait (AF): identified in row 1 of characteristics dictionary\n", "trait_row = 1\n", "# Age and gender information is not provided in the sample characteristics\n", "age_row = None\n", "gender_row = None\n", "\n", "# 2.2 Data Type Conversion Functions\n", "def convert_trait(x):\n", " \"\"\"Convert trait data to binary format (0: no AF, 1: AF)\"\"\"\n", " if ':' in x:\n", " value = x.split(':', 1)[1].strip()\n", " if 'sinus rhythm' in value.lower():\n", " return 0 # No atrial fibrillation\n", " elif 'atrial fibrillation' in value.lower() or 'poaf' in value.lower():\n", " return 1 # Has atrial fibrillation\n", " return None\n", "\n", "def convert_age(x):\n", " \"\"\"Convert age data to continuous format\"\"\"\n", " # Not applicable for this dataset\n", " return None\n", "\n", "def convert_gender(x):\n", " \"\"\"Convert gender data to binary format (0: female, 1: male)\"\"\"\n", " # Not applicable for this dataset\n", " return None\n", "\n", "# 3. Save Metadata\n", "# Initial filtering on dataset usability\n", "is_trait_available = trait_row is not None\n", "validate_and_save_cohort_info(\n", " is_final=False,\n", " cohort=cohort,\n", " info_path=json_path,\n", " is_gene_available=is_gene_available,\n", " is_trait_available=is_trait_available\n", ")\n", "\n", "# 4. Clinical Feature Extraction\n", "if trait_row is not None:\n", " # Create a DataFrame from the sample characteristics dictionary\n", " # Convert the sample characteristics dictionary to a DataFrame format\n", " sample_chars = {0: ['tissue: epicardial adipose tissue'], \n", " 1: ['patient diagnosis: sinus rhythm after surgery', \n", " 'patient diagnosis: postoperative atrial fibrillation after surgery (POAF)']}\n", " \n", " # Create a DataFrame from the sample characteristics\n", " # First, find the maximum number of values in any row\n", " max_values = max(len(values) for values in sample_chars.values())\n", " \n", " # Create a dictionary to hold the transformed data\n", " clinical_dict = {}\n", " for row_idx, values in sample_chars.items():\n", " for col_idx, value in enumerate(values):\n", " col_name = f\"characteristic_{col_idx+1}\"\n", " if col_name not in clinical_dict:\n", " clinical_dict[col_name] = [None] * len(sample_chars)\n", " clinical_dict[col_name][row_idx] = value\n", " \n", " # Convert to DataFrame\n", " clinical_data = pd.DataFrame(clinical_dict)\n", " \n", " # Extract clinical features using the geo_select_clinical_features function\n", " selected_clinical_df = geo_select_clinical_features(\n", " clinical_df=clinical_data,\n", " trait=trait,\n", " trait_row=trait_row,\n", " convert_trait=convert_trait,\n", " age_row=age_row,\n", " convert_age=convert_age,\n", " gender_row=gender_row,\n", " convert_gender=convert_gender\n", " )\n", " \n", " # Preview the extracted clinical features\n", " preview = preview_df(selected_clinical_df)\n", " print(\"Preview of selected clinical features:\")\n", " print(preview)\n", " \n", " # Create directory if it doesn't exist\n", " os.makedirs(os.path.dirname(out_clinical_data_file), exist_ok=True)\n", " \n", " # Save the extracted clinical features as a CSV file\n", " selected_clinical_df.to_csv(out_clinical_data_file, index=False)\n", " print(f\"Clinical data saved to {out_clinical_data_file}\")\n" ] }, { "cell_type": "markdown", "id": "bb1b9198", "metadata": {}, "source": [ "### Step 3: Gene Data Extraction" ] }, { "cell_type": "code", "execution_count": 4, "id": "4022db7b", "metadata": { "execution": { "iopub.execute_input": "2025-03-25T06:47:03.025614Z", "iopub.status.busy": "2025-03-25T06:47:03.025502Z", "iopub.status.idle": "2025-03-25T06:47:03.072894Z", "shell.execute_reply": "2025-03-25T06:47:03.072557Z" } }, "outputs": [ { "name": "stdout", "output_type": "stream", "text": [ "Matrix file found: ../../input/GEO/Atrial_Fibrillation/GSE143924/GSE143924_series_matrix.txt.gz\n", "Gene data shape: (8596, 30)\n", "First 20 gene/probe identifiers:\n", "Index(['A2M-AS1', 'AACS', 'AADAC', 'AADACL2', 'AADACP1', 'AAK1', 'AARD',\n", " 'AASS', 'ABCA1', 'ABCA10', 'ABCA11P', 'ABCA3', 'ABCA5', 'ABCA9-AS1',\n", " 'ABCB1', 'ABCB11', 'ABCB4', 'ABCB7', 'ABCC3', 'ABCC6'],\n", " dtype='object', name='ID')\n" ] } ], "source": [ "# 1. Get the SOFT and matrix file paths again \n", "soft_file, matrix_file = geo_get_relevant_filepaths(in_cohort_dir)\n", "print(f\"Matrix file found: {matrix_file}\")\n", "\n", "# 2. Use the get_genetic_data function from the library to get the gene_data\n", "try:\n", " gene_data = get_genetic_data(matrix_file)\n", " print(f\"Gene data shape: {gene_data.shape}\")\n", " \n", " # 3. Print the first 20 row IDs (gene or probe identifiers)\n", " print(\"First 20 gene/probe identifiers:\")\n", " print(gene_data.index[:20])\n", "except Exception as e:\n", " print(f\"Error extracting gene data: {e}\")\n" ] }, { "cell_type": "markdown", "id": "f0044668", "metadata": {}, "source": [ "### Step 4: Gene Identifier Review" ] }, { "cell_type": "code", "execution_count": 5, "id": "40ec19c9", "metadata": { "execution": { "iopub.execute_input": "2025-03-25T06:47:03.073972Z", "iopub.status.busy": "2025-03-25T06:47:03.073859Z", "iopub.status.idle": "2025-03-25T06:47:03.075735Z", "shell.execute_reply": "2025-03-25T06:47:03.075428Z" } }, "outputs": [], "source": [ "# Review the gene identifiers from the output provided\n", "# The identifiers shown (A2M-AS1, AACS, AADAC, etc.) appear to be \n", "# standard human gene symbols rather than probe IDs or other identifiers\n", "\n", "# These match the standard HGNC (HUGO Gene Nomenclature Committee) gene symbol format\n", "# Examples like ABCA1, ABCC3 are well-known human gene symbols\n", "# Identifiers like A2M-AS1 and ABCA9-AS1 follow the antisense RNA naming convention\n", "\n", "# No mapping needed as these are already proper human gene symbols\n", "requires_gene_mapping = False\n" ] }, { "cell_type": "markdown", "id": "0707ce8b", "metadata": {}, "source": [ "### Step 5: Data Normalization and Linking" ] }, { "cell_type": "code", "execution_count": 6, "id": "0478a4c2", "metadata": { "execution": { "iopub.execute_input": "2025-03-25T06:47:03.076778Z", "iopub.status.busy": "2025-03-25T06:47:03.076671Z", "iopub.status.idle": "2025-03-25T06:47:05.421047Z", "shell.execute_reply": "2025-03-25T06:47:05.420660Z" } }, "outputs": [ { "name": "stdout", "output_type": "stream", "text": [ "Gene data shape before normalization: (8596, 30)\n", "Gene data shape after normalization: (7873, 30)\n" ] }, { "name": "stdout", "output_type": "stream", "text": [ "Normalized gene expression data saved to ../../output/preprocess/Atrial_Fibrillation/gene_data/GSE143924.csv\n" ] }, { "name": "stdout", "output_type": "stream", "text": [ "Original clinical data preview:\n", " !Sample_geo_accession GSM4276706 \\\n", "0 !Sample_characteristics_ch1 tissue: epicardial adipose tissue \n", "1 !Sample_characteristics_ch1 patient diagnosis: sinus rhythm after surgery \n", "\n", " GSM4276707 \\\n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: sinus rhythm after surgery \n", "\n", " GSM4276708 \\\n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: sinus rhythm after surgery \n", "\n", " GSM4276709 \\\n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: sinus rhythm after surgery \n", "\n", " GSM4276710 \\\n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: sinus rhythm after surgery \n", "\n", " GSM4276711 \\\n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: sinus rhythm after surgery \n", "\n", " GSM4276712 \\\n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: sinus rhythm after surgery \n", "\n", " GSM4276713 \\\n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: sinus rhythm after surgery \n", "\n", " GSM4276714 ... \\\n", "0 tissue: epicardial adipose tissue ... \n", "1 patient diagnosis: sinus rhythm after surgery ... \n", "\n", " GSM4276726 \\\n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: postoperative atrial fibril... \n", "\n", " GSM4276727 \\\n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: postoperative atrial fibril... \n", "\n", " GSM4276728 \\\n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: postoperative atrial fibril... \n", "\n", " GSM4276729 \\\n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: postoperative atrial fibril... \n", "\n", " GSM4276730 \\\n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: postoperative atrial fibril... \n", "\n", " GSM4276731 \\\n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: postoperative atrial fibril... \n", "\n", " GSM4276732 \\\n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: postoperative atrial fibril... \n", "\n", " GSM4276733 \\\n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: postoperative atrial fibril... \n", "\n", " GSM4276734 \\\n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: postoperative atrial fibril... \n", "\n", " GSM4276735 \n", "0 tissue: epicardial adipose tissue \n", "1 patient diagnosis: postoperative atrial fibril... \n", "\n", "[2 rows x 31 columns]\n", "Selected clinical data shape: (1, 30)\n", "Clinical data preview:\n", " GSM4276706 GSM4276707 GSM4276708 GSM4276709 \\\n", "Atrial_Fibrillation 0.0 0.0 0.0 0.0 \n", "\n", " GSM4276710 GSM4276711 GSM4276712 GSM4276713 \\\n", "Atrial_Fibrillation 0.0 0.0 0.0 0.0 \n", "\n", " GSM4276714 GSM4276715 ... GSM4276726 GSM4276727 \\\n", "Atrial_Fibrillation 0.0 0.0 ... 1.0 1.0 \n", "\n", " GSM4276728 GSM4276729 GSM4276730 GSM4276731 \\\n", "Atrial_Fibrillation 1.0 1.0 1.0 1.0 \n", "\n", " GSM4276732 GSM4276733 GSM4276734 GSM4276735 \n", "Atrial_Fibrillation 1.0 1.0 1.0 1.0 \n", "\n", "[1 rows x 30 columns]\n", "Linked data shape before processing: (30, 7874)\n", "Linked data preview (first 5 rows, 5 columns):\n", " Atrial_Fibrillation A2M-AS1 AACS AADAC \\\n", "GSM4276706 0.0 508.207839 381.384960 111.908777 \n", "GSM4276707 0.0 892.370816 1172.689215 20000.000000 \n", "GSM4276708 0.0 821.254817 264.082320 384.802720 \n", "GSM4276709 0.0 925.448628 253.827399 209.135691 \n", "GSM4276710 0.0 489.628264 363.278332 3299.517699 \n", "\n", " AADACL2 \n", "GSM4276706 69.831635 \n", "GSM4276707 78.385174 \n", "GSM4276708 102.435318 \n", "GSM4276709 58.320162 \n", "GSM4276710 64.239732 \n" ] }, { "name": "stdout", "output_type": "stream", "text": [ "Data shape after handling missing values: (30, 7874)\n", "For the feature 'Atrial_Fibrillation', the least common label is '0.0' with 15 occurrences. This represents 50.00% of the dataset.\n" ] }, { "name": "stdout", "output_type": "stream", "text": [ "Linked data saved to ../../output/preprocess/Atrial_Fibrillation/GSE143924.csv\n" ] } ], "source": [ "# 1. Normalize gene symbols in the gene expression data\n", "# Use normalize_gene_symbols_in_index to standardize gene symbols\n", "normalized_gene_data = normalize_gene_symbols_in_index(gene_data)\n", "print(f\"Gene data shape before normalization: {gene_data.shape}\")\n", "print(f\"Gene data shape after normalization: {normalized_gene_data.shape}\")\n", "\n", "# Save the normalized gene data to file\n", "os.makedirs(os.path.dirname(out_gene_data_file), exist_ok=True)\n", "normalized_gene_data.to_csv(out_gene_data_file)\n", "print(f\"Normalized gene expression data saved to {out_gene_data_file}\")\n", "\n", "# Load the actual clinical data from the matrix file that was previously obtained in Step 1\n", "soft_file, matrix_file = geo_get_relevant_filepaths(in_cohort_dir)\n", "background_info, clinical_data = get_background_and_clinical_data(matrix_file)\n", "\n", "# Get preview of clinical data to understand its structure\n", "print(\"Original clinical data preview:\")\n", "print(clinical_data.head())\n", "\n", "# 2. If we have trait data available, proceed with linking\n", "if trait_row is not None:\n", " # Extract clinical features using the original clinical data\n", " selected_clinical_df = geo_select_clinical_features(\n", " clinical_df=clinical_data,\n", " trait=trait,\n", " trait_row=trait_row,\n", " convert_trait=convert_trait,\n", " age_row=age_row,\n", " convert_age=convert_age,\n", " gender_row=gender_row,\n", " convert_gender=convert_gender\n", " )\n", "\n", " print(f\"Selected clinical data shape: {selected_clinical_df.shape}\")\n", " print(\"Clinical data preview:\")\n", " print(selected_clinical_df.head())\n", "\n", " # Link the clinical and genetic data\n", " linked_data = geo_link_clinical_genetic_data(selected_clinical_df, normalized_gene_data)\n", " print(f\"Linked data shape before processing: {linked_data.shape}\")\n", " print(\"Linked data preview (first 5 rows, 5 columns):\")\n", " print(linked_data.iloc[:5, :5] if not linked_data.empty else \"Empty dataframe\")\n", "\n", " # 3. Handle missing values\n", " try:\n", " linked_data = handle_missing_values(linked_data, trait)\n", " print(f\"Data shape after handling missing values: {linked_data.shape}\")\n", " except Exception as e:\n", " print(f\"Error handling missing values: {e}\")\n", " linked_data = pd.DataFrame() # Create empty dataframe if error occurs\n", "\n", " # 4. Check for bias in features\n", " if not linked_data.empty and linked_data.shape[0] > 0:\n", " # Check if trait is biased\n", " trait_type = 'binary' if len(linked_data[trait].unique()) <= 2 else 'continuous'\n", " if trait_type == \"binary\":\n", " is_biased = judge_binary_variable_biased(linked_data, trait)\n", " else:\n", " is_biased = judge_continuous_variable_biased(linked_data, trait)\n", " \n", " # Remove biased demographic features\n", " if \"Age\" in linked_data.columns:\n", " age_biased = judge_continuous_variable_biased(linked_data, 'Age')\n", " if age_biased:\n", " linked_data = linked_data.drop(columns='Age')\n", " \n", " if \"Gender\" in linked_data.columns:\n", " gender_biased = judge_binary_variable_biased(linked_data, 'Gender')\n", " if gender_biased:\n", " linked_data = linked_data.drop(columns='Gender')\n", " else:\n", " is_biased = True\n", " print(\"Cannot check for bias as dataframe is empty or has no rows after missing value handling\")\n", "\n", " # 5. Validate and save cohort information\n", " note = \"\"\n", " if linked_data.empty or linked_data.shape[0] == 0:\n", " note = \"Dataset contains gene expression data related to atrial fibrillation after cardiac surgery, but linking clinical and genetic data failed, possibly due to mismatched sample IDs.\"\n", " else:\n", " note = \"Dataset contains gene expression data for atrial fibrillation after cardiac surgery, which is relevant to arrhythmia research.\"\n", " \n", " is_usable = validate_and_save_cohort_info(\n", " is_final=True,\n", " cohort=cohort,\n", " info_path=json_path,\n", " is_gene_available=True,\n", " is_trait_available=True,\n", " is_biased=is_biased,\n", " df=linked_data,\n", " note=note\n", " )\n", "\n", " # 6. Save the linked data if usable\n", " if is_usable:\n", " os.makedirs(os.path.dirname(out_data_file), exist_ok=True)\n", " linked_data.to_csv(out_data_file)\n", " print(f\"Linked data saved to {out_data_file}\")\n", " else:\n", " print(\"Dataset is not usable for analysis. No linked data file saved.\")\n", "else:\n", " # If no trait data available, validate with trait_available=False\n", " is_usable = validate_and_save_cohort_info(\n", " is_final=True,\n", " cohort=cohort,\n", " info_path=json_path,\n", " is_gene_available=True,\n", " is_trait_available=False,\n", " is_biased=True, # Set to True since we can't use data without trait\n", " df=pd.DataFrame(), # Empty DataFrame\n", " note=\"Dataset contains gene expression data but lacks proper clinical trait information for arrhythmia analysis.\"\n", " )\n", " \n", " print(\"Dataset is not usable for arrhythmia analysis due to lack of clinical trait data. No linked data file saved.\")" ] } ], "metadata": { "language_info": { "codemirror_mode": { "name": "ipython", "version": 3 }, "file_extension": ".py", "mimetype": "text/x-python", "name": "python", "nbconvert_exporter": "python", "pygments_lexer": "ipython3", "version": "3.10.16" } }, "nbformat": 4, "nbformat_minor": 5 }