Label-free Raman spectroscopy reveals tumor microenvironmental changes induced by intermittent fasting for the prevention of breast cancer in animal model.

The development of tools that can provide a holistic picture of the evolution of the tumor microenvironment in response to intermittent fasting on the prevention of breast cancer is highly desirable. Here, we show, for the first time, the use of label-free Raman spectroscopy to reveal biomolecular alterations induced by intermittent fasting in the tumor microenvironment of breast cancer using a dimethyl-benzanthracene induced rat model. To quantify biomolecular alterations in the tumor microenvironment, chemometric analysis of Raman spectra obtained from untreated and treated tumors was performed using multivariate curve resolution-alternative least squares and support vector machines. Raman measurements revealed remarkable and robust differences in lipid, protein, and glycogen content prior to morphological manifestations in a dynamically changing tumor microenvironment, consistent with the proteomic changes observed by quantitative mass spectrometry. Taken together with its non-invasive nature, this research provides prospective evidence for the clinical translation of Raman spectroscopy to identify biomolecular variations in the microenvironment induced by intermittent fasting for the prevention of breast cancer, providing new perspectives on the specific molecular effects in the tumorigenesis of breast cancer.

Imaging the levator ani and the puborectalis muscle: implications in understanding regional anatomy, physiology and pathology.

Purpose:To review the findings of recent dynamic imaging of the levator ani muscle in order to explain its function during defecation. Historical anatomical studies have suggested that the levator ani initiates defecation by lifting the anal canal, with conventional dissections and static radiologic imagery having been equated with manometry and electromyography.Materials and methods:An analysis of the literature was made concerning the chronological development of imaging modalities specifically designed to assess pelvic floor dynamics. Comparisons are made between imaging and electromyographic data at rest and during provocative manoeuvres including squeeze and strain.Results:The puborectalis muscle is shown distinctly separate from the levator ani and the deep external anal sphincter. In contrast to conventional teaching that the levator ani initiates defecation by lifting the anus, dynamic illustration defecography (DID) has confirmed that the abdominal musculature and the diaphragm instigate defecation with the transverse and vertical component portions of the levator ani resulting in descent of the anus. Current imaging has shown a tendinous peripheral structure to the termination of the conjoint longitudinal muscle, clarifying the anatomy of the perianal spaces. Planar oXy defecography has established patterns of movement of the anorectal junction that separate controls from those presenting with descending perineum syndrome or with anismus (paradoxical puborectalis spasm).Conclusions:Dynamic imaging of the pelvic floor (now mostly with MR proctography) has clarified the integral role of the levator ani during defecation. Rather than lifting the rectum, the muscle ensures descent of the anal canal.

Microcrystalline cellulose grafted hyperbranched polyester with roll comb structure for synergistic toughening and strengthening of microbial PHBV/bio-based polyester elastomer composites.

The brittle feature of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is the major challenge that strongly restricts its application at present. Successfully synthesized bio-based engineering polyester elastomers (BEPE) were combined with PHBV to create entirely bio-composites with the intention of toughening PHBV. Herein, the 2,2-Bis(hydroxymethyl)-propionic acid (DMPA) was grafted onto microcrystalline cellulose (MCC) and then further transformed into hyperbranched polyester structure via polycondensation. The modified MCC, named MCHBP, had plenty of terminal hydroxyl groups, which get dispersed between PHBV and BEPE. Besides, a large number of terminal hydroxyl groups of MCHBP can interact with the carbonyl groups of PHBV or BEPE in a wide range of hydrogen bonds, and subsequently increase the adhesion and stress transfer between the PHBV and BEPE. The tensile toughness and the elongation at break of the PHBV/BEPE composites with 0.5phr MCHBP were improved by 559.7 % and 221.8 % in comparison to those of PHBV/BEPE composites. Results also showed that MCHBP can play a heterogeneous nucleation effect on the crystallization of PHBV. Therefore, this research can address the current issue of biopolymers' weak mechanical qualities and may have uses in food packaging.

Highly selective fluorescent probe with an ideal pH profile for the rapid and unambiguous determination of subcellular labile iron (III) pools in human cells.

A convenient tool for detecting iron ions in subcellular structures is desired for better understanding its roles in biological systems. In this work, a new Fe3+ sensor, 2-(2-((1-(6-acetylpyridin-2-yl)ethylidene)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthen]-3-one (RhPK), which operates across the entire cellular pH range and is capable of unambiguously detecting Fe3+ ion in live human cells at subcellular resolution, is reported. The sensor exhibits high selectivity and sensitivity toward Fe3+ with a rapid fluorescence response and a 12-fold increase in intensity upon the addition of 1 equivalent Fe3+ at pH 7.3. RhPK forms a 1:1 complex with Fe3+ with an apparent binding constant 1.54 × 107 M-1 and a detection limit of 50 nM. The sensor is stable between pH 4.2 and 9.0 and operates across the whole cellular pH range. Cell imaging demonstrates the ability of the sensor to unambiguously detect basal level Fe3+ as well as its dynamic changes in real-time in live cells at subcellular resolution, with one labile Fe3+ pool identified in mitochondria in human primary fibroblast (ws1) cells for the first time and two Fe3+ pools confirmed in mitochondria and endo/lysosomes in human SH-SY5Y neuroblastoma cells, suggesting different cell types have distinctive Fe3+ storage in subcellular compartments. The RhPK probe is powerful for rapid and sensitive bioimaging of Fe3+ at subcellular level, enabling the unambiguous detection of labile Fe3+ pools at the entire cellular pH range, which is of great significance to understand the biological chemistry of Fe3+ and its roles in physiological processes and diseases.

Facile synthesis of a novel genetically encodable fluorescent α-amino acid emitting greenish blue light.

We report the facile synthesis and characterization of a novel fluorescent α-amino acid 4-phenanthracen-9-yl-l-phenylalanine (Phen-AA) (5) that emits greenish blue light in the visible region. This genetically encodable l-α-amino acid has excellent photostability with a 75% quantum yield. It readily gets into human cells, being clearly imaged upon 405 nm laser excitation. The synthetic procedure is resistant to racemization and only involves three simple steps which use mild conditions and generate the Phen-AA in reasonably good yield. It may find broad applications in research, biotechnology, and the pharmaceutical industry.

A novel near-infrared turn-on and ratiometric fluorescent probe capable of copper(ii) ion determination in living cells.

A near-infrared ratiometric fluorescent probe CR-Ac based on a coumarin-benzopyrylium platform has been developed for selective detection of Cu2+. The cell imaging data revealed the capabilities of CR-Ac in monitoring the dynamic changes of subcellular Cu2+ and the quantification of Cu2+ levels in living cells.

Reaction-based turn-on fluorescent probes with magnetic responses for Fe(2+) detection in live cells.

Iron is the most abundant nutritionally essential transition metal found in the human body. It plays important roles in various biological processes such as oxygen delivery, electron transport, enzymatic reactions and DNA synthesis and repair. However, iron can also catalyze the production of free radicals, which are linked to quite a few diseases such as cancer, neurodegenerative diseases, and cardiovascular diseases. Both iron deficiency and iron overload are related to various health problems. Thus, precisely monitoring iron ions (Fe(2+) and Fe(3+)) in biological systems is important in understanding the detailed biological functions of iron and its trafficking pathways. However, effective tools for monitoring labile Fe(2+) in biological systems have not yet been established. Reported herein are turn on, reaction-based coumarin and rhodamine-linked nitroxide probes (Cou-T and Rh-T) for selective detection of Fe(2+) in solution and in living cells. Rh-T displayed a unique change in the EPR signal as well as enhancement of the fluorescence signal resulting from a specific redox reaction between the probe and Fe(2+). The turn-on fluorescence response towards Fe(2+) allows the subcellular imaging of endogenous Fe(2+) as well as imaging under conditions of external iron supplementation or depletion, with a labile Fe(2+) pool located in the mitochondria of human fibroblast primary cells. The detection and mechanism were verified by the magnetic properties of the probe via electron paramagnetic resonance (EPR) spectroscopy in solution and in cells.

Zinc-binding sites on selected flavonoids.

Flavonoids have attracted increased attention due to their broad bioactivities related to health and diseases. Modulating metal homeostasis may play an important role in their bioactivities. Recent studies have suggested that dietary flavonoids may affect zinc homeostasis, uptake, and transport. In this work, the zinc-binding sites on a few selected flavonoids have been investigated by (1)H NMR spectroscopy under physiological relevant pH and the species formed were verified by mass spectrometry. Zinc binding induces distinct changes in the proton resonances on the flavonoid rings, providing useful information to locate the Zn-binding sites. No Zn-binding was observed with flavone which lacks a chelation site. Zinc was found to bind to the 3-hydroxyl-4-keto, catechol, and 5-hydroxyl-4-keto chelation sites of flavonol, 3',4'-dihydroxylflavone and chrysin, respectively. Kaempferol and myricetin chelate zinc at the 3-hydroxyl-4-keto site while rutin binds zinc preferentially at the 5-hydroxyl-4-keto site. However, morin appears to bind zinc at the 1-ether-2-hydroxyl site.

A novel protein extracted from foxtail millet bran displays anti-carcinogenic effects in human colon cancer cells.

Millet is an important cereal food and exhibits multiple biological activities, including immunodulatory, antioxidant, antifungal and anti-hyperglycemia effects. Herein, we describe a novel 35kDa protein with anti-cancer properties, named FMBP, which was extracted and purified from foxtail millet bran by cell-based screening. FMBP is highly homologous to peroxidase as revealed by mass spectrometry and gene sequencing analysis. Furthermore, in vivo anti-tumor results implicated that the novel protein FMBP had the ability to suppress xenografted tumor growth in nude mice. Mechanistically, FMBP is able to suppress colon cancer cell growth through induction of G1 phase arrest and also causes a loss of mitochondrial transmembrane potential which results in caspase-dependent apoptosis in colon cancer cells. Notably, FMBP has much lower toxicity in normal colon epithelial cells. Taken together, FMBP has targeted anti-colon cancer effects and may serve as a therapeutic agent against colon cancer.

Direct observation of internalization and ROS generation of amyloid ß-peptide in neuronal cells at subcellular resolution.

Seeing in many colors: Confocal images acquired using fluorescently labeled amyloid ß-peptide revealed its efficient internalization by endocytosis into endosomes/lysosomes of human neuronal cells with a small portion reaching mitochondria, inducing marked cellular and mitochondrial reactive oxygen species production.

A turn-on fluorescent sensor for imaging labile Fe(3+) in live neuronal cells at subcellular resolution.

An eye for an iron: A highly sensitive, selective and reversible turn-on Fe(3+) sensor for imaging labile Fe(3+) in live cells at subcellular resolution is reported. The sensor can respond to changes in intracellular Fe(3+) levels and was used to image endogenous chelatable Fe(3+) in live human neuroblastoma SH-SY5Y cells, with two Fe(3+) pools being identified in mitochondria and endosomes/ lysosomes for the first time.

Exploration of biguanido-oxovanadium complexes as potent and selective inhibitors of protein tyrosine phosphatases.

The inhibitory effects of three biguanido-oxovanadium complexes ([VO(L(1-3))(2)]·nH(2)O: HL(1) = metformin, HL(2) = phenformin, HL(3) = moroxydine) against four protein tyrosine phosphatases (PTPs) and an alkaline phosphatase (ALP) were investigated. The complexes display strong inhibition against PTP1B and TCPTP (IC(50), 80-160 nM), a bit weaker inhibition against HePTP (IC(50), 190-410 nM) and SHP-1(IC(50), 0.8-3.3 µM) and much weaker inhibition against ALP (IC(50), 17-35 µM). Complex 3 is about twofold less potent against PTP1B, TCPTP and HePTP than complexes 1 and 2, while complex 2 inhibits SHP-1 more strongly (about three to fourfold) than the other two complexes. These results suggest that the structures of the ligands slightly influence the potency and selectivity against PTPs. The complexes inhibit PTP1B and ALP with a typical competitive type.

Ternary oxovanadium(IV) complexes with amino acid-Schiff base and polypyridyl derivatives: synthesis, characterization, and protein tyrosine phosphatase 1B inhibition.

To investigate the structure-activity relationship of vanadium complexes in inhibiting protein tyrosine phosphatase1B (PTP1B), eight mixed-ligand oxovanadium(IV) complexes, [V(IV)O(SalAla)(NN)] (H(2)SalAla for salicylidene alanine, NN for N,N'-donor heterocyclic base, namely, 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 3), dipyrido[3,2-a:2',3'-c]phenazine (dppz, 4)), [V(IV)O(SalLys)(dpq)] (5), [V(IV)O(SalLys)(dppz)] (6), [V(IV)O(SalAsp)(dppz)], (7) and [V(IV)O(SalTrp)(dppz)] (8)), of which 3-8 are new, have been prepared and characterized by elemental analysis, infrared, UV-visible, electrospray ionization mass spectrometry and conductivity. The molar conductance data confirmed the non-electrolytic nature of the complexes in DMSO solution. The coordination in [V(IV)O (SalAla)(phen)] (2) was confirmed by X-ray crystal structure analysis. The oxidation state of V(IV) with d(1) configuration in 2 was confirmed by EPR. The speciation of VO-SalAla-phen in aqueous solution was investigated by potentiometric pH titrations. The results indicate that the main species are two ternary complexes at the pH range 7.0-7.4. Biochemical assays demonstrate that the mixed-ligand oxovanadium(IV) complexes are potent inhibitors of PTP1B with IC(50) values in the range of 62-597nM, approximately 3-10 fold weaker in potency than those of similar mixed-ligand oxovanadium(IV) complexes of salicylidene anthranilic acid (SAA) derivative with polypyridyl ligands, except complex 8, which exhibits comparable or better inhibition activity than those of the mixed-ligand oxovanadium(IV) complexes of SAA derivative with polypyridyl ligands. The results demonstrate that the structures of vanadium complexes influence the PTP1B inhibition activity. Kinetics assays reveal that complex 2 inhibits PTP1B in a competitive manner.

MRI anatomy of the anal region.

PURPOSE: The aim of this study was to identify the normal anatomy of the anal region on magnetic resonance images. METHODS: T1-weighted turbo spin-echo images of anal sagittal sections, anal coronal sections, and oblique anal transverse planes were obtained with a body coil in 60 normal volunteers (30 women and 30 men, aged 19-25 years) at rest in the supine position. RESULTS: T1-weighted images showed fat spaces and muscles simultaneously, allowing visualization of 7 image layers, including the mucosa, submucosa, anal smooth muscle, inner (intersphincteric) space, vertical levator, outer (intersphincteric) space, and external anal sphincter. The anal smooth muscle was derived from the rectal smooth muscle, and the inner space originated from the perirectal space. The outer space lay between the vertical levator and the external sphincters. The puborectalis did not have a longitudinal portion. The deep, superficial, and SC sphincters were 3 separate muscle bundles. The perianal spaces had a complex interconnection. CONCLUSIONS: Multiplanar body-coil MRI studies can show anorectal fat spaces and musculature simultaneously, allowing fat spaces and musculature to serve as mutual referents. The results of imaging of the anal region with this method are different from previous imaging descriptions and may provide a more accurate and systemic description of the anal region structures than was previously available.

Inhibition protein tyrosine phosphatases by an oxovanadium glutamate complex, Na2[VO(Glu)2(CH3OH)](Glu = glutamate).

The insulin-sensitizing effect of vanadium complexes has been linked to their ability to inhibit protein tyrosine phosphatases (PTPs). Considering that vanadium complexes may exchange in vivo with amino acids, forming in situ vanadium-amino acid complexes, we have synthesized and characterized an oxovanadium glutamate complex, Na(2)[V(IV)O(Glu)(2)(CH(3)OH)]H(2)O (1·H(2)O). The complex showed potent inhibition against four human PTPs (PTP1B, TCPTP, HePTP, and SHP-1) with IC(50) in the 0.21-0.37 µM ranges. Fluorescence titration studies suggest that the complex binds to PTP1B with the formation of a 2:1 complex. Enzyme kinetics analysis using Lineweaver-Burk plots indicates a typical competitive inhibition mode.

Synthesis, characterization, and protein tyrosine phosphatases inhibition activities of oxovanadium(IV) complexes with Schiff base and polypyridyl derivatives.

Seven new mixed-ligand vanadyl complexes, [V(IV)O(5-Br-SAA)(NN)] and [V(IV)O(2-OH-NAA)(NN)] (1-7) (5-Br-SAA for 5-bromosalicylidene anthranilic acid, 2-OH-NAA for 2-hydroxy-1-naphthaldehyde anthranilic acid and NN for N,N'-donor heterocyclic base, namely, 2,2'-bipyridine (bpy, 1 and 5), 1,10-phenanthroline (phen, 2 and 6), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 3 and 7), dipyrido[3,2-a:2',3'-c]phenazine (dppz, 4)), were synthesized and characterized. X-ray crystal structure of [V(IV)O(5-Br-SAA)(phen)] revealed a distorted octahedral geometry with the Schiff base ligand coordinated in a tridentate ONO-fashion and the phenanthroline ligand in a bidentate fashion. Density-functional theory (DFT) calculations suggest a similar structure and the same coordination mode for all the other oxovanadium complexes synthesized. Biochemical assays demonstrate that the mixed-ligand oxovanadium(IV) complexes are potent inhibitors of protein tyrosine phosphatase 1B (PTP1B), with IC(50) values approximately 41-75 nM. Kinetics assays suggest that the complexes inhibit PTP1B in a competitive manner. Notably, they had moderate selectivity of PTP1B over T-cell protein tyrosine phosphatase (TCPTP) (about 2-fold) and good selectivity over Src homology phosphatase 1 (SHP-1) (about 4 approximately 7-fold). Thus, these mixed-ligand complexes represent a promising class of PTP1B inhibitors for future development as anti-diabetic agents.

A novel profluorescent probe for detecting oxidative stress induced by metal and H(2)O(2) in living cells.

A profluorescent probe that has no fluorescent response to H(2)O(2), iron or copper ions but can be readily activated in the presence of both H(2)O(2) and Fe (or Cu) ion has been developed; the probe is capable of detecting oxidative stress promoted by Fe (or Cu) and H(2)O(2) (i.e. the Fenton reaction conditions) in living cells.

Potent inhibition of protein tyrosine phosphatase 1B by copper complexes: implications for copper toxicity in biological systems.

A new dinuclear copper complex and several Cu-amino acid complexes inhibit protein tyrosine phosphatase 1B competitively at submicromolar levels, suggesting that copper complexes may interfere with cellular signaling pathways by inhibiting protein tyrosine phosphatases.

Ternary oxovanadium(IV) complexes of ONO-donor Schiff base and polypyridyl derivatives as protein tyrosine phosphatase inhibitors: synthesis, characterization, and biological activities.

A series of oxovanadium complexes with mixed ligands, a tridentate ONO-donor Schiff base ligand [viz., salicylidene anthranilic acid (SAA)], and a bidentate NN ligand [viz., 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), dipyrido[3,2-a:2',3'-c]phenazine (dppz), or 7-methyldipyrido[3,2-a:2',3'-c]phenazine (dppm)], have been synthesized and characterized by elemental analysis, electrospray ionization mass spectrometry, UV-vis spectroscopy, Fourier transform IR spectroscopy, EPR spectroscopy, and X-ray crystallography. Crystal structures of both complexes, [V(IV)O(SAA)(bpy)].0.25bpy and [V(IV)O(SAA)(phen)].0.33H(2)O, reveal that oxovanadium(IV) is coordinated with one nitrogen and two oxygen atoms from the Schiff base and two nitrogen atoms from the bidentate planar ligands, in a distorted octahedral geometry (VO(3)N(3)). The oxidation state of V(IV) with d(1) configuration was confirmed by EPR spectroscopy. The speciation of VO-SAA-bpy in aqueous solution was investigated by potentiomtreic pH titrations, and the results revealed that the main species are two ternary complexes at a pH range of 7.0-7.4, and one is the isolated crystalline complex. The complexes have been found to be potent inhibitors against human protein tyrosine phosphatase 1B (PTP1B) (IC(50) approximately 30-61 nM), T-cell protein tyrosine phosphatase (TCPTP), and Src homology phosphatase 1 (SHP-1) in vitro. Interestingly, the [V(IV)O(SAA)(bpy)] complex selectively inhibits PTP1B over the other two phosphatases (approximate ninefold selectivity against SHP-1 and about twofold selectivity against TCPTP). Kinetics assays suggest that the complexes inhibit PTP1B in a competitive and reversible manner. These suggest that the complexes may be promising candidates as novel antidiabetic agents.

A novel H2O2-triggered anti-Fenton fluorescent pro-chelator excitable with visible light.

A chelator and a pro-chelator that can be activated by H(2)O(2) and subsequently sequesters iron and attenuates the Fenton reaction have been developed; both molecules are fluorescent excitable by visible light, and H(2)O(2)-activation, as well as iron-chelation, induces remarkable changes in fluorescence.