EM-transcriptomic signature predicts drug response in advanced stages of high-grade serous ovarian carcinoma based on ascites-derived primary cultures.

Introduction: High-grade serous ovarian carcinoma (HGSOC) remains a medical challenge despite considerable improvements in the treatment. Unfortunately, over 75% of patients have already metastasized at the time of diagnosis. Advances in understanding the mechanisms underlying how ascites cause chemoresistance are urgently needed to derive novel therapeutic strategies. This study aimed to identify the molecular markers involved in drug sensitivity and highlight the use of ascites as a potential model to investigate HGSOC treatment options. Methods: After conducting an in silico analysis, eight epithelial-mesenchymal (EM)-associated genes related to chemoresistance were identified. To evaluate differences in EM-associated genes in HGSOC samples, we analyzed ascites-derived HGSOC primary cell culture (AS), tumor (T), and peritoneal nodule (NP) samples. Moreover, in vitro experiments were employed to measure tumor cell proliferation and cell migration in AS, following treatment with doxorubicin (DOX) and cisplatin (CIS) and expression of these markers. Results: Our results showed that AS exhibits a mesenchymal phenotype compared to tumor and peritoneal nodule samples. Moreover, DOX and CIS treatment leads to an invasive-intermediate epithelial-to-mesenchymal transition (EMT) state of the AS by different EM-associated marker expression. For instance, the treatment of AS showed that CDH1 and GATA6 decreased after CIS exposure and increased after DOX treatment. On the contrary, the expression of KRT18 has an opposite pattern. Conclusion: Taken together, our study reports a comprehensive investigation of the EM-associated genes after drug exposure of AS. Exploring ascites and their associated cellular and soluble components is promising for understanding the HGSOC progression and treatment response at a personalized level.

The importance of the enzyme Gamma-glutamyltransferase in the pathogenic cluster in type2 diabetic patient.

Introduction. Gamma-glutamyltransferase (GGT) is a liver enzyme involved in inflammation and oxidative stress. It is already known that MCP-1 (Monocyte Chemoattractant Protein-1) and TNF-α (tumour necrosis factor) as inflammatory markers, ICAM-1 (Intercellular Adhesion Molecule-1) as an endothelial dysfunctional marker, and glutathione, as an antioxidant, have abnormal levels in type 2 diabetic patients. The aim of this study was to evaluate the specific biological picture of type 2 diabetic patients that also associate higher GGT activity. Methods. Eighty-five type 2 diabetes, aged 40-70 years with a duration of diabetes less than 6 years without infections, epilepsy, chronic liver or cardiac diseases, without alcohol consumption (>20 g/day) were divided in two subgroups, those with normal and those with high abnormal GGT. Results. The diabetic patients with high GGT (n=31) had dysglycaemia, dyslipidemia, higher inflammatory markers (CRP, TNF-α, MCP-1) and endothelial dysfunction (high leptin and sICAM). sICAM, serum MCP-1 and TNF-α levels had significant correlations with GGT activity (r= 0.38, r=0.30 and 0.26 respectively, p<0.05). Conclusion. This study underlines that in non-alcoholic diabetic patients, with a duration of the metabolic disease less than 6 years, sICAM, serum MCP-1 and TNF-α might play an important role in dysmetabolism, and higher level for GGT represents the "red flag" for this condition.

EDEM1 regulates the insulin mRNA level by inhibiting the endoplasmic reticulum stress-induced IRE1/JNK/c-Jun pathway.

Pancreatic beta cells produce and secrete insulin as a response to rises in blood glucose. Despite the advances in understanding glucose-regulated insulin transcription and translation the mechanisms triggering the synthesis of new insulin molecules are still incompletely described. In this report, we identify EDEM1 as a new modulator of insulin synthesis and secretion. In the presence of EDEM1, INS-1E cells secrete significantly more insulin upon glucose stimulation compared to control cells. We found that overexpression of EDEM1 inhibited the IRE1/JNK/c-Jun pathway, leading to an increase in the insulin mRNA level. Similarly, EDEM1 transduced human islets secreted significantly more insulin upon stimulation. Furthermore, EDEM1 improved insulin secretion restoring normoglycemia and glucose tolerance in diabetic rats. We propose EDEM1 as a regulator of the UPR via IRE1/XBP1s and IRE1/JNK/c-Jun signaling cascades and insulin transcription in pancreatic ß-cells, supporting EDEM1 as a potential target for the treatment of diabetes.

miRNAs as Biomarkers in Diabetes: Moving towards Precision Medicine.

Diabetes mellitus (DM) is a complex metabolic disease with many specifically related complications. Early diagnosis of this disease could prevent the progression to overt disease and its related complications. There are several limitations to using existing biomarkers, and between 24% and 62% of people with diabetes remain undiagnosed and untreated, suggesting a large gap in current diagnostic practices. Early detection of the percentage of insulin-producing cells preceding loss of function would allow for effective therapeutic interventions that could delay or slow down the onset of diabetes. MicroRNAs (miRNAs) could be used for early diagnosis, as well as for following the progression and the severity of the disease, due to the fact of their pancreatic specific expression and stability in various body fluids. Thus, many studies have focused on the identification and validation of such groups or "signatures of miRNAs" that may prove useful in diagnosing or treating patients. Here, we summarize the findings on miRNAs as biomarkers in diabetes and those associated with direct cellular reprogramming strategies, as well as the relevance of miRNAs that act as a bidirectional switch for cell therapy of damaged pancreatic tissue and the studies that have measured and tracked miRNAs as biomarkers in insulin resistance are addressed.

The role of DNA demethylation in liver to pancreas transdifferentiation.

BACKGROUND: Insulin producing cells generated by liver cell transdifferentiation, could serve as an attractive source for regenerative medicine. The present study assesses the relationship between DNA methylation pTFs induced liver to pancreas transdifferentiation. RESULTS: The transdifferentiation process is associated with DNA demethylation, mainly at gene regulatory sites, and with increased expression of these genes. Active inhibition of DNA methylation promotes the pancreatic transcription factor-induced transdifferentiation process, supporting a causal role for DNA demethylation in this process. CONCLUSIONS: Transdifferentiation is associated with global DNA hypomethylation, and with increased expression of specific demethylated genes. A combination of epigenetic modulators may be used to increase chromatin accessibility of the pancreatic transcription factors, thus promoting the efficiency of the developmental process.

Phenotypic assessment of liver-derived cell cultures during in vitro expansion.

Background: Liver cells represent an attractive source of cells for autologous regenerative medicine. The present study assesses the liver cells' stability during in vitro expansion, as a prerequisite for therapeutic use. Results: The human liver cell cultures in this study were propagated efficiently in vitro for at least 12 passages. No significant changes in morphology, intracellular ultrastructures and characteristic markers expression were found during in vitro expansion of cells from all analyzed donors. However, expanded cells derived from male donors of >60 years old, lost the Y chromosome. Conclusion: Liver-derived cell cultures adopt a proliferative, stable mesenchymal phenotype, through an epithelial to mesenchymal transition process. The molecular and phenotypic changes of the cells during propagation are uniform, despite the heterogeneity of the different donors. Loss of Y chromosome occurs after cells' propagation in elder male donors.

The effect of liver donors' age, gender and metabolic state on pancreatic lineage activation.

Autologous cells replacement therapy by liver to pancreas transdifferentiation (TD) allows diabetic patients to be also the donors of their own therapeutic tissue. Aim: To analyze whether the efficiency of the process is affected by liver donors' heterogeneity with regard to age, gender and the metabolic state. Materials & methods: TD of liver cells derived from nondiabetic and diabetic donors at different ages was characterized at molecular and cellular levels, in vitro. Results: Neither liver cells proliferation nor the propagated cells TD efficiency directly correlate with the age (3-60 years), gender or the metabolic state of the donors. Conclusion: Human liver cells derived from a wide array of ages and metabolic states can be used for autologous cells therapies for diabetics.

COVID-19 and diabetes mellitus: Unraveling the hypotheses that worsen the prognosis (Review).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the caused disease - coronavirus disease 2019 (COVID-19), has affected so far >6,000,000 people worldwide, with variable grades of severity, and has already inflicted >350,000 deaths. SARS-CoV-2 infection seems severely affected by background diseases such as diabetes mellitus and its related complications, that seem to be favoring the most severe manifestations of SARS-CoV-2 and, therefore, require special attention in clinical care units. The present literature review focus on addressing several hypotheses explaining why diabetic patients could develop multi-organ failure in severe acute respiratory syndrome coronavirus (SARS-CoV) infections. Undoubtedly, as diabetes related complications are present it is expected to emphasize the severity of the COVID-19. Dermatological complications can occur and worsen in diabetic patients, and diseases such as acanthosis nigricans and psoriasis are prone to more severe manifestations of COVID-19. Approaches to treat SARS-CoV-2 infected patients, based on different solutions i.e. plasma therapy, use of antiviral compounds, development of vaccines or new therapeutic agents are ongoing.

Evolution of Inflammatory and Oxidative Stress Markers in Romanian Obese Male Patients with Type 2 Diabetes Mellitus after Laparoscopic Sleeve Gastrectomy: One Year Follow-Up.

Geography is one of the key drivers of the significant variation in the etiopathogenic profile and prevalence of type 2 diabetes mellitus (T2DM) and obesity, therefore geographically based data are fundamental for implementing the appropriate interventions. Presently, the selection criteria of T2DM and obesity patients for laparoscopic sleeve gastrectomy (LSG) have not reached a worldwide consensus-highlighting the need for sharing experts' guidance in the preoperative evaluation, choice of the interventional procedure, perioperative management and patient long-term care. The aim of the current study was to evaluate the impact of LSG on T2DM (T2DM) remission in Romanian obese male patients, based on a multiparametric, prospective investigation. We have conducted a randomized controlled study on 41 obese male participants with the body mass index (BMI) ≥ 30 kg/m2, aged 30-65 years, which were randomly divided in two study groups: one receiving conventional treatment and the second undergoing LSG. The clinical and anthropometrical parameters, resting metabolic rate, general biochemical status, adipocytes profile, gastrointestinal hormones levels, proinflammatory, oxidant and antioxidant profiles were determined at three time points: V1 (baseline), V2 (after six months) and V3 (after 12 months). Glycated hemoglobin (HbA1c), blood glucose levels, BMI, weight, visceral fat level, HDL-cholesterol, incretin hormones, proinflammatory and the oxidative stress status were significantly improved in the LSG versus conventional treatment group. This is the first study reporting on the evaluation of metabolic surgery impact on Romanian obese male patients with T2DM. Our results confirm that LSG could contribute to T2DM remission in patients with diabesity, but this beneficial effect seems to be critically influenced by the duration of T2DM rather than by the obesity status. Our results show that, in addition to the parameters included in the prediction algorithm, the proinsulin levels, proinsulin/insulin ratio and the visceral fat percentage could bring added value to the assessment of metabolic status.

In vivo Diagnosis of Primary Cutaneous Amyloidosis -the Role of Reflectance Confocal Microscopy.

Primary cutaneous amyloidosis (PCA) is a form of localized amyloidosis. It is characterized by the deposition of a fibrillar material in the superficial dermis, without affecting other systems or organs. The diagnosis can be made clinically, but usually a skin biopsy is performed in order to exclude other skin diseases with similar appearance. Reflectance confocal microscopy (RCM) is a novel imaging tool that enables in vivo characterization of various skin changes with a high, quasi-microscopic resolution. This technique might have an important role in the differential diagnosis of cutaneous amyloidosis, by the in vivo assessment of epidermal changes and dermal amyloid deposition. Moreover, it is completely non-invasive and can be safely repeated on the same skin area. However, to date, there is only one published paper presenting the confocal features of primary cutaneous amyloidosis. Hereby, we describe the in vivo RCM features of PCA lesions from a patient with diabetes and correlate them with histologic findings. This strengthens the clinical usefulness of in vivo RCM examination for the non-invasive diagnosis of cutaneous amyloidosis, especially in patients that might associate diseases with impaired wound healing.

Current and future applications of confocal laser scanning microscopy imaging in skin oncology.

Confocal laser scanning microscopy (CLSM) is a modern imaging technique that enables the in vivo or ex vivo characterization of skin lesions located in the epidermis and superficial dermis with a high quasi-microscopic resolution. Currently, it is considered to be the most promising imaging tool for the evaluation of superficial skin tumors. The in vivo mode adds the advantage of noninvasive, dynamic, in real-time assessment of the tumor associated vasculature and inflammation. It offers the possibility to repeatedly examine the same skin area without causing any damage and to monitor disease progression and treatment outcome. Furthermore, this novel technology allows the evaluation of the entire lesion and can be used to guide biopsies and to define tumor margins before surgical excision or other invasive therapies. CLSM diagnostic features may differentiate between the various histologic subtypes of skin tumors and therefore helps in choosing the best therapeutic approach. In this study, we present the CLSM characteristic features of the most common melanocytic and non-melanocytic skin tumors, as well as future possible CLSM applications in the study of experimental skin tumorigenesis on animal models.

In vivo confocal laser scanning microscopy imaging of skin inflammation: Clinical applications and research directions.

In vivo confocal laser scanning microscopy (CLSM) is a novel imaging technique that provides noninvasive, morphological characterization of skin structures with a resolution that is very close to that of light microscopy. Moreover, as it allows repeated imaging of the same skin area at different time-points, it is an excellent method for monitoring disease course, response to treatment or specific stimuli and a path to study dynamic phenomena in real-time. To date, two different variants of in vivo CLSM have been authorized in dermatological field, namely the reflectance confocal microscopy predominantly for clinical diagnosis and the fluorescence confocal microscopy mainly for research purposes. This study describes the principles of in vivo CLSM technique, its role in the diagnosis and monitoring of inflammatory skin diseases, as well as some promising research directions to study the dynamics of skin inflammation using this method. In vivo CLSM evaluation of inflammatory dermatoses and of the skin inflammatory component in various diseases has an undoubted potential with broad applications ranging from clinical, morphological to experimental, functional studies involving the skin.

Markers of Oxidative Stress and Antioxidant Defense in Romanian Patients with Type 2 Diabetes Mellitus and Obesity.

Type 2 diabetes mellitus (T2DM) is strongly associated with obesity. The adipose tissue secretes bioactive adipokines leading to low grade inflammation, amplified by oxidative stress, which promotes the formation of advanced glycation end products and eventually leads to dyslipidemia and vascular complications. The aim of this study was to correlate anthropometric, biochemical and oxidative stress parameters in newly diagnosed (ND) T2DM patients and to investigate the role of oxidative stress in T2DM associated with obesity. A group of 115 ND- T2DM patients was compared to a group of 32 healthy subjects in terms of clinical, anthropometric, biochemical and oxidative stress parameters. ND-T2DM patients had significantly lower adiponectin, glutathione (GSH) and gluthatione peroxidase (GPx) and elevated insulin, proinsulin, HOMA-IR index, proinsulin/insulin (P/I) and proinsulin/adiponectin (P/A) ratio, fructosamine, and total oxidant status (TOS). The total body fat mass was positively correlated with total oxidant status (TOS). Positive correlations were found between TOS and glycated hemoglobin (HbA1c), and between TOS and glycaemia. Negative correlations were identified between: GPx and glycaemia, GPx and HbA1c, and also between GSH and fructosamine. The total antioxidant status was negatively correlated with the respiratory burst. The identified correlations suggest the existence of a complex interplay between diabetes, obesity and oxidative stress.

Estimating the yin-yang nature of Western herbs: a potential tool based on antioxidation-oxidation theory.

BACKGROUND: One of the biggest obstacles to progress in traditional Chinese medicine (TCM) development in Western countries is the difficulty of applying the traditional concepts to the Western medicinal plants, which are not traditionally described in ancient literature. During recent years, new advances in the field of understanding Yin/Yang aspects from a modern bioscientific point of view have led to the conclusion that antioxidationoxidation concepts might mirror a Yin-Yang relationship. METHODS: This study was intended to integrate the Yin-Yang theory of the traditional Chinese medicine with modern antioxidation-oxidation theory, and to propose a biochemical tool based on redox parameters (e.g. antioxidant capacity, chemiluminescence-CL signal inducing capacity), usable for the classification of Western medicinal plants from Yin/Yang perspective. Trolox equivalent antioxidant capacity (TEAC) of six vegetal aqueous extracts (Symphitum officinalae (radix)-SYM, Inula helenium (radix)-INU, Calendula officinalis (flores)-CAL, Angelica arhanghelica (folium)ANG(F), Angelica arhanghelica (radix)-ANG(R), Ecbalium Elaterium (fruits)-ECB) and luminol-enhanced chemiluminescence of PMNL on addition of these vegetal extracts were measured. Percentages from the maximal or minimal values obtained were calculated for each extract (TEAC%, PMNL stimulation%, PMNL inhibition%, relative speed of action% (RSA%%)), specific Yin-Yang significance was assigned to each relative parameter. In the end, an integration of all the relative values was done, in order to find a global "Yin" or a "Yang" trait of each vegetal extract. RESULTS: TEAC decreased in the following order: SYM > INU > CAL >ANG(F) > ANG(R > ECB. Three vegetal extracts (SYM > INU > ECB) decreased the luminol-enhanced chemiluminescence of PMNL, two (ANG(R) > ANG(F)) increased it, while one (CAL) had a dual effect. After the integration of the percentages, CAL was found to have a global "Yang" trait, while the rest of the plants had a global "Yin" trait. CONCLUSIONS: TEAC% and PMNL inhibition% appears to correlate with the Yin properties of herbs, while PMNL stimulation% and RSA% might correlate with Yang aspects within the formal TCM classification system, and may be useful criteria in describing the Western herbs from a TCM point of view.

Erythrocyte membrane stability to hydrogen peroxide is decreased in Alzheimer disease.

The brain and erythrocytes have similar susceptibility toward free radicals. Therefore, erythrocyte abnormalities might indicate the progression of the oxidative damage in Alzheimer disease (AD). The aim of this study was to investigate erythrocyte membrane stability and plasma antioxidant status in AD. Fasting blood samples (from 17 patients with AD and 14 healthy controls) were obtained and erythrocyte membrane stability against hydrogen peroxide and 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH), serum Trolox equivalent antioxidant capacity (TEAC), residual antioxidant activity or gap (GAP), erythrocyte catalase activity (CAT), erythrocyte superoxide dismutase (SOD) activity, erythrocyte nonproteic thiols, and total plasma thiols were determined. A significant decrease in erythrocyte membrane stability to hydrogen peroxide was found in AD patients when compared with controls (P<0.05). On the contrary, CAT activity (P<0.0001) and total plasma thiols (P<0.05) were increased in patients with AD compared with controls. Our results indicate that the most satisfactory measurement of the oxidative stress level in the blood of patients with AD is the erythrocyte membrane stability to hydrogen peroxide. Reduced erythrocyte membrane stability may be further evaluated as a potential peripheral marker for oxidative damage in AD.

Effect of diet and omega-3 fatty acids in NAFLD.

Nonalcoholic fatty liver disease (NAFLD) ranges from simple hepatic steatosis to steatohepatitis (NASH) and cirrhosis. The aim of this study is to test beneficial effects of omega-3 fatty acids (DHA 130 mg, EPA 25 mg) treatment in NAFLD, in a mouse model of non-alcoholic fatty liver disease. As pretreatment, 50 mice were fed for one month with a high-fat diet to induce NAFLD. Then, the mice were divided in different groups according to diet (normo- or hypercaloric), with and without treatment with omega-3 fatty acids, for another month, forming the post-treatment group. The liver and blood samples were collected for biochemical and histopathological analysis. Biochemical parameters including: glycemia, total cholesterol, triglycerides, uric acid, albumin, total plasma antioxidant capacity (TEAC) was measured in serum. Glutathione (GSH), total thiols and malonyldialdehyde (MDA) were determined in mouse liver homogenates. Mice from post-treatment group, on hypercaloric diet with or without omega-3 fatty acids treatment, had medium hepatopathy (granular and vacuolar degeneration of the hepatocytes) and hypertriglyceridemia. Omega-3 fatty acid treatment lowered the rise of triglycerides (p<0.03), glycemia (p<0.01) and cholesterol (p<0.02) in serum and MDA level of the liver (p<0.05). Mice from post-treatment group, on normocaloric diet with or without omega-3 fatty acid had different histopathological and biochemical results. Those with normocaloric/normolipidic diet and omega-3 fatty acids treatment had reversed liver histopathological results from NASH to normal aspect and had the best metabolic parameters results. In conclusion, omega-3 fatty acids treatment associated with a normocaloric/normolipidic diet has hepatoprotective action in nonalcoholic fatty liver disease.

Diet and paraoxonase 1 enzymatic activity in diabetic foot patients from Romania and Belgium: favorable association of high flavonoid dietary intake with arylesterase activity.

BACKGROUND/AIMS: The antiatherosclerotic enzyme paraoxonase (PON1) is affected by disease and lifestyle. We investigated the impact of diet in diabetic foot patients from 2 European countries. METHODS: Dietary intake and serum PON1 activity, using as substrate paraoxon (paraoxonase) or phenylacetate (arylesterase), were assessed in patients from Bucharest (n = 40) and Antwerp (n = 30) and in 34 healthy controls. RESULTS: The diabetic patients had lower paraoxonase and arylesterase activities than the controls. Arylesterase was lowest in the Bucharest patients, 116 +/- 42 U/ml, versus 141 +/- 43 and 184 +/- 49 U/ml in the Antwerp patients and controls, respectively (p < 0.0005). The Bucharest patients had worse glycemic control, higher blood pressure, lower HDL cholesterol and a diet richer in cholesterol and poorer in monounsaturated fats and fish. In contrast, their median intake of vitamins E and C, folic acid and flavonoids was higher, 82 mg (range: 4-259 mg), versus 28 mg (range: 5-169 mg) aglycone units in Antwerp (p = 0.005). Flavonoid intake predicted arylesterase independently of HDL cholesterol, region and sex (beta = 0.27; p = 0.03), and patients with high intake achieved normal levels of arylesterase (30.1 +/- 10.0 U/micromol in the highest versus 21.0 +/- 8.2 U/micromol total cholesterol in the lowest tertile; p = 0.02). CONCLUSION: A flavonoid-rich diet is positively associated with PON1 arylesterase activity in diabetic foot patients.

Protection of erythrocyte membrane against oxidative damage by geriforte in healthy human subjects.

The influence of Geriforte, an Ayurvedic natural supplement, on the antioxidant defense systems in human erythrocytes and plasma was investigated in an open human clinical study. The ability of Geriforte to inhibit the azo-bis 2-amidinopropane dihydrochloride (AAPH) dependent lysis of erythrocytes was also evaluated. Geriforte supplementation increased the activity of erythrocyte catalase (265.745 +/- 15.768 vs. 352,329 +/- 18.480 K/g Hb/mL, p < 0.01) and reduced the free radical mediated cytotoxicity induced by azo-bis 2-amidinopropane dihydrochloride (AAPH), which was measured by erythrocyte membrane stability assay (0.0439 +/- 0.0069 vs. 0.1291 +/- 0.0396 C50- AAPH (mM), p < 0.05). The intervention did not change significantly the activity of erythrocyte superoxide dismutase and the plasma levels of antioxidant agents. The results indicate that Geriforte possesses cytoprotective properties on erythrocytes against oxidative challenge and specific antioxidant activity, which involves mainly the intracellular protective systems and the membranes.

Antioxidant defense capacity in scleroderma patients.

BACKGROUND: Oxidative stress is associated with scleroderma (systemic sclerosis) and is supposed to favor disease progression by complex effects on the vascular endothelium and on fibroblasts. METHODS: Plasma oxidative process marker, thiobarbituric acid-reactive substances, and several markers of antioxidant defense capacity (plasma total antioxidant activity, serum albumin, uric acid and glutathione, superoxide dismutase and catalase) were evaluated by spectrophotometric methods using blood samples collected from 23 scleroderma patients and 21 healthy controls. RESULTS: In scleroderma patients, thiobarbituric acid-reactive substances levels (mmol/L plasma) were significantly elevated (29.3+/-5.8) compared with healthy controls (16.6+/-3.1, p<0.001). Total antioxidant activity (mmol Trolox/L) was significantly lower in scleroderma patients than in controls (1.29+/-0.13 vs. 1.55+/-0.23, p<0.001), as well as the antioxidant gap (mmol Trolox/L) (0.57+/-0.18 vs. 0.92+/-0.22, p<0.001). Superoxide dismutase activity (IU/g hemoglobin) was markedly decreased in patients as compared with controls (395+/-184 vs. 659+/-211, p<0.001). CONCLUSIONS: Lower plasma total antioxidant activity and plasma antioxidant gap in scleroderma patients show that plasma antioxidant defense is deficient in scleroderma patients. As previous studies on this issue are controversial, the decreased erythrocyte superoxide dismutase activity found in the patients in this study needs further investigation.