High‐throughput instant quantification of protein expression and purity based on photoactive yellow protein turn off/on label

Quantifying the concentration and purity of a target protein is essential for high‐throughput protein expression test and rapid screening of highly soluble proteins. However, conventional methods such as PAGE and dot blot assay generally involve multiple time‐consuming tasks requiring hours or do not allow instant quantification. Here, we demonstrate a new method based on the Photoactive yellow protein turn Off/On Label (POOL) system that can instantly quantify the concentration and purity of a target protein. The main idea of POOL is to use Photoactive Yellow Protein (PYP), or its miniaturized version, as a fusion partner of the target protein. The characteristic blue light absorption and the consequent yellow color of PYP is absent when initially expressed without its chromophore, but can be turned on by binding its chromophore, p‐coumaric acid. The appearance of yellow color upon adding a precursor of chromophore to the co‐expressed PYP can be used to check the expression amount of the target protein via visual inspection within a few seconds as well as to quantify its concentration and purity with the aid of a spectrometer within a few minutes. The concentrations measured by the POOL method, which usually takes a few minutes, show excellent agreement with those by the BCA Kit, which usually takes ～1 h. We demonstrate the applicability of POOL in E. coli, insect, and mammalian cells, and for high‐throughput protein expression screening.     

RhoJ modulates melanoma invasion by altering actin cytoskeletal dynamics

Rho family GTPases regulate diverse processes in human melanoma ranging from tumor formation to metastasis and chemoresistance. In this study, a combination of in vitro and in vivo approaches was utilized to determine whether RHOJ, a CDC42 homologue that regulates melanoma chemoresistance, also controls melanoma migration. Depletion or overexpression of RHOJ altered cellular morphology, implicating a role for RHOJ in modulating actin cytoskeletal dynamics. RHOJ depletion inhibited melanoma cell migration and invasion in vitro and melanoma tumor growth and lymphatic spread in mice. Molecular studies revealed that RHOJ alters actin cytoskeletal dynamics by inducing the phosphorylation of LIMK, cofilin, and p41‐ARC (ARP2/3 complex subunit) in a PAK1‐dependent manner in vitro and in tumor xenografts. Taken together, these observations identify RHOJ as a melanoma linchpin determinant that regulates both actin cytoskeletal dynamics and chemoresistance by activating PAK1.     

Metabotropic Glutamate Receptor 1 Expression and Its Polymorphic Variants Associate with Breast Cancer Phenotypes

Several epidemiological studies have suggested a link between melanoma and breast cancer. Metabotropic glutamate receptor 1 (GRM1), which is involved in many cellular processes including proliferation and differentiation, has been implicated in melanomagenesis, with ectopic expression of GRM1 causing malignant transformation of melanocytes. This study was undertaken to evaluate GRM1 expression and polymorphic variants in GRM1 for associations with breast cancer phenotypes. Three single nucleotide polymorphisms (SNPs) in GRM1 were evaluated for associations with breast cancer clinicopathologic variables. GRM1 expression was evaluated in human normal and cancerous breast tissue and for in vitro response to hormonal manipulation. Genotyping was performed on genomic DNA from over 1,000 breast cancer patients. Rs6923492 and rs362962 genotypes associated with age at diagnosis that was highly dependent upon the breast cancer molecular phenotype. The rs362962 TT genotype also associated with risk of estrogen receptor or progesterone receptor positive breast cancer. In vitro analysis showed increased GRM1 expression in breast cancer cells treated with estrogen or the combination of estrogen and progesterone, but reduced GRM1 expression with tamoxifen treatment. Evaluation of GRM1 expression in human breast tumor specimens demonstrated significant correlations between GRM1 staining with tissue type and molecular features. Furthermore, analysis of gene expression data from primary breast tumors showed that high GRM1 expression correlated with a shorter distant metastasis-free survival as compared to low GRM1 expression in tamoxifen-treated patients. Additionally, induced knockdown of GRM1 in an estrogen receptor positive breast cancer cell line correlated with reduced cell proliferation. Taken together, these findings suggest a functional role for GRM1 in breast cancer.     

Semaphorin 3A Is a New Early Diagnostic Biomarker of Experimental and Pediatric Acute Kidney Injury

Background

Semaphorin 3A is a secreted protein that regulates cell motility and attachment in axon guidance, vascular growth, immune cell regulation and tumor progression. However, nothing is known about its role in kidney pathophysiology. Here, we determined whether semaphorin3A is induced after acute kidney injury (AKI) and whether urinary semaphorin 3A can predict AKI in humans undergoing cardiopulmonary bypass (CPB).

Methods and Principal Findings

In animals, semaphorin 3A is localized in distal tubules of the kidney and excretion increased within 3 hr after reperfusion of the kidney whereas serum creatinine was significantly raised at 24 hr. In humans, using serum creatinine, AKI was detected on average only 48 hours after CPB. In contrast, urine semaphorin increased at 2 hours after CPB, peaked at 6 hours (2596±591 pg/mg creatinine), and was no longer significantly elevated 12 hours after CPB. The predictive power of semaphorin 3A as demonstrated by area under the receiver-operating characteristic curve for diagnosis of AKI at 2, 6, and 12 hours after CPB was 0.88, 0.81, and 0.74, respectively. The 2-hour urine semaphorin measurement strongly correlated with duration and severity of AKI, as well as length of hospital stay. Adjusting for CPB time and gender, the 2-hour semaphorin remained an independent predictor of AKI, with an odds ratio of 2.19.

Conclusion

Our results suggest that semaphorin 3A is an early, predictive biomarker in experimental and pediatric AKI, and may allow for the reliable early diagnosis and prognosis of AKI after CPB, much before the rise in serum creatinine.     

Broad spectrum identification of SUMO substrates in melanoma cells

Like phosphorylation, protein sumoylation likely represents a dynamic PTM to alter protein function in support of cell regulatory systems. The broad-spectrum impact of transient or chronic engagement of signal transduction cascades on protein sumoylation has not been explored. Here, we find that epidermal growth factor (EGF) stimulation evokes a rapid alteration in small ubiquitin modifier (SUMO) target selection, while oncogene expression alters steady-state SUMO-protein profiles. A proteomic SUMO target analysis in melanoma cells identified proteins involved in cellular signaling, growth control, and neural differentiation.     

Investigation of structural stability and functionality of homodimeric gramicidin towards peptide-based drug: a molecular simulation approach

Increasing death rates due to antibiotic resistance deteriorate the existing treatment measures. Antimicrobial peptides have turned into the emerging cure for multidrug resistance. However, the stability and functionality determine an antimicrobial peptide as a drug. Analyses of the homodimeric β-helical peptide, gramicidin have suggested the significant role of gramicidin-A, gramicidin-B, and gramicidin-C as antimicrobial compounds, but the structural basis for understanding the stability and functionality is insufficient to resolve multidrug resistance. To identify the best template among gramicidin types as a therapeutic product, we combined a detailed comparative static analysis and dynamic analysis along with conformational free energy and secondary structure prediction. We observed that the high intramolecular interactions and the geometrical features favored gramicidin-A among other types of gramicidin. Our analyses further revealed that the secondary structure of gramicidin-A showed β sheets with coils along the conformations without any disruption, thereby enhanced its membrane interactions in terms of binding free energy. In conclusion, gramicidin-A has definitely showed enhanced structural stability and functionality; this could be considered the best template for a potential therapeutic product.     

Natural products as a hunting ground for combinatorial chemistry

Natural products have a long history of success as biologically active leads for therapeutic agents. The ability to prepare analogues and to discover structure-activity relationships is necessary to truly harness the potential of natural products. Recently, combinatorial chemistry has risen to this challenge, and even fairly complex natural products can be targeted for parallel synthesis. Academic and industrial efforts have employed natural products from the peptide, alkaloid, polyketide, and terpenoid and steroid classes in combinatorial chemistry approaches for the production of medicinally important compounds.     

Fatty acid production from amino acids and alpha-keto acids by Brevibacterium linens BL2

Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and alpha-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of alpha-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and alpha-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from alpha-keto acids only. BL2 also converted alpha-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of B. linens ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and alpha-keto acids and that carbon metabolism is important in regulating this event.     

Natural products and combinatorial chemistry: back to the future

The introduction of high-throughput synthesis and combinatorial chemistry has precipitated a global decline in the screening of natural products by the pharmaceutical industry. Some companies terminated their natural products program, despite the unproven success of the new technologies. This was a premature decision, as natural products have a long history of providing important medicinal agents. Furthermore, they occupy a complementary region of chemical space compared with the typical synthetic compound library. For these reasons, the interest in natural products has been rekindled. Various approaches have evolved that combine the power of natural products and organic chemistry, ranging from the combinatorial total synthesis of analogues to the exploration of natural product scaffolds and the design of completely unnatural molecules that resemble natural products in their molecular characteristics.     

Renal artery stenosis and ipsilateral renal cell carcinoma: description of an in situ partial nephrectomy and splenorenal arterial bypass

A case of a renal artery stenosis and ipsilateral renal cell carcinoma with long term results is reported. A 65-year-old man with renovascular hypertension, renal insufficiency, and nephrotic range proteinuria presented with an incidental renal cell carcinoma. Concomitant in situ left partial nephrectomy and splenorenal arterial bypass was achieved. The patient is doing well without evidence of malignancy, stable renal function, markedly improved proteinuria and stable blood pressure more than three years later. The techniques of this procedure are detailed and underscore the possibility of successful removal of a renal cell carcinoma with preservation of renal function despite renal artery stenosis.