Understanding FAHFAs: From structure to metabolic regulation

The discovery of branched fatty acid esters of hydroxy fatty acids (FAHFAs) in humans draw attention of many researches to their biological effects. Although FAHFAs were originally discovered in insects and plants, their introduction into the mammalian realm opened new horizons in bioactive lipid research. Hundreds of isomers from different families have been identified so far and their role in (patho) physiological processes is currently being explored.The family of palmitic acid esters of hydroxy stearic acids (PAHSAs), especially 5-PAHSA and 9-PAHSA regioisomers, stands out in the crowd of other FAHFAs for their anti-inflammatory and anti-diabetic effects. Beneficial effects of PAHSAs have been linked to metabolic disorders such as type 1 and type 2 diabetes, colitis, and chronic inflammation. Besides PAHSAs, a growing family of polyunsaturated FAHFAs exerts mainly immunomodulatory effects and biological roles of many other FAHFAs remain currently unknown. Therefore, FAHFAs represent unique lipid messengers capable of affecting many immunometabolic processes.The objective of this review is to summarize the knowledge concerning the diversity of FAHFAs, nomenclature, and their analysis and detection. Special attention is paid to the total syntheses of FAHFAs, optimal strategies, and to the formation of the stereocenter required for optically active molecules. Biosynthetic pathways of saturated and polyunsaturated FAHFAs in mammals and plants are reviewed together with their metabolism and degradation. Moreover, an overview of biological effects of branched FAHFAs is provided and many unanswered questions regarding FAHFAs are discussed.     

FAHFA footprint in the visceral fat of mice across their lifespan

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of endogenous lipids with anti-inflammatory and anti-diabetic effects, having the potential to treat type 2 diabetes (T2D). In view of the important regulatory and therapeutic actions of FAHFAs on age-related diseases such as T2D, we hypothesized that they may also play crucial roles in the growth, development, and aging process. Here, we investigated the FAHFA footprint in the visceral adipose tissue (VAT) of mice across lifespan to attain potential clues for understanding the roles of FAHFAs in growth, development, and aging using chemical isotope labeling assisted liquid chromatography-mass spectrometry. VAT samples were harvested from 80 C57BL/6J male mice of nine different ages (1, 2, 3, 6, 9, 12, 15, 18, and 24 months). The results showed that a total of 51 FAHFA families, including 301 regioisomers, were detected in the VAT of mice of all ages, and the number of FAHFAs (both family and regioisomer) in VAT increased with age, from 35 families (186 ± 0 regioisomers) at 1-month-old mice to 46 families (278 ± 6 regioisomers) in 18-month-old mice. Furthermore, the content of 12 FAHFA families per 100 mg of VAT of mice was highly correlated with age, and was usually low in the middle-age (3–15 months). However, because the VAT mass was 4–5 fold higher in middle-aged mice compared to younger or older mice, the total amount of most of the FAHFA regioisomers in VAT was increased in middle-aged mice. To the best of our knowledge, this is the first study to show that the number of regioisomers from 51 FAHFA families and abundance of 15 FAHFA families are strongly dependent on age, which would be helpful for understanding the mechanisms underlying the effects of FAHFAs on growth, development, and aging.     

High intensity interval exercise decreases IL-8 and enhances the immunomodulatory cytokine interleukin-10 in lean and overweight–obese individuals

PurposeTo compare the effects of two interval exercises with different intensities on acute inflammatory response in lean and overweight–obese subjects.MethodsTen lean (BMI < 24.9 kg/m²) and 12 overweight–obese (BMI 25 to <34.9 kg/m²) males performed two conditions in randomly assigned: (1) high intensity interval exercise (HIIE) 10 × 60 s (85–90%P_Max)/75 s (50%P_Max); (2) moderate intensity interval exercise (MIIE) 10 × 60 s (70–75%P_Max)/60 s (50%P_Max), with blood collections at pre, immediately and 30 min post each exercise bouts to evaluate total and differential leukocyte counts, serum creatine kinase (CK), lactate dehydrogenase (LDH) and systemic levels of IL-1ra, IL-6, IL-8, IL-10, IL-17a and CCL2.ResultsIn lean group, HIIE induced a significant increase in total leukocytes and monocyte, while MIIE session did not change the number of leukocytes. Overweight–obese group presented similar increase in leukocytes, monocytes and lymphocytes in both HIIE and MIIE sessions. At baseline, overweight–obese group showed high levels of CK, IL-8, IL-6 and CCL2 and lower concentrations of IL-10 compared to lean group. The MIIE did not alter the cytokine concentrations in both groups, independently of the time analysis. The HIIE induced significant decrease in IL-8 levels 30 min post session in both the groups, and a progressive elevation in IL-10 levels immediately and 30 min post in lean and overweight–obese. Regarding IL-6, overweight–obese subjects presented progressive increase either immediately and 30 min after HIIE, while lean individuals presented significant increase only 30 min after exercise.ConclusionsThe acute inflammatory response to interval exercise is intensity-dependent. Although obesity influences the basal concentrations of several cytokines, only HIIE induced important alterations in IL-8 and IL-10 levels, which may have important implications in the control of chronic low-grade inflammation in obesity.     

Determination of iodine in human milk and infant formulas

The aim of this study was to develop a method to determine iodine in human milk and infant formulas using ICP-MS. The milk samples were digested using an alkaline digestion (5% NH₃, 45 W, 2 min and 30 s), and the method was validated using a certified reference material (CRM) BCR CRM151. On the other hand the milk was separated in three fractions, whey, fat and caseins using ultracentrifugation (15 min, 4 °C, 50,000 rpm) and the iodine was determined in the different fractions. About 27 samples of different infant formulas and 14 samples of human milk have been studied. In the human milk the values found were between 144±93.2 μg kg⁻¹, whereas in the infant formulas the values were 53.3±19.5. For both types of samples the bigger amount of iodine is in the whey fraction, between 80% and 90%, whereas in the fat there is about a 2% of the total iodine and in the casein fraction the levels are between 5% and 10% depending on the type of sample.     

Validation of an optimized method for the determination of iodine in human breast milk by inductively coupled plasma mass spectrometry (ICPMS) after tetramethylammonium hydroxide extraction

In this study a novel method to determine iodine concentrations in human breast milk was developed and validated. The iodine was analyzed by inductively coupled plasma mass spectrometry (ICPMS) following tetramethylammonium hydroxide (TMAH) extraction at 90 °C in disposable polypropylene tubes. While similar approaches have been used previously, this method adopted a shorter extraction time (1 h vs. 3 h) and used antimony (Sb) as the internal standard, which exhibited greater stability in breast milk and milk powder matrices compared to tellurium (Te). Method validation included: defining iodine linearity up to 200 μg L⁻¹; confirming recovery of iodine from NIST 1549 milk powder. A recovery of 94–98% was also achieved for the NIST 1549 milk powder and human breast milk samples spiked with sodium iodide and thyroxine (T4) solutions. The method quantitation limit (MQL) for human breast milk was 1.6 μg L⁻¹. The intra-assay and inter-assay coefficient of variation for the breast milk samples and NIST powder were <1% and <3.5%, respectively. NIST 1549 milk powder, human breast milk samples and calibration standards spiked with the internal standard were all stable for at least 2.5 months after extraction. The results of the validation process confirmed that this newly developed method provides greater accuracy and precision in the assessment of iodine concentrations in human breast milk than previous methods and therefore offers a more reliable approach for assessing iodine concentrations in human breast milk.     

Metabolomic markers of fatigue: Association between circulating metabolome and fatigue in women with chronic widespread pain

Background

Fatigue is a sensation of unbearable tiredness that frequently accompanies chronic widespread musculoskeletal pain (CWP) and inflammatory joint disease. Its mechanisms are poorly understood and there is a lack of effective biomarkers for diagnosis and onset prediction. We studied the circulating metabolome in a population sample characterised for CWP to identify biomarkers showing specificity for fatigue.

Synthesis and biological evaluation of novel hydroxybenzaldehyde-based kojic acid analogues as inhibitors of mushroom tyrosinase

Two series of novel kojic acid analogues (4a–j) and (5a–d) were designed and synthesized, and their mushroom tyrosinase inhibitory activities was evaluated. The result indicated that all the synthesized derivatives exhibited excellent tyrosinase inhibitory properties having IC₅₀ values in the range of 1.35 ± 2.15–17.50 ± 2.75 μM, whereas standard inhibitor kojic acid have IC₅₀ values 20.00 ± 1.08 μM. Specifically, 5-phenyl-3-[5-hydroxy-4-pyrone-2-yl-methylmercap-to]-4-(2,4-dihydroxyl-benzylamino)-1,2,4-triazole (4f) exhibited the most potent tyrosinase inhibitory activity with IC₅₀ value of 1.35 ± 2.15 μM. The kinetic studies of the compound (4f) demonstrated that the inhibitory effects of the compound on the tyrosinase were belonging to competitive inhibitors. Meanwhile, the structure-activity relationship was discussed.     

Feasibility study of detecting surface electromyograms in severely obese patients

The aims of this study were to examine if surface EMG signals can be detected from the quadriceps femoris muscle of severely obese patients and to investigate if differences exist in quadriceps force and myoelectric manifestations of fatigue between obese patients and lean controls.Fourteen severely obese patients (body mass index, BMI, mean ± SD: 44.9 ± 6.3 kg/m²) and fourteen healthy controls (BMI: 23.7 ± 2.5 kg/m²) were studied. The vastus medialis and lateralis of the dominant thigh were concurrently investigated during voluntary isometric contractions (10-s long at submaximal and maximal intensities and intermittent submaximal contractions until exhaustion) and sustained (120-s long) electrically elicited contractions.We found that the detection of surface EMG signals from the quadriceps is feasible also in severely obese subjects presenting increased thickness of the subcutaneous fat tissue. In addition, we confirmed and extended previous findings showing that the volume conductor properties determine the amplitude and spectral features of the detected surface EMG signals: the lower the subcutaneous tissue thickness, the higher the amplitude and mean frequency estimates. Further, we found no differences in the mechanical and myoelectric manifestations of fatigue during intermittent voluntary and sustained electrically elicited contractions between obese patients and lean controls.     

Proliferation and apoptosis in subcutaneous adipose tissue of lactating cows with different genetic merit for milk yield

The aim of this study was to investigate the adipocyte size and fate in subcutaneous fat (scAT) of cows diverging for genetic merit at mid lactation stage, when anabolic activity increases and animals are in a state of positive energy balance. Twenty mid lactation cows (180 ± 20 days in milk) grouped according to the Estimated Breeding Values (EBV) for milk yield in plus (EBVp) and minus (EBVm) variants were selected. Average of adipocytes area, proliferation and apoptotic labelling index as well as DLK-1 expression, a marker of pre-adipocytes, were immunohistochemically evaluated in scAT biopsies. In EBVp cows, the BCS was lower (P < 0.01) whereas milk yield, protein, fat yield (P < 0.001) and plasma free fatty acid concentration (P < 0.05) were higher. The scAT of EBVp cows showed a significantly (P < 0.001) higher frequency between 500 and 3000 μm² classes in comparison to EBVm cows, that showed a significantly (P < 0.01) higher apoptotic labeling index. The immunohistochemical reaction showed DLK-1 positivity in scAT of EBVp cows. Taking together, the data indicate a link between milk yield genetic merit of cows, scAT morphology and function, suggesting greater dynamics and metabolic flexibility in EBVp cows.     

Contrast Enhancement on Cone-Beam Breast-CT for Discrimination of Breast Cancer Immunohistochemical Subtypes

PURPOSE: To evaluate whether contrast enhancement on cone-beam breast-CT (CBBCT) could aid in discrimination of breast cancer subtypes and receptor status. METHODS: This study included female patients age >40 years with malignant breast lesions identified on contrast-enhanced CBBCT. Contrast enhancement of malignant breast lesions was standardized to breast fat tissue contrast enhancement. All breast lesions were approved via image-guided biopsy or surgery. Immunohistochemical staining was conducted for expression of estrogen (ER), progesterone (PR), human epidermal growth factor receptor-2 (HER2) and Ki-67 index. Contrast enhancement of breast lesions was correlated with immunohistochemical breast cancer subtypes (Luminal A, Luminal B, HER2 positive, triple negative), receptor status and Ki-67 expression. RESULTS: Highest contrast enhancement was seen for Luminal A lesions (93.6 HU) compared to Luminal B lesions (47.6 HU, P = .002), HER2 positive lesions (83.5 HU, P = .359) and triple negative lesions (45.3 HU, P = .005). Contrast enhancement of HER2 positive lesions was higher than Luminal B lesions (P = .044) and triple negative lesions (P = .039). No significant difference was evident between Luminal B and triple negative lesions (P = .439). Lesions with high Ki-67 index showed lower contrast enhancement than those with low Ki-67 index (P = .0043). ER, PR and HER2 positive lesions demonstrated higher contrast enhancement than their receptor negative counterparts, although differences did not reach statistical significance (P = .1714; P = .3603; P = .2166). CONCLUSIONS: Contrast enhancement of malignant breast lesions on CBBCT correlates with immunohistochemical subtype and proliferative potential. Thereby, CBBCT might aid in selecting individualized treatment strategies for breast cancer patients based on pre-operative imaging.     

The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells

BACKGROUND: MDM2 is a negative regulator of p53 and is upregulated in numerous human cancers. While different MDM2 splice variants have been observed in both normal tissues and malignant cells, their functions are poorly understood. METHODS: We evaluated the effect of MDM2 splice variants by overexpression in MCF-7 cells and analyses of expression of downstream genes (qPCR and Western blot), subcellular localization (immunofluorescence), cell cycle assays (Nucleocounter3000), apoptosis analysis (Annexin V detection), and induction of senescence (β-galactosidase analysis). RESULTS: In a screen for MDM2 splice variants in MCF-7 breast cancer cells, extended with data from healthy leukocytes, we found P2-MDM2-10 and MDM2-Δ5 to be the splice variants expressed at highest levels. Contrasting MDM2 full-length protein, we found normal tissue expression levels of P2-MDM2-10 and MDM2-Δ5 to be highest in individuals harboring the promoter SNP309TT genotype. While we detected no protein product coded for by MDM2-Δ5, the P2-MDM2-10 variant generated a protein markedly more stable than MDM2-FL. Both splice variants were significantly upregulated in stressed cells (P = 4.3 × 10^?4 and P = 7.1 × 10^?4, respectively). Notably, chemotherapy treatment and overexpression of P2-MDM2-10 or MDM2-Δ5 both lead to increased mRNA levels of the endogenous MDM2-FL (P = .039 and P = .070, respectively) but also the proapoptotic gene PUMA (P = .010 and P = .033, respectively), accompanied by induction of apoptosis and repression of senescence. CONCLUSION: We found P2-MDM2-10 and MDM2-Δ5 to have distinct biological functions in breast cancer cells. GENERAL SIGNIFICANCE: Alternative splicing may influence the oncogenic effects of the MDM2 gene.     

Obesity and spinal loads; a combined MR imaging and subject-specific modeling investigation

Epidemiological studies have identified obesity as a possible risk factor for low back disorders. Biomechanical models can help test such hypothesis and shed light on the mechanism involved. A novel subject-specific musculoskeletal-modelling approach is introduced to estimate spinal loads during static activities in five healthy obese (BMI > 30 kg/m²) and five normal-weight (20 < BMI < 25 kg/m²) individuals. Subjects underwent T1 through S1 MR imaging thereby measuring cross-sectional-area (CSA) and moment arms of trunk muscles together with mass and center of mass (CoM) of T1-L5 segments. MR-based subject-specific models estimated spinal loads using a kinematics/optimization-driven approach. Average CSAs of muscles, moment arms of abdominal muscles, mass and sagittal moment arm of CoM of T1-L5 segments were larger in obese individuals (p < 0.05 except for the moment arm of CoMs) but moment arms of their back muscles were similar to those of normal-weight individuals (p > 0.05). Heavier subjects did not necessarily have larger muscle moment arms (e.g., they were larger in 64 kg (BMI = 20.7 kg/m²) subject than 78 kg (BMI = 24.6 kg/m²) subject) or greater T1-L5 trunk weight (e.g., the 97 kg (BMI = 31 kg/m²) subject had similar trunk weight as 109 kg (BMI = 33.3 kg/m²) subject). Obese individuals had in average greater spinal loads than normal-weight ones but heavier subjects did not necessarily have greater spinal loads (117 kg (BMI = 40.0 kg/m²) subject had rather similar L5-S1 compression as 105 kg (BMI = 34.7 kg/m²) subject). Predicted L4-L5 intradiscal pressures for the normal-weight subjects ranged close to the measured values (R² = 0.85–0.92). Obese individuals did not necessarily have greater IDPs than normal-weight ones.     

Impact of lactation stage on milk composition and blood biochemical and hematological parameters of dairy Baladi goats

The objective of this study was to elucidate the impact of lactation stage on milk composition, hematological and biochemical parameters of dairy Baladi goats under Egyptian conditions. Forty-eight Baladi goats (32.8 ± 2.9 kg of BW) were enrolled in the current study. The lactation period has been divided into three stages; early (DIM less than 80 days), Mid (DIM 80–140 days), and Late (DIM over 140 days). Baladi goats had decreased daily-MY at a rate of 18.4% and 31.9% at mid and late stages of lactation, compared with early stage, respectively (p = 0.001). Furthermore, lactose% decreased significantly with progress of lactation (p = 0.017). Total solids%, however, decreased significantly at early stage of lactation in comparison with mid and late stages (p = 0.022). On the contrary, no significant differences were found in protein, fat and SNF percentages at different stages of lactation (p = 0.836, 0.625 and 0.281, respectively). Serum glucose and total protein were significantly reduced at late stage of lactation in comparison with early and mid stages (p = 0.001 and 0.001, respectively). On the contrary, no significant differences were found for erythrocytes count, hemoglobin, serum cholesterol, catalase and triiodothyronine at different stages of lactation. There were high and positive correlations between daily-MY and serum total protein (r = 0.87, P < 0.01) and triiodothyronine (r = 0.41, P < 0.01). However, negative estimates were reported between daily-MY and triglycerides (r = ?0.55, P < 0.01) and cholesterol (r = ?0.33, P < 0.05). Our results indicate that dairy Baladi goats produce milk with relatively stable protein, fat and solid not fat (SNF) contents at the different stages of lactation, encouraging the continuous utilization of their milk in processing. Also, dairy Baladi goats seem able to maintain the most vital biochemical parameters.     

Seasonal variations in fatty acid composition of pasture forage plants and CLA content in ewe milk fat

The relations between fatty acids (FAs) composition of pasture forage plants and the content of conjugated linoleic acid (CLA) as a total content of cis-9, trans-11 + trans-7, cis-9 + trans-8, cis-10 CLA isomers in ewes’ milk fat during natural pasture season (April–September) were investigated. The extracts of ewes’ milk fat samples as well as the pasture samples were analyzed for fatty acid composition by capillary gas chromatography with flame ionization and mass spectrometric detection. α-Linolenic, linoleic, and palmitic acids were predominant in pasture plants, and their contents varied during pasture season. The most abundant and most varied fatty acid compound in pasture plants was α-linolenic acid. Its content significantly decreased from 62% to 39% (of total FA) (P < 0.001) from May to August, and subsequently it slightly (57%) increased from August to September (P < 0.05), compared with the beginning of pasture season. Similarly, the content of CLA in ewes’ milk fat decreased from 2.4% in May to 1.3% in August (P < 0.001), and subsequently it rose to 2.6% in September (P < 0.001). The α-linolenic/linoleic acid ratio in the pasture sample decreased from 4.36 in May to 1.97 in August (P < 0.001), and subsequently it increased to 3.14 in September (P < 0.001); thus, it reached the level approaching to that at the beginning of pasture season. The pasture seasonal variations in the ratio were directly proportional to the corresponding content of CLA and indirectly proportional to the ratio in ewes’ milk fat. The results suggest that the seasonal variations in CLA content in ewes’ milk fat are related primarily to the seasonal variation in α-linolenic acid content in grass lipids.     

Serum MicroRNAs Related with Chemoradiotherapy Resistance in Advanced-Stage Cervical Squamous Cell Carcinoma

OBJECTIVE: To investigate the serum microRNAs as biomarkers in predicting chemoradiotherapy resistance in advanced-stage cervical squamous cell carcinoma (ACSCC) patients. METHODS: Serum samples were collected from International Federation of Gynecology and Obstetrics (FIGO) stage IIB to IIIB cervical squamous cell carcinoma patients treated with platinum based Concomitant Chemoradiotherapy (CCRT) in our hospital during September 2013 to November 2015. Twenty well-matched samples (10 resistant and 10 sensitive) were chosen to screen the miRNA expression profile using serum samples pooled with microarrays. miRNAs expressed significantly different between two groups were further verified in 131 patients (29 resistant and 102 sensitive) serum samples with TaqMan Real-time PCR. The AUC was used to evaluate the accuracy of the biomarkers for prediction. RESULTS: MiR-136-5, miR-152-3p and miR-206 were expressed significantly different between sensitive and resistant groups. Results of 131 patients verification showed that the levels of miR-206 in sensitive samples and resistant samples were 2.715 ± 0.2115 and 14.64 ± 1.184, respectively, which was significantly different (P < .0001), while miR-136-5p and miR-152-3p could not be tested without pre-amplification reactions. Univariate analysis revealed that miR-206 expression was significantly associated with patients' DFS. Multivariate analysis demonstrated that miR-206 expression, tumor differentiation and pelvic lymph nodes metastasis were the independent prognostic factors associated with DFS in this cohort (P = .008, 0.000, 0.000, respectively). The probability of the prognostic accuracy of miR-206 expression in predicting chemoradiotherapy sensitivity of ACSCC patients was 91.3% (79.3% sensitivity and 92.2% specificity). CONCLUSION: Serum miR-206 is a powerful tool in predicting chemoradiotherapy sensitivity in ACSCC patients.     

A Long Noncoding RNA Signature That Predicts Pathological Complete Remission Rate Sensitively in Neoadjuvant Treatment of Breast Cancer

BACKGROUND: Mounting evidence suggests that long noncoding RNAs (lncRNAs) are closely related to pathological complete response (pCR) in neoadjuvant treatment of breast cancer. Here, we construct lncRNA associated models to predict pCR rate. METHODS: LncRNA expression profiles of breast cancer patients treated with neoadjuvant chemotherapy (NAC) were obtained from Gene Expression Omnibus by repurposing existing microarray data. The prediction model was firstly built by analyzing the correlation between pCR and lncRNA expression in the discovery dataset GSE 25066 (n = 488). Another three independent datasets, GSE20194 (n = 278), GSE20271 (n = 178), and GSE22093 (n = 97), were integrated as the validation cohort to assess the prediction efficiency. RESULTS: A novel lncRNA signature (LRS) consisting of 36 lncRNAs was identified. Based on this LRS, patients with NAC treatment were divided into two groups: LRS-high group and LRS-low group, with positive correlation of pCR rate in the discovery dataset. In the validation cohort, univariate and multivariate analyses both demonstrated that high LRS was associated with higher pCR rate. Subgroup analysis confirmed that this model performed well in luminal B [odds ratio (OR) = 5.4; 95% confidence interval (CI) = 2.7-10.8; P = 1.47e-06], HER2-enriched (OR = 2.5; 95% CI = 1.1-5.7; P = .029), and basal-like (OR = 5.5; 95% CI = 2.3-16.2; P = 5.32e-04) subtypes. Compared with other preexisting prediction models, LRS demonstrated better performance with higher area under the curve. Functional annotation analysis suggested that lncRNAs in this signature were mainly involved in cancer proliferation process. CONCLUSION: Our findings indicated that our lncRNA signature was sensitive to predict pCR rate in the neoadjuvant treatment of breast cancer, which deserves further evaluation.     

Prognostic Value of Molecular Breast Cancer Subtypes based on Her2, ESR1, PGR and Ki67 mRNA-Expression in Muscle Invasive Bladder Cancer

INTRODUCTION: Gene expression analyses have identified similarities between bladder and breast cancer, where clinical risk stratification is based on Her2, ESR1, PGR and Ki67 expression. The aim of the study was to assess the respective marker gene expression in patients treated with radical cystectomy for muscle-invasive bladder cancer (MIBC) and to evaluate the applicability of breast cancer subtypes for MIBC risk stratification. MATERIALS & METHODS: 102 patients treated with radical cystectomy for MIBC were assessed. Using routine FFPE tissue and an IVD validated kit, mRNA expression was measured by single step RT-qPCR. Partition test were employed to define cut-off values for high or low marker gene expression. Association of expression with outcome was assessed using Kaplan-Meier analysis and multivariate cox regression analysis. Finally, we performed validation of our results in the MD-Anderson cohort (n = 57). RESULTS: Cancer specific survival (CSS) was impaired in patients with high gene expression of Her2 (P = 0.0009) and ESR1 (P = 0.04). In the multivariate regression model Her2 expression remained significant for the prediction of CSS (HR = 2.11, CI 1.11-4.21, P = 0.024). Furthermore, molecular stratification by breast cancer subgroups was significant (P = 0.023) for CSS prediction. Especially the differentiation between Her2-positive and Luminal A (HR = 4.41, CI 1.53-18.71, P = 0.004) and Luminal B (HR = 1.96, CI 0.99-4.08, P = 0.053) respectively was an independent prognostic parameter for CSS. External validation resulted in comparable risk stratification with differences in fractional subgroups distribution. CONCLUSION: Gene expression of Her2, ESR1, PGR, Ki67 and corresponding breast cancer subtypes allow a risk-stratification in MIBC, whereby Her2 overexpressing tumors reveal a particularly poor prognosis.     

Study the influence of culture conditions on rennin production by Rhizomucor miehei using solid-state fermentations

Investigations were conducted on the production of Rennin enzyme from the fungi Rhizomucor miehei 3420 NRRL using Solid-State fermentation. Wheat bran was used as a substrate. The influence of moisture content, incubation temperature, and the initial pH of fermentation medium were studied. The protein content, milk clotting activity (MCA), specific activity, proteolytic activity (PA), and (MCA/PA) ratio of the extracted enzyme were calculated after 4 days of incubation to evaluate the quality of the enzyme. The results showed that the optimal conditions for production were as follows: incubation temperature of 40 °C, moisture content of 60%, and pH of (3). Under these conditions, a production process of Rennin enzyme was established, and the values of protein content, milk clotting activity, specific activity, proteolytic activity, and (MCA/PA) ratio reached to 4 mg/mL, 600 SU/mL, 150 SU/mg, 45 PU/mL, 13.3 respectively.