A comprehensive evaluation of arginine and its derivatives as protein formulation stabilizers.

Arginine and its derivatives (such as arginine ethyl ester and acetyl arginine) have varying degrees of protein aggregation suppressor effect across different protein solutions. To understand this performance ambiguity, we evaluated the activity of arginine, acetyl arginine, and arginine ethyl ester for aggregation suppressor effect against human intravenous immunoglobulin G (IgG) solution at pH 4.8. Both arginine and its cationic derivative arginine ethyl ester in their hydrochloride salt forms significantly reduced the colloidal and conformational stability (reduced kd and Tm) of IgG. Consequently, the monomer content was decreased with an increase in subvisible particulates after agitation or thermal stress. Furthermore, compared to arginine, arginine ethyl ester with one more cationic charge and hydrochloride salt form readily precipitated IgG at temperatures higher than 25 °C. On the contrary, acetyl arginine, which mostly exists in a neutral state at pH 4.8, efficiently suppressed the formation of subvisible particles retaining a high amount of monomer owing to its higher colloidal and conformational stability. Concisely, the charged state of additives significantly impacts protein stability. This study demonstrated that contrary to popular belief, arginine and its derivatives may either enhance or suppress protein aggregation depending on their net charge and concentration.

LC/MS-Based Polar Metabolite Profiling Identified Unique Biomarker Signatures for Cervical Cancer and Cervical Intraepithelial Neoplasia Using Global and Targeted Metabolomics.

Cervical cancer remains one of the most prevalent cancers among females worldwide. Therefore, it is important to discover new biomarkers for early diagnosis of cervical intraepithelial neoplasia (CIN) and cervical cancer, preferably non-invasive ones. In the present study, we aimed to identify unique metabolic signatures for CINs and cervical cancers using global and targeted metabolomic profiling. Plasma samples (69 normal, 55 CIN1, 42 CIN2/3, and 60 cervical cancer) were examined by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-QTOF-MS) coupled with multivariate statistical analysis. Metabolic pathways were analyzed using the integrated web-based tool MetaboAnalyst. A multivariate logistic regression analysis was conducted to evaluate the combined association of metabolites and human papillomavirus (HPV) status with the risk of cervical carcinogenesis. A total of 28 metabolites exhibiting discriminating levels among normal, CIN, and cervical cancer patients (Kruskal-Wallis test p < 0.05) were identified in the global profiling analysis. The pathway analysis showed significantly altered alanine, aspartate, and glutamate metabolic pathways (FDR p-value < 0.05) in both the discovery and validation phases. Seven metabolites (AMP, aspartate, glutamate, hypoxanthine, lactate, proline, and pyroglutamate) were discriminated between CINs and cervical cancer versus normal (area under the curve (AUC) value > 0.8). The levels of these metabolites were significantly high in patients versus normal (p < 0.0001) and were associated with increased risk of developing CIN2/3 and cervical cancer. Additionally, elevated levels of the seven metabolites combined with positive HPV status were correlated with substantial risk of cancer progression. These results demonstrated that metabolomics profiling is capable of distinguishing CINs and cervical cancers from normal and highlighted potential biomarkers for the early detection of cervical carcinogenesis.

Prediction of Recurrence With KRAS Mutational Burden Using Ultrasensitive Digital Polymerase Chain Reaction of Radial Resection Margin of Resected Pancreatic Ductal Adenocarcinoma.

OBJECTIVE: Although complete surgical resection is the only curative method for pancreatic cancer, the radial resection margins of pylorus-preserving pancreaticoduodenectomy specimens might be underevaluated. METHODS: KRAS mutation was assessed with droplet digital polymerase chain reaction on cells collected from the radial resection margins of 81 patients, and the results were compared with those of conventional pathologic resection margin (pRM) evaluation. RESULTS: KRAS mutation was detected in 76 patients (94%), and molecular resection margin (mRM) positivity defined by a KRAS mutation rate of 4.19% or greater was observed in 18 patients (22%). Patients with mRM-positive had significantly worse recurrence-free survival (RFS) than those with mRM-negative in entire groups (P = 0.008) and in subgroups without chemotherapy or radiation therapy (all, P < 0.001). When combined pRMs-mRMs were evaluated, patients with combined pRM-mRM-positive (either pRM- or mRM-positive) had significantly worse RFS than those with combined resection margin-negative (both pRM and mRM negative) by univariate (P = 0.002) and multivariate (P = 0.03) analyses. CONCLUSIONS: KRAS mutational analysis with ultrasensitive droplet digital polymerase chain reaction of the radial resection margin in pancreatic cancer patients who underwent pylorus-preserving pancreaticoduodenectomy can provide more accurate information on RFS by using alone or in combination with conventional pRM evaluation, especially in patients without chemotherapy or radiation therapy.

Metabolic Alterations Associated with Atorvastatin/Fenofibric Acid Combination in Patients with Atherogenic Dyslipidaemia: A Randomized Trial for Comparison with Escalated-Dose Atorvastatin.

In the current study, the metabolic effects of atorvastatin dose escalation versus atorvastatin/fenofibric acid combination were compared using metabolomics analyses. Men and women with combined hyperlipidaemia were initially prescribed atorvastatin (10 mg, ≥4 weeks). Patients who reached low-density lipoprotein-cholesterol targets, but had triglyceride and high-density lipoprotein-cholesterol levels ≥150 mg/dL and <50 mg/dL, respectively, were randomized to receive atorvastatin 20 mg or atorvastatin 10 mg/fenofibric acid 135 mg for 12 weeks. Metabolite profiling of serum was performed and changes in metabolites after drug treatment in the two groups were compared. Analysis was performed using patients' samples obtained before and after treatment. Of 89 screened patients, 37 who met the inclusion criteria were randomized, and 34 completed the study. Unlike that in the dose-escalation group, distinct clustering of both lipid and aqueous metabolites was observed in the combination group after treatment. Most lipid metabolites of acylglycerols and many of ceramides decreased, while many of sphingomyelins increased in the combination group. Atorvastatin dose escalation modestly decreased lysophosphatidylcholines; however, the effect of combination therapy was variable. Most aqueous metabolites decreased, while L-carnitine remarkably increased in the combination group. In conclusion, the atorvastatin/fenofibric acid combination induced distinct metabolite clustering. Our results provide comprehensive information regarding metabolic changes beyond conventional lipid profiles for this combination therapy.

Successful Treatment with a High-dose Rifampin-containing Regimen for Pulmonary Tuberculosis with a Disputed rpoB Mutation.

Mutations in the rpoB gene of Mycobacterium tuberculosis can result in resistance to rifampin. Among various mutations in the rpoB gene, some known as disputed rpoB mutations can cause low-level rifampin resistance. It has been suggested that a high-dose rifampin (20 mg/kg)-based regimen might be effective in treating tuberculosis (TB) caused by M. tuberculosis with disputed rpoB mutations exhibiting low-level resistance. We herein report the first two cases of pulmonary TB caused by M. tuberculosis with a disputed rpoB mutation (CTG511CCG) that showed successful treatment outcomes with a high-dose rifampin-based regimen.

MS-Based Metabolite Profiling of Aboveground and Root Components of Zingiber mioga and Officinale.

Zingiber species are members of the Zingiberaceae family, and are widely used for medicinal and food purposes. In this study aboveground and root parts of Zingiber mioga and Zingiber officinale were subjected to metabolite profiling by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) in order to characterize them by species and parts and also to measure bioactivities. Both primary and secondary metabolites showed clear discrimination in the PCA score plot and PLS-DA by species and parts. Tetrahydrocurcumin, diarylheptanoid, 8-gingerol, and 8-paradol were discriminating metabolites between Z. mioga and Z. officinale that were present in different quantities. Eleven flavonoids, six amino acids, six organic acids, four fatty acids, and gingerenone A were higher in the aboveground parts than the root parts. Antioxidant activities were measured and were highest in the root part of Z. officinale. The relatively high contents of tetrahydrocurcumin, diarylheptanoid, and galanganol C in the root part of Z. officinale showed highly positive correlation with bioactivities based on correlation assay. On the basis of these results, we can suggest different usages of structurally different parts of Zingiber species as food plants.

Features of atherosclerosis in hemodialysis patients.

BACKGROUND: Cardiovascular disease is the main cause of mortality in dialysis patients. Carotid intima-media thickness (CIMT) is used as a surrogate marker of early atherosclerosis. Atherosclerosis can cause vascular access failure. The purpose of this study was to define the clinical features of atherosclerosis in hemodialysis patients based on CIMT and to define the relationship between CIMT and access failure. METHODS: In this cross-sectional study, the CIMT of 60 patients on hemodialysis was examined using B-mode Doppler ultrasonography between May 2012 and November 2012. Carotid atherosclerosis was defined as a CIMT≥0.9 mm or the incidence of atherosclerotic plaques. RESULTS: The patients' mean age was 54.5±10.6 years, and 60% of the patients were male. The CIMT was 0.81±0.47 mm (range, 0.35-2.50 mm). The group with atherosclerosis was characterized by older age compared with those without atherosclerosis. Patients with atherosclerosis showed much shorter durations of access patency than their counterparts in the nonatherosclerosis group (hazard ratio, 2.822; 95% confidence interval, 1.113-7.156; P=0.029). Moreover, being overweight was associated with a 2.47-fold (95% confidence interval, 1.101-5.548) increased primary access failure. CONCLUSION: This study shows that atherosclerosis is associated with older age. Patients who are overweight and have atherosclerosis may have shortened access patency.

Molecular analysis of congenital scoliosis: a candidate gene approach.

The etiology of congenital scoliosis is largely unknown. The severe vertebral disorder, spondylocostal dysostosis type 1, is associated with a homozygous delta-like 3 (DLL3) mutation. Scoliosis has been observed in a heterozygous DLL3 carrier, raising the possibility of its involvement in congenital scoliosis. We present the first molecular study of congenital scoliosis by analysis of the candidate gene DLL3 and demonstrate one novel missense variant. However, no novel or previously described mutations are present in our cohort, indicating that DLL3 mutations may not be a major cause of congenital scoliosis. Additionally, we have evaluated patients with congenital scoliosis not diagnosed with a known syndrome and identified a significant number of associated renal and cardiac anomalies and familial incidence of idiopathic scoliosis in this group.

Regulation of Cyr61/CCN1 gene expression through RhoA GTPase and p38MAPK signaling pathways.

Cysteine-rich protein 61 (Cyr61/CCN1) is an angiogenic factor and a member of a family of growth factor-inducible immediate-early genes with functions in cell adhesion, proliferation and differentiation. We investigated the regulatory mechanisms and signaling pathways involved in Cyr61/CCN1gene activation in smooth muscle cells. Treatment of these cells with sphingosine 1-phosphate (S1P), a bioactive lysolipid, increased rapidly but transiently the expression of the Cyr61/CCN1 gene at both the mRNA and protein levels. Cyr61/CCN1 mRNA stability was not altered but the transcription rate of the Cyr61/CCN1 gene was increased fivefold in isolated nuclei from S1P-stimulated cells indicating that the level of control is primarily transcriptional. Transfection experiments showed that a 936-bp promoter fragment of the human Cyr61/CCN1 gene is functional and induces a reporter gene activity in S1P-treated cells. Using a combination of cis-element mutagenesis and expression of dominant negative inhibitors of transcription factors, we showed that both a CRE and AP-1 site and their cognate transcription factors, cAMP response element binding protein (CREB) and AP-1, were responsible for the promoter activity in S1P-stimulated cells. Furthermore, by using either pharmacological inhibitors or active forms of known signaling molecules, we showed that inducible Cyr61/CCN1 gene expression occurs through RhoA GTPase and that additional signaling through the p38 pathway is required. In particular, p38 seems to regulate Cyr61/CCN1 promoter activity through modulation of phosphorylation of CREB and the CREB kinase, MSK1. These findings demonstrate the transcriptional regulation of the Cyr61/CCN1 gene and provide clues to the signaling molecules and transcription factors involved in such regulation.

Cyr61 and CTGF are molecular markers of bladder wall remodeling after outlet obstruction.

Cysteine-rich protein (Cyr61) and connective tissue growth factor (CTGF) are key immediate early growth factors with functions in cell proliferation, differentiation, and extracellular matrix synthesis. Studies were performed to assess the gene expression profile of Cyr61 and CTGF in rat urinary bladder during growth in response to partial outlet obstruction. The mRNA levels of Cyr61 as determined by ribonuclease protection assay increased sharply after 1 day and remained elevated throughout the time period of the obstruction. This correlates well with increased bladder weight. The CTGF mRNA levels seemed to peak within the second week of the urethral obstruction and correlate well with increased type I collagen mRNA. The expression pattern of either Cyr61 or CTGF proteins corroborated that of their respective mRNAs. Immunohistochemical analyses showed that immunoreactivity of Cyr61 was confined to detrusor smooth muscle and that of CTGF was detected within both detrusor muscle and lamina propria layers. These data strongly indicate the involvement of Cyr61 and CTGF in bladder wall remodeling as a result of the outlet obstruction.

Mechanical regulation of IGF-I and IGF-binding protein gene transcription in bladder smooth muscle cells.

Mechanical forces are well known to modulate smooth muscle cell growth and synthetic phenotype. The signals controlling this process are complex and potentially involve changes in the expression of peptide growth factor genes such as those of the insulin-like growth factor (IGF) system. This study was designed to investigate the mechanical regulation of IGF-I and the binding proteins for IGF (IGFBPs) in smooth muscle cells cultured on a deformable surface and subjected to cyclic stretch. Using the RNase protection assay, we found that the application of a cyclic biaxial strain to cells induced a 2.5- to 4-fold increase in IGF-I mRNA levels after 8 h and an even greater increase after 16-24 h of stretch. This change was not affected by variations in the magnitude of the applied strain but was attenuated ( approximately 40%) when cells were treated with antagonists for angiotensin II receptors. Furthermore, the transcript levels of the three major IGF binding proteins produced in smooth muscle cells, e.g., IGFBP-2, IGFBP-4, and IGFBP-5, varied between stretched and control cells. Both IGFBP-2 and IGFBP-4 mRNA levels were consistently reduced in stretched cells but remained comparable to those of the control cells when the angiotensin II transducing pathway was blocked by inhibitors prior to the application of mechanical strain. Conversely, the gene expression of IGFBP-5 was upregulated in stretched cells, and neutralizing antibodies to IGF-I blocked this activation. Similarly, pharmacologic inhibition of the phosphatidylinositol 3-kinase, an important component of the IGF receptor transduction pathway, inhibited IGFBP-5 gene expression in stretched cells. These results suggest that the downstream effects of mechanical strain on IGF-I and IGFBP transcript levels are mediated, to greater or lesser extent, either through an angiotensin II tranducing pathway or via a feedback loop involving the autocrine secretion of IGF-I itself.