miRNA-based electrochemical biosensors for ovarian cancer.

Ovarian cancer, a prevalent and deadly cancer among women, presents a significant challenge for early detection due to its heterogeneous nature. MicroRNAs, short non-coding regulatory RNA fragments, play a role in various cellular processes. Aberrant expression of these microRNAs has been observed in the carcinogenesis-related processes of many cancer types. Numerous studies highlight the critical role of microRNAs in the initiation and progression of ovarian cancer. Given their clinical importance and predictive value, there has been considerable interest in developing simple, prompt, and sensitive miRNA biosensor strategies. Among these, electrochemical sensors have demonstrated advantageous characteristics such as simplicity, sensitivity, low cost, and scalability. These microRNA-based electrochemical biosensors are valuable tools for early detection and point-of-care applications. This article discusses the potential role of microRNAs in ovarian cancer and recent advances in the development of electrochemical biosensors for miRNA detection in ovarian cancer samples.

Aptamer biosensors for thrombin.

Thrombin, a key factor in the coagulation cascade, is a valuable biomarker of great importance for the prognosis, diagnosis, and monitoring of various diseases, including cancer and heart disease. Due to the increasing attention to the development of point-of-care testing (POCT) options, various types of biosensors have been invented to enhance the accuracy and speed of detection of important biomarkers such as thrombin. Implementation of aptamers in biosensors (aptasensors) improves the target recognition capacity due to the high-affinity binding nature of aptamers. Herein, this review presents recent studies of aptasensors for thrombin detection based on different detection mechanisms encompassing optical biosensors, surface-enhanced Raman spectroscopy (SERS), electrochemical detection, piezoelectric detection, and lateral flow assay.

Designing a Secretory form of RTX-A as an Anticancer Toxin: An In Silico Approach.

BACKGROUND: Cancer is a leading cause of death and a significant public health issue worldwide. Standard treatment methods such as chemotherapy, radiotherapy, and surgery are only sometimes effective. Therefore, new therapeutic approaches are needed for cancer treatment. Sea anemone actinoporins are pore-forming toxins (PFTs) with membranolytic activities. RTX-A is a type of PFT that interacts with membrane phospholipids, resulting in pore formation. The synthesis of recombinant proteins in a secretory form has several advantages, including protein solubility and easy purification. In this study, we aimed to discover suitable signal peptides for producing RTX-A in Bacillus subtilis in a secretory form. METHODS: Signal peptides were selected from the Signal Peptide Web Server. The probability and secretion pathways of the selected signal peptides were evaluated using the SignalP server. ProtParam and Protein-sol were used to predict the physico-chemical properties and solubility. AlgPred was used to predict the allergenicity of RTX-A linked to suitable signal peptides. Non-allergenic, stable, and soluble signal peptides fused to proteins were chosen, and their secondary and tertiary structures were predicted using GOR IV and I-TASSER, respectively. The PROCHECK server performed the validation of 3D structures. RESULTS: According to bioinformatics analysis, the fusion forms of OSMY_ECOLI and MALE_ECOLI linked to RTX-A were identified as suitable signal peptides. The final proteins with signal peptides were stable, soluble, and non-allergenic for the human body. Moreover, they had appropriate secondary and tertiary structures. CONCLUSION: The signal above peptides appears ideal for rationalizing secretory and soluble RTX-A. Therefore, the signal peptides found in this study should be further investigated through experimental researches and patents.

MicroRNA biosensors for detection of glioblastoma.

Glioblastoma (GBM) is the most common type of malignant brain tumor.The discovery of microRNAs and their unique properties have made them suitable tools as biomarkers for cancer diagnosis, prognosis, and evaluation of therapeutic response using different types of nanomaterials as sensitive and specific biosensors. In this review, we discuss microRNA-based electrochemical biosensing systems and the use of nanoparticles in the evolving development of microRNA-based biosensors in glioblastoma.

MicroRNA biosensors for the detection of liver cancer.

Liver cancer is one of the deadliest types worldwide and early diagnosis is highly important for successful treatment. Therefore, it is necessary to develop rapid, sensitive, simple, and inexpensive analytical tools for its detection. MicroRNAs (miRNA) represent unique biomarkers whose expression in biofluids is strongly associated with cancer in general and miR-21, -31, -122, -145, -146a, -200c, -221, -222, and -223 in liver cancer, specifically. Various biosensors for miRNA detection have been developed. These include electrochemical biosensors based on amperometric, potentiometric, conductometric and impedimetric technology. Furthermore, the use of advanced nanomaterials with enhanced chemical stability, conductivity and electrocatalytic activity have greatly increased the sensitivity and specificity of these devices. The present review focuses on recent advances in electrochemical biosensors for miRNA detection in liver cancer.

An Oxidative Stress Study on Curcumin and Nanocurcumin against Aluminum phosphide-induced Kidney Injury in Rats: The Role of SIRT1/FOXO3 Signaling Pathway in Nephrotoxicity.

INTRODUCTION: In this study, we have investigated the aluminium phosphide (ALP) toxicity on Renal Function and oxidative stress in kidney tissue of male rats and the possible protective role of Curcumin and nanoCurcumin against ALP-induced nephrotoxicity. METHODS: Thirty-six adult male rats were divided into 6 groups (n=6). ALP (2 mg/kg oral administration) and control groups received Curcumin and nanoCurcumin (oral administration 100 mg/kg ( or without it. After seven days of treatment, kidney parameters, oxidative stress biomarkers, and expression level of sirtuins1 (SIRT1)/Forkhead box protein O1 (FoxO1) pathway genes were evaluated in kidney tissue. In addition, histopathological changes in the kidney tissues were assayed. RESULTS: In the ALP group, compared to the control group, lipid peroxidation levels, urea, and creatinine were increased, and total antioxidant capacity and thiol groups decreased significantly P<0.05. In Curcumin and nanoCurcumin groups compared to the ALP group, lipid peroxidation and creatinine decreased significantly P<0.05. Also, Curcumin and nanoCurcumin improved the tissue damage caused by ALP. NanoCurcumin modulated the effect of ALP on the gene expression levels in SIRT1/FoxO1. CONCLUSION: The present study showed that ALP intoxication in kidney tissue can induce oxidative damage. Moreover, Curcumin and nanocurcumin, as potential antioxidants, can be effective therapeutics in ALP-induced nephrotoxicity.

How can nanomicelle-curcumin modulate aluminum phosphide-induced neurotoxicity?: Role of SIRT1/FOXO3 signaling pathway.

Aluminum phosphide (ALP) is among the most significant causes of brain toxicity and death in many countries. Curcumin (CUR), a major turmeric component, is a potent protective agent against many diseases, including brain toxicity. This study aimed to examine the probable protection potential of nanomicelle curcumin (nanomicelle-CUR) and its underlying mechanism in a rat model of ALP-induced brain toxicity. A total of 36 Wistar rats were randomly divided into six groups (n = 6) and exposed to ALP (2 mg/kg/day, orally) + CUR or nanomicelle-CUR (100 mg/kg/day, orally) for 7 days. Then, they were anesthetized, and brain tissue samples were dissected to evaluate histopathological alterations, oxidative stress biomarkers, gene expression of SIRT1, FOXO1a, FOXO3a, CAT and GPX in brain tissue via hematoxylin and eosin (H&E) staining, biochemical and enzyme-linked immunosorbent assay (ELISA) methods and Real-Time PCR analysis. CUR and nanomicelle-CUR caused significant improvement in ALP-induced brain damage by reducing the MDA levels and induction of antioxidant capacity (TTG, TAC and SOD levels) and antioxidant enzymes (CAT, GPX), modulation of histopathological changes and up-regulation of gene expression of SIRT1 in brain tissue. It was concluded that nanomicelle-CUR treatment ameliorated the harmful effects of ALP-induced brain toxicity by reducing oxidative stress. Therefore, it could be considered a suitable therapeutic choice for ALP poisoning.

MicroRNA biosensors for detection of gastrointestinal cancer.

Gastrointestinal (GI) cancers are one of the most common causes of cancer-related mortality. The discovery of microRNAs (miRs) and their unique role in cancer and other diseases has prompted the development of highly sensitive molecular diagnostic tools using nanomaterials as sensitive and specific biosensors. Among these, electrochemical biosensors, which are based on a simple and inexpensive design, make them desirable in clinical applications as well as a mass-produced point-of-care device. We review miR-based electrochemical biosensors in GI cancer and examine the use of nanoparticles in the evolving development of miR-based biosensors. Among these, a number of approaches including redox labeled probes, catalysts, redox intercalating agents and free redox indicators are highlighted for use in electrochemical biosensor technology.

Non-coding RNAs in gynecologic cancer.

The term "gynecologic cancer" pertains to neoplasms impacting the reproductive tissues and organs of women encompassing the endometrium, vagina, cervix, uterus, vulva, and ovaries. The progression of gynecologic cancer is linked to various molecular mechanisms. Historically, cancer research primarily focused on protein-coding genes. However, recent years have unveiled the involvement of non-coding RNAs (ncRNAs), including microRNAs, long non-coding RNAs (LncRNAs), and circular RNAs, in modulating cellular functions within gynecological cancer. Substantial evidence suggests that ncRNAs may wield a dual role in gynecological cancer, acting as either oncogenic or tumor-suppressive agents. Numerous clinical trials are presently investigating the roles of ncRNAs as biomarkers and therapeutic agents. These endeavors may introduce a fresh perspective on the diagnosis and treatment of gynecological cancer. In this overview, we highlight some of the ncRNAs associated with gynecological cancers.

Exosomal noncoding RNAs in prostate cancer.

Prostate cancer (PCa) is the second most common cancer and the fifth leading cause of mortality among men. The recurrent reports of false-positive results of common PCa biomarkers have led to the introduction of some promising biomarkers for PCa, such as exosomal non-coding RNAs (ncRNAs). Exosomes contain various components, such as several ncRNAs (miRNAs and lncRNAs), which are important in the initiation and progression of PCa. These ncRNAs also reflect the state of the origin cell. In this article, we reviewed research on the importance and roles of ncRNAs in PCa, focusing on exosomal ncRNAs. We highlighted plasma exosomal miRNAs (8 miRNAs), urine exosomal miRNAs (19miRNAs), serum miRNAs (2 miRNAs), and five miRNAs in semen used for PCa diagnosis. Also, four exosomal lncRNAs in plasma and urine can be used as biomarkers for PCa diagnosis.

Nanocurcumin Improves Lipid Status, Oxidative Stress, and Function of the Liver in Aluminium Phosphide-Induced Toxicity: Cellular and Molecular Mechanisms.

Background: The present study aimed to evaluate the effect of nanocurcumin and curcumin on liver transaminases, lipid profile, oxidant and antioxidant system, and pathophysiological changes in aluminium phosphide (ALP) induced hepatoxicity. Material and Methods. In this experimental study, thirty-six male Wistar rats were randomly divided into six groups curcumin (Cur), nanocurcumin (Nanocur), ALP, ALP+Cur, and ALP+Nanocur. All treatments were performed by oral gavage for seven days. After treatment, animals were sacrificed, and liver and blood samples were taken. Serum levels of aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (AP), total bilirubin, cholesterol, triglyceride, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) were measured by photometric methods. Total antioxidant capacity (TAC) and malondialdehyde (MDA) as parameters of oxidative stress and mRNA expression of the nonenzyme protein including Sirtuin 1 (STR1), Forkhead box protein O1 (FOXO1) and protein O3 (FOXO3), catalase (CAT), and glutathione peroxidase (GPX) as the enzyme protein in homogenized tissues have been investigated. A histologist analyzed liver tissue sections after staining with hematoxylin-eosin. Results: In the aluminium phosphide group, there was a significant increase in MDA, ALT, AST, and AP and total bilirubin, cholesterol, triglyceride, LDL, and VLDL; AST, ALT, total bilirubin, LDL, VLDL, cholesterol, and MDA were significantly decreased; and HDL and TAC were significantly increased compared to ALP (P < 0.05). In the ALP+Nanocur group, ALT, AST, ALP, total bilirubin, cholesterol, LDL, VLDL, triglyceride, and MDA were significantly decreased and HDL and TAC were increased significantly (P < 0.05). The effect of nanocurcumin on controlling serum levels of LDL, VLDL, triglyceride, and MDA in ALP-poisoned rats was significantly more than curcumin (P < 0.05). The ALP group had significant changes in genes SIRT1, FOXO1a, FOXO3a, CAT, and GPX compared to healthy controls (P < 0.05). Nanocurcumin mice expressed more SIRT1, FOXO1a, CAT, and GPX genes than controls, and curcumin-treated mice expressed more SIRT1 and FOXO1a genes (P < 0.05). Histopathological findings also indicated a more significant protective effect of nanocurcumin relative to curcumin against ALP-induced hepatotoxicity. Conclusion: Nanocurcumin significantly protects the liver against aluminum phosphide toxicity. It is suggested that nanocurcumin-based drugs be developed to reduce the toxic effects of ALP in poisoned patients.

Exosomal microRNAs and long noncoding RNAs: Novel mediators of drug resistance in lung cancer.

Lung cancer therapeutic resistance, especially chemoresistance, is a key issue in the management of this malignancy. Despite the development of novel molecularly targeted drugs to promote therapeutic efficacy, 5-year survival of lung cancer patients is still dismal. Molecular studies through the recent years have fortunately presented multiple genes and signaling pathways, which contribute to lung cancer chemoresistance, providing a better perception of the biology of tumor cells, as well as the molecular mechanisms involved in their resistance to chemotherapeutic agents. Among those mechanisms, transfer of extracellular vesicles, such as exosomes, between cancer cells and the surrounding noncancerous ones is considered as an emerging route. Exosomes can desirably function as signaling vesicles to transmit multiple molecules from normal cells to cancer cells and their microenvironment, or vice versa. Using this ability, exosomes may affect the cancer cells' chemoresistance/chemosensitivity. Recently, noncoding RNAs (esp. microRNAs and long noncoding RNAs), as key molecules transferred by exosomes, have been reported to play a substantial role in the process of drug resistance, through modulation of various proteins and their corresponding genes. Accordingly, the current review principally aims to highlight exosomal micro- and long noncoding RNAs involved in lung cancer chemoresistance. Moreover, major molecular mechanisms, which connect corresponding RNA molecules to drug resistance, will briefly be addressed, for better clarifying of possible roles of exosomal noncoding RNAs in promoting the effectiveness of lung cancer therapy.

Laccase: Various types and applications.

Laccase belongs to the polyphenol oxidase family and is very important in removing environmental pollutants due to its structural and functional properties. Recently, the ability of laccase to oxidize phenolic and nonphenolic substances has been considered by many researchers. This enzyme's application scope includes a broad range of chemical processes and industrial usages, such as bioremediation, nanobiotechnology, woodworking industries, bleaching of paper pulp, dyeing in the textile industry, biotechnological uses in food industries, biorefining, detoxification from wastewater, production of organic matter from phenolic and amine substrates, and biofuels. Although filamentous fungi produce large amounts of laccase, high-yield industrial-scale production of laccase is still faced with many problems. At present, researchers are trying to increase the efficiency and productivity and reduce the final price of laccase by finding suitable microorganisms and improving the process of production and purification of laccase. This article reviews the introduction of laccase, its properties, production processes, and the effect of various factors on the enzyme's stability and activity, and some of its applications in various industries.

Evaluation of Protective Effects of Curcumin and Nanocurcumin on Aluminium Phosphide­Induced Subacute Lung Injury in Rats: Modulation of Oxidative Stress through SIRT1/FOXO3 Signalling Pathway.

OBJECTIVE: Aluminum phosphide (AlP) is widely used to protect stored food products and grains from pests and rodents. The availability of AlP, especially in Asian countries it has become a desirable factor to commit suicide. The phosphine produced from ALP is a very reactive radical and a respiratory inhibitor that causes oxidative damage. There is no dedicated antidote or effective drug to manage AlP-induced lung toxicity. The present study aims to evaluate and compare the protective effects of curcumin and nanocurcumin on ALP­induced subacute lung injury and determine the underlying mechanism. METHODS: Rats were exposed to AlP (2 mg/kg/day, orally)+curcumin or nanocurcumin (100 mg/kg/day, orally) for 7 days. Then rats were anesthetized and lung tissues were collected. Oxidative stress biomarkers, genes expression of antioxidant enzymes, participated genes in the SIRT1/FOXO3 pathway, and lung histopathology were assessed by biochemical and ELISA methods, Real-Time PCR analysis, and H&E staining. RESULTS: Curcumin and nanocurcumin produced a remarkable improvement in AlP-induced lung damage through reduction of MDA, induction of antioxidant capacity (TAC, TTG) and antioxidant enzymes (CAT, GPx), modulation of histopathological changes, and up-regulation of genes expression of SIRT1, FOXO3, FOXO1 in lung tissue. CONCLUSION: Nanocurcumin had a significantly more protective effect than curcumin to prevent AlP-induced lung injury via inhibition of oxidative stress. Nanocurcumin could be considered a suitable therapeutic choice for AlP poisoning.

PPARγ activation by pioglitazone enhances the anti-proliferative effects of doxorubicin on pro-monocytic THP-1 leukemia cells via inducing apoptosis and G2/M cell cycle arrest.

PURPOSE: Doxorubicin (DOX) is a common chemotherapeutic agent, with toxic side effects, and chemoresistance. Combination chemotherapy is a successful approach to overcome these limitations. Here, we investigated the effects of pioglitazone (PGZ), a PPARγ agonist, and/or DOX on the viability, cell cycle, apoptosis on THP-1 cells and normal human monocytes (NHMs). METHODS: MTT assay was used to evaluate the cytotoxicity of DOX and/or PGZ. Cell cycle progression and apoptosis induction were examined by PI or Annexin V-PI double staining, and analyzed by flow cytometry. Quantitative RT-PCR was used to evaluate the changes in the mRNA expression of cell cycle progression or apoptosis-associated genes including P27, P21, CDK2, P53, BCL2 and FasR. RESULTS: DOX, PGZ and DOX + PGZ exerted their cytotoxic effects in a dose- and time-dependent manner with low toxicity on NHMs. The cell growth inhibitory effects of DOX were in association with G2/M arrest, while PGZ executed S phase arrest. PGZ treatment enhanced G2/M among DOX-treated combinations with moderate elevation in the S phase. DOX, PGZ and combined treatments induced apoptosis (mostly late phase) in a dose-dependent manner. All treatments resulted in the significant overexpression of p21, p27, p53 and FasR genes and downregulation of CDK2. DOX + PGZ combined treatments exhibited the most significant changes in mRNA expression. CONCLUSION: We demonstrated that the antiproliferative, cell cycle regulation and apoptosis-inducing capacity of DOX was enhanced by PGZ in THP-1 leukemia cells in a dose-dependent manner. Therefore, the combination of DOX + PGZ could be used as a novel combination to target AML.

Gastrointestinal cancer drug resistance: the role of exosomal miRNAs.

Resistance of gastrointestinal (GI) cancer cells to therapeutic agents are one of the major problems in treating this type of cancer. Although the exact mechanism of drug resistance has not yet been fully elucidated, various factors have been identified as contributing factors involved in this process. Several studies have revealed the role of exosomes, especially exosomal microRNAs (miRNAs), in GI tumorigenesis, invasion, angiogenesis, and drug resistance. Exosomes, a type of small extracellular vesicles (EVs), are originated from endosomes and are released into the extracellular environment and body fluids by different cell types. Exosomes mediate cell-cell communication by transferring different cargos, including miRNAs, between parent and recipient cells. Therefore, identifying these exosomal miRNAs and their functions in GI cancers might provide new clues to further explore the secret of this process and thus help in drug-resistance management. This review article will discuss the roles of exosomal miRNAs and their mechanisms of action in drug resistance of different types of GI cancer cells (e.g., stomach, esophagus, liver, pancreas, and colon) to therapeutic agents.

microRNAs in female infertility: An overview.

Infertility impacts a considerable number of women worldwide, and it affects different aspects of family life and society. Although female infertility is known as a multifactorial disorder, there are strong genetic and epigenetic bases. Studies revealed that miRNAs play critical roles in initiation and development of female infertility related disorders. Early diagnosis and control of these diseases is an essential key for improving disease prognosis and reducing the possibility of infertility and other side effects. Investigating the possible use of miRNAs as biomarkers and therapeutic options is valuable, and it merits attention. Thus, in this article, we reviewed research associated with female diseases and highlighted microRNAs that are related to the polycystic ovary syndrome (up to 30 miRNAs), premature ovarian failure (10 miRNAs), endometriosis (up to 15 miRNAs), uterine fibroids (up to 15 miRNAs), endometrial polyp (3 miRNAs), and pelvic inflammatory (6 miRNAs), which are involved in one or more ovarian or uterine disease-causing processes.

Chlorella vulgaris is an effective supplement in counteracting non-alcoholic fatty liver disease-related complications through modulation of dyslipidemia, insulin resistance, and inflammatory pathways.

This study was aimed to investigate the effect of microalgae Chlorella vulgaris (C. vulgaris) on nonalcoholic fatty liver disease (NAFLD)-related complications induced by high-fat diet (HFD). Fifty adult male rats were divided into six groups. Control group and HFD group treated with or without C. vulgaris 5% and 10%. Biochemical parameters in serum were measured by spectrophotometric and enzyme-linked immunosorbent assay (ELISA) methods. The relative gene expression levels of Tumor Necrosis Factor-alpha (TNF-α), NF-kappa B (NF-ƙB), and p38 Mitogen-Activated Protein Kinases (p38 MAPK) in the liver were assessed by using quantitative real-time PCR, while the protein levels of NF-ƙB and TNF-α in the liver homogenate were determined by ELISA. The effects of HFD significantly were reversed by C. vulgaris, especially at a 10% dose. Therefore, it can be concluded that C. vulgaris therapeutically could be useful to improve NAFLD and its complications. PRACTICAL APPLICATIONS: It is established that NAFLD is associated with the resistance to insulin, dyslipidemia, and inflammation. Accordingly, modulating of these conditions may be useful in the management of NAFLD. Our results showed the effectiveness of C. vulgaris against NAFLD-related complication through the alleviating insulin resistance, dyslipidemia and also down-regulation of inflammatory genes in p38 MAPK/TNF-α/NF-ƙB pathway. The results of our study may be useful for scientist to prepare an effective supplement from C. vulgaris to overcoming NAFLD-related complications.

An updated review of the H19 lncRNA in human cancer: molecular mechanism and diagnostic and therapeutic importance.

Accumulating evidence has reported that H19 long non-coding RNA (lncRNA) expression level is deregulated in human cancer. It has been also demonstrated that de-regulated levels of H19 could affect cancer biology by various mechanisms including microRNA (miRNA) production (like miR-675), miRNA sponging and epigenetic modifications. Furthermore, lncRNA could act as a potential diagnosis and prognosis biomarkers and also a candidate therapeutic approach for different human cancers. In this narrative review, we shed light on the molecular mechanism of H19 in cancer development and pathogenesis. Moreover, we discussed the expression pattern and diagnostic and therapeutic importance of H19 as a potential biomarker in a range of human malignancies from breast to osteosarcoma cancer.

Alteration in inflammatory mediators in seriously eye-injured war veterans, long-term after sulfur mustard exposure.

BACKGROUND: Sulfur mustard (SM) exposure produces extensive systemic and ocular adverse effects on the victims. One of the most important effects is immunological insults that can lead to other organ damages, including the eyes. METHODS: In this descriptive study, 128 SM-exposed veterans with severe eye injury were compared with 31 healthy controls. Tear levels of tumor necrosis factor (TNF)-α and serum concentrations of interleukin (IL)-1α, IL-1ß, IL1Ra, IL-6, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), and Fas Ligand (FasL) were compared between the two groups. RESULTS: Meibomian gland dysfunction (MGD); tear breakup time (TBUT < 10″); and conjunctival, limbal, and corneal abnormalities were more frequent among the cases (MS-exposed veterans) than the controls. Ocular involvement was mild in 14.8%, moderate in 24.2%, and severe in 60.9% of the cases. Serum levels of IL-1α and FasL were significantly higher among the cases than among the controls (P < 0.001 and P = 0.037, respectively). Also, a significant decrease was observed in serum and tear levels of TNF-α in the cases as compared with controls (P < 0.001, P < 0.001, respectively). Serum levels of FasL were significantly higher in cases with severe ocular involvement than in the controls (P = 0.03). Nonetheless, serum levels of IL-1ß, IL-1Ra, IL-1α/IL-1Ra, and IL-6 were not significantly different between the two groups. CONCLUSION: Serum levels of IL-1α and FasL may cause different ocular surface abnormalities in SM-exposed patients. Lower tear TNF-α concentration may be due to lower serum levels of this cytokine in these patients.