Proinflammatory cytokines suppress nonsense-mediated RNA decay to impair regulated transcript isoform processing in pancreatic ß cells.

Introduction: Proinflammatory cytokines are implicated in pancreatic ß cell failure in type 1 and type 2 diabetes and are known to stimulate alternative RNA splicing and the expression of nonsense-mediated RNA decay (NMD) components. Here, we investigate whether cytokines regulate NMD activity and identify transcript isoforms targeted in ß cells. Methods: A luciferase-based NMD reporter transiently expressed in rat INS1(832/13), human-derived EndoC-ßH3, or dispersed human islet cells is used to examine the effect of proinflammatory cytokines (Cyt) on NMD activity. The gain- or loss-of-function of two key NMD components, UPF3B and UPF2, is used to reveal the effect of cytokines on cell viability and function. RNA-sequencing and siRNA-mediated silencing are deployed using standard techniques. Results: Cyt attenuate NMD activity in insulin-producing cell lines and primary human ß cells. These effects are found to involve ER stress and are associated with the downregulation of UPF3B. Increases or decreases in NMD activity achieved by UPF3B overexpression (OE) or UPF2 silencing raise or lower Cyt-induced cell death, respectively, in EndoC-ßH3 cells and are associated with decreased or increased insulin content, respectively. No effects of these manipulations are observed on glucose-stimulated insulin secretion. Transcriptomic analysis reveals that Cyt increases alternative splicing (AS)-induced exon skipping in the transcript isoforms, and this is potentiated by UPF2 silencing. Gene enrichment analysis identifies transcripts regulated by UPF2 silencing whose proteins are localized and/or functional in the extracellular matrix (ECM), including the serine protease inhibitor SERPINA1/α-1-antitrypsin, whose silencing sensitizes ß-cells to Cyt cytotoxicity. Cytokines suppress NMD activity via UPR signaling, potentially serving as a protective response against Cyt-induced NMD component expression. Conclusion: Our findings highlight the central importance of RNA turnover in ß cell responses to inflammatory stress.

Proinflammatory Cytokines Suppress Nonsense-Mediated RNA Decay to Impair Regulated Transcript Isoform Processing in Pancreatic ß-Cells.

Proinflammatory cytokines are implicated in pancreatic ß-cell failure in type 1 and type 2 diabetes and are known to stimulate alternative RNA splicing and the expression of Nonsense-Mediated RNA Decay (NMD) components. Here, we investigate whether cytokines regulate NMD activity and identify transcript isoforms targeted in ß-cells. A luciferase-based NMD reporter transiently expressed in rat INS1(832/13), human-derived EndoC-ßH3 or dispersed human islet cells is used to examine the effect of proinflammatory cytokines (Cyt) on NMD activity. Gain- or loss-of function of two key NMD components UPF3B and UPF2 is used to reveal the effect of cytokines on cell viability and function. RNA-sequencing and siRNA-mediated silencing are deployed using standard techniques. Cyt attenuate NMD activity in insulin-producing cell lines and primary human ß-cells. These effects are found to involve ER stress and are associated with downregulation of UPF3B. Increases or decreases in NMD activity achieved by UPF3B overexpression (OE) or UPF2 silencing, raises or lowers Cyt-induced cell death, respectively, in EndoC-ßH3 cells, and are associated with decreased or increased insulin content, respectively. No effects of these manipulations are observed on glucose-stimulated insulin secretion. Transcriptomic analysis reveals that Cyt increase alternative splicing (AS)-induced exon skipping in the transcript isoforms, and this is potentiated by UPF2 silencing. Gene enrichment analysis identifies transcripts regulated by UPF2 silencing whose proteins are localized and/or functional in extracellular matrix (ECM) including the serine protease inhibitor SERPINA1/α-1-antitrypsin, whose silencing sensitises ß-cells to Cyt cytotoxicity. Cytokines suppress NMD activity via UPR signalling, potentially serving as a protective response against Cyt-induced NMD component expression. Our findings highlight the central importance of RNA turnover in ß-cell responses to inflammatory stress.

Fetal Programming of the Endocrine Pancreas: Impact of a Maternal Low-Protein Diet on Gene Expression in the Perinatal Rat Pancreas.

In rats, the time of birth is characterized by a transient rise in beta cell replication, as well as beta cell neogenesis and the functional maturation of the endocrine pancreas. However, the knowledge of the gene expression during this period of beta cell expansion is incomplete. The aim was to characterize the perinatal rat pancreas transcriptome and to identify regulatory pathways differentially regulated at the whole organ level in the offspring of mothers fed a regular control diet (CO) and of mothers fed a low-protein diet (LP). We performed mRNA expression profiling via the microarray analysis of total rat pancreas samples at embryonic day (E) 20 and postnatal days (P) 0 and 2. In the CO group, pancreas metabolic pathways related to sterol and lipid metabolism were highly enriched, whereas the LP diet induced changes in transcripts involved in RNA transcription and gene regulation, as well as cell migration and apoptosis. Moreover, a number of individual transcripts were markedly upregulated at P0 in the CO pancreas: growth arrest specific 6 (Gas6), legumain (Lgmn), Ets variant gene 5 (Etv5), alpha-fetoprotein (Afp), dual-specificity phosphatase 6 (Dusp6), and angiopoietin-like 4 (Angptl4). The LP diet induced the downregulation of a large number of transcripts, including neurogenin 3 (Neurog3), Etv5, Gas6, Dusp6, signaling transducer and activator of transcription 3 (Stat3), growth hormone receptor (Ghr), prolactin receptor (Prlr), and Gas6 receptor (AXL receptor tyrosine kinase; Axl), whereas upregulated transcripts were related to inflammatory responses and cell motility. We identified differentially regulated genes and transcriptional networks in the perinatal pancreas. These data revealed marked adaptations of exocrine and endocrine in the pancreas to the low-protein diet, and the data can contribute to identifying novel regulators of beta cell mass expansion and functional maturation and may provide a valuable tool in the generation of fully functional beta cells from stem cells to be used in replacement therapy.

Trefoil factor 3 in perinatal pancreas is increased by gestational low protein diet and associated with accelerated ß-cell maturation.

The endocrine pancreas expands markedly in the first postnatal days and the insulin producing ß-cells initiate a functional maturation preceded by a morphological change of the islets of Langerhans. Trefoil factor 3 (TFF3) is a secreted peptide expressed in intestinal epithelia, where it promotes migration, but its role in the pancreas is not characterized. The aim of this study was to examine the expression and function of TFF3 in perinatal rat pancreas, ex vivo cultured fetal rat pancreas and in the rat ß-cell line INS-1E. Control or gestational low-protein diet perinatal rat pancreas was harvested at embryonic day 20 (E20), day of birth (P0) and postnatal day 2 (P2). TFF3 mRNA was upregulated 4.5-fold at P0 vs. E20 and downregulated again at P2. In protein-undernourished pups induction of TFF3 at P0 was further increased to 9.7-fold and was increased at P2. TFF3 caused tyrosine phosphorylation of EGFR in INS-1E ß-cells, and purified recombinant TFF3 increased both attachment and spreading of INS-1E ß-cells. In ex vivo cultures of collagenase digested fetal rat pancreas, a model of perinatal ß-cell maturation, TFF3 increased cellular spreading as well as insulin mRNA levels. TFF3 also increased the expression of Pref1/Dlk1 that shares similarities in expression and regulation with TFF3. These results suggest that TFF3 may promote adhesion and spreading of cells to accelerate ß-cell maturation. This study indicates a functional role for TFF3 in pancreatic ß-cell maturation in the perinatal period, which is altered by low protein diet during gestation.

abYsis: Integrated Antibody Sequence and Structure-Management, Analysis, and Prediction.

abYsis is a web-based antibody research system that includes an integrated database of antibody sequence and structure data. The system can be interrogated in numerous ways-from simple text and sequence searches to sophisticated queries that apply 3D structural constraints. The publicly available version includes pre-analyzed sequence data from the European Molecular Biology Laboratory European Nucleotide Archive (EMBL-ENA) and Kabat as well as structure data from the Protein Data Bank. A researcher's own sequences can also be analyzed through the web interface. A defining characteristic of abYsis is that the sequences are automatically numbered with a series of popular schemes such as Kabat and Chothia and then annotated with key information such as complementarity-determining regions and potential post-translational modifications. A unique aspect of abYsis is a set of residue frequency tables for each position in an antibody, allowing "unusual residues" (those rarely seen at a particular position) to be highlighted and decisions to be made on which mutations may be acceptable. This is especially useful when comparing antibodies from different species. abYsis is useful for any researcher specializing in antibody engineering, especially those developing antibodies as drugs. abYsis is available at www.abysis.org.

Communication: Three-fold covariance imaging of laser-induced Coulomb explosions.

We apply a three-fold covariance imaging method to analyse previously acquired data [C. S. Slater et al., Phys. Rev. A 89, 011401(R) (2014)] on the femtosecond laser-induced Coulomb explosion of spatially pre-aligned 3,5-dibromo-3',5'-difluoro-4'-cyanobiphenyl molecules. The data were acquired using the "Pixel Imaging Mass Spectrometry" camera. We show how three-fold covariance imaging of ionic photofragment recoil trajectories can be used to provide new information about the parent ion's molecular structure prior to its Coulomb explosion. In particular, we show how the analysis may be used to obtain information about molecular conformation and provide an alternative route for enantiomer determination.

Research Resource: A Dual Proteomic Approach Identifies Regulated Islet Proteins During ß-Cell Mass Expansion In Vivo.

Diabetes is characterized by insulin insufficiency due to a relative paucity of functional ß-cell mass. Thus, strategies for increasing ß-cell mass in situ are sought-after for therapeutic purposes. Pregnancy is a physiological state capable of inducing robust ß-cell mass expansion, however, the mechanisms driving this expansion are not fully understood. Thus, the aim of this study was to characterize pregnancy-induced changes in the islet proteome at the peak of ß-cell proliferation in mice. Islets from pregnant and nonpregnant littermates were compared via 2 proteomic strategies. In vivo pulsed stable isotope labeling of amino acids in cell culture was used to monitor de novo protein synthesis during the first 14.5 days of pregnancy. In parallel, protein abundance was determined using ex vivo dimethyl labelling at gestational day 14.5. Comparison of the 2 datasets revealed 170 islet proteins to be up regulated as a response to pregnancy. These included several proteins, not previously associated with pregnancy-induced islet expansion, such as CLIC1, STMN1, MCM6, PPIB, NEDD4, and HLTF. Confirming the validity of our approach, we also identified proteins encoded by genes known to be associated with pregnancy-induced islet expansion, such as CHGB, IGFBP5, MATN2, EHHADH, IVD, and BMP1. Bioinformatic analyses demonstrated enrichment and activation of the biological functions: "protein synthesis" and "proliferation," and predicted the transcription factors HNF4α, MYC, MYCN, E2F1, NFE2L2, and HNF1α as upstream regulators of the observed expressional changes. As the first characterization of the islet-proteome during pregnancy, this study provides novel insight into the mechanisms involved in promoting pregnancy-induced ß-cell mass expansion and function.

Implications for the offspring of circulating factors involved in beta cell adaptation in pregnancy.

OBJECTIVE: Several studies have shown an increase in beta cell mass during pregnancy. Somatolactogenic hormones are known to stimulate the proliferation of existing beta cells in rodents whereas the mechanism in humans is still unclear. We hypothesize that in addition to somatolactogenic hormones there are other circulating factors involved in beta cell adaptation to pregnancy. This study aimed at screening for potential pregnancy-associated circulating beta cell growth factors. SAMPLES: Serum samples from nonpregnant and pregnant women. METHODS: The effect of serum from pregnant women on the proliferation of rat beta cells was studied using [3H]thymidine incorporation and 5-ethynyl-2'-deoxyuridine proliferation assays. In addition, serum from pregnant and nonpregnant women was fractionated by gel filtration and high performance liquid chromatography. The fractionated serum was screened for mitogenic activity in INS-1E cells. Proteins and peptides in mitogenic active serum fractions were identified by amino acid sequencing and mass spectrometry. MAIN OUTCOME MEASURES: Presence of circulating beta cell proliferating factors. RESULTS: Late gestational pregnancy serum significantly increased proliferation of rat beta cells compared with early pregnancy and nonpregnancy. The mitogenic active serum fractions contained proteins and peptides derived from kininogen-1, fibrinogen-α, α1-antitrypsin, apolipoprotein-A1, placental lactogen, angiotensinogen and serum albumin. CONCLUSION: Pregnancy serum is able to stimulate proliferation of rat beta cells. We have identified several circulating factors that may contribute to beta cell adaptation to pregnancy. Further studies are needed to elucidate their possible role in glucose homeostasis in the mother and her offspring.

Impact of fetal and neonatal environment on beta cell function and development of diabetes.

The global epidemic of diabetes is a serious threat against health and healthcare expenses. Although genetics is important it does not explain the dramatic increase in incidence, which must involve environmental factors. Two decades ago the concept of the thrifty phenotype was introduced, stating that the intrauterine environment during pregnancy has an impact on the gene expression that may persist until adulthood and cause metabolic diseases like obesity and type 2 diabetes. As the pancreatic beta cells are crucial in the regulation of metabolism this article will describe the influence of normal pregnancy on the beta cells in both the mother and the fetus and how various conditions like diabetes, obesity, overnutrition and undernutrition during and after pregnancy may influence the ability of the offspring to adapt to changes in insulin demand later in life. The influence of environmental factors including nutrients and gut microbiota on appetite regulation, mitochondrial activity and the immune system that may affect beta cell growth and function directly and indirectly is discussed. The possible role of epigenetic changes in the transgenerational transmission of the adverse programming may be the most threatening aspect with regard to the global diabetes epidemics. Finally, some suggestions for intervention are presented.

Co-ordinated regulation of neurogenin-3 expression in the maternal and fetal pancreas during pregnancy.

OBJECTIVE: Several studies have shown increased beta cell mass during pregnancy in both rodents and humans. Proliferation of existing beta cells seems to be the predominant mechanism in rodents, whereas the mechanism in humans is unclear. We hypothesized that neogenesis contributes to the increased beta cell mass in pregnancy and that circulating factors are involved. SAMPLES: Pancreatic tissue from mice and rat and serum from pregnant women. METHOD: Morphometric analysis of pancreas of pregnant and nonpregnant mice was carried out by immunocytochemical staining for the neogenic marker neurogenin-3. Messenger RNA levels of neurogenin-3 and the transcription factor musculoaponeurotic fibrosarcoma oncogene family protein B in fetal rat pancreas cells, cultured with serum from pregnant women, were measured by quantitative polymerase chain reaction. MAIN OUTCOME MEASURES: The number of neurogenin-3-positive cells present in pregnant mice was increased compared with nonpregnant mice. Neurogenin-3 and musculoaponeurotic fibrosarcoma oncogene family protein B mRNA was detected in fetal rat pancreas exposed to serum from pregnant women. RESULTS: In pregnant mice we found a 3.6-fold increase in beta cell volume at day 18 compared with nonpregnant mice and a 3.5-fold increase in neurogenin-3 volume at day 14, mainly located in the acinar compartment where it was eightfold higher than in nonpregnant mice. In fetal rat pancreatic cells exposed to serum from pregnant women we found a marked increase in both neurogenin-3 and musculoaponeurotic fibrosarcoma oncogene family protein B mRNA levels in fibroblast-like cells. CONCLUSION: These results suggest that neogenesis contributes to the increased beta cell mass in pregnancy and that circulating factors are involved in beta cell formation in both the maternal and fetal pancreas during pregnancy.

Computer simulations reveal complex distribution of haemodynamic forces in a mouse retina model of angiogenesis.

There is currently limited understanding of the role played by haemodynamic forces on the processes governing vascular development. One of many obstacles to be overcome is being able to measure those forces, at the required resolution level, on vessels only a few micrometres thick. In this paper, we present an in silico method for the computation of the haemodynamic forces experienced by murine retinal vasculature (a widely used vascular development animal model) beyond what is measurable experimentally. Our results show that it is possible to reconstruct high-resolution three-dimensional geometrical models directly from samples of retinal vasculature and that the lattice-Boltzmann algorithm can be used to obtain accurate estimates of the haemodynamics in these domains. We generate flow models from samples obtained at postnatal days (P) 5 and 6. Our simulations show important differences between the flow patterns recovered in both cases, including observations of regression occurring in areas where wall shear stress (WSS) gradients exist. We propose two possible mechanisms to account for the observed increase in velocity and WSS between P5 and P6: (i) the measured reduction in typical vessel diameter between both time points and (ii) the reduction in network density triggered by the pruning process. The methodology developed herein is applicable to other biomedical domains where microvasculature can be imaged but experimental flow measurements are unavailable or difficult to obtain.

Dynamic stark control of torsional motion by a pair of laser pulses.

The torsional motion of a molecule composed of two substituted benzene rings, linked by a single bond, is coherently controlled by a pair of strong (3×10^{13} W cm^{-2}), nonresonant (800 nm) 200-fs-long laser pulses-both linearly polarized perpendicular to the single-bond axis. If the second pulse is sent at the time when the two benzene rings rotate toward (away from) each other the amplitude of the torsion is strongly enhanced (reduced). The torsional motion persists for more than 150 ps corresponding to approximately 120 torsional oscillations. Our calculations show that the key to control is the strong transient modification of the natural torsional potential by the laser-induced dynamic Stark effect.

CTSH regulates ß-cell function and disease progression in newly diagnosed type 1 diabetes patients.

Over 40 susceptibility loci have been identified for type 1 diabetes (T1D). Little is known about how these variants modify disease risk and progression. Here, we combined in vitro and in vivo experiments with clinical studies to determine how genetic variation of the candidate gene cathepsin H (CTSH) affects disease mechanisms and progression in T1D. The T allele of rs3825932 was associated with lower CTSH expression in human lymphoblastoid cell lines and pancreatic tissue. Proinflammatory cytokines decreased the expression of CTSH in human islets and primary rat ß-cells, and overexpression of CTSH protected insulin-secreting cells against cytokine-induced apoptosis. Mechanistic studies indicated that CTSH exerts its antiapoptotic effects through decreased JNK and p38 signaling and reduced expression of the proapoptotic factors Bim, DP5, and c-Myc. CTSH overexpression also up-regulated Ins2 expression and increased insulin secretion. Additionally, islets from Ctsh(-/-) mice contained less insulin than islets from WT mice. Importantly, the TT genotype was associated with higher daily insulin dose and faster disease progression in newly diagnosed T1D patients, indicating agreement between the experimental and clinical data. In line with these observations, healthy human subjects carrying the T allele have lower ß-cell function, which was evaluated by glucose tolerance testing. The data provide strong evidence that CTSH is an important regulator of ß-cell function during progression of T1D and reinforce the concept that candidate genes for T1D may affect disease progression by modulating survival and function of pancreatic ß-cells, the target cells of the autoimmune assault.

Mixed-field orientation of molecules without rotational symmetry.

The mixed-field orientation of an asymmetric-rotor molecule with its permanent dipole moment nonparallel to the principal axes of polarizability is investigated experimentally and theoretically. We find that for the typical case of a strong, nonresonant laser field and a weak static electric field complete 3D orientation is induced if the laser field is elliptically polarized and if its major and minor polarization axes are not parallel to the static field. For a linearly polarized laser field solely the dipole moment component along the most polarizable axis of the molecule is relevant resulting in 1D orientation even when the laser polarization and the static field are nonparallel. Simulations show that the dipole moment component perpendicular to the most-polarizable axis becomes relevant in a strong dc electric field combined with the laser field. This offers an alternative approach to 3D orientation by combining a linearly polarized laser field and a strong dc electric field arranged at an angle equal to the angle between the most polarizable axis of the molecule and its permanent dipole moment.

Impulsive laser induced alignment of molecules dissolved in helium nanodroplets.

We show that a 450 fs nonresonant, moderately intense, linearly polarized laser pulse can induce field-free molecular axis alignment of methyliodide (CH(3)I) molecules dissolved in a helium nanodroplet. Time-resolved measurements reveal rotational dynamics much slower than that of isolated molecules and absence of the sharp transient alignment recurrences characteristic of gas phase molecules. Our results presage a range of new opportunities for exploring both molecular dynamics in a dissipative environment and the properties of He nanodroplets.

MicroRNA-29a is up-regulated in beta-cells by glucose and decreases glucose-stimulated insulin secretion.

Chronically elevated levels of glucose impair pancreatic beta-cell function while inducing beta-cell proliferation. MicroRNA-29a (miR-29a) levels are increased in several tissues in diabetic animals and mediate decreased insulin-stimulated glucose-transport of adipocytes. The aim was to investigate the impact of glucose on miR-29a levels in INS-1E beta-cells and in human islets of Langerhans and furthermore to evaluate the impact of miR-29a on beta-cell function and proliferation. Increased glucose levels up-regulated miR-29a in beta-cells and human and rat islets of Langerhans. Glucose-stimulated insulin-secretion (GSIS) of INS-1E beta-cells was decreased by forced expression of miR-29a, while depletion of endogenous miR-29a improved GSIS. Over-expression of miR-29a increased INS-1E proliferation. Thus, miR-29a up-regulation is involved in glucose-induced proliferation of beta-cells. Furthermore, as depletion of miR-29a improves beta-cell function, miR-29a is a mediator of glucose-induced beta-cell dysfunction. Glucose-induced up-regulation of miR-29a in beta-cells could be implicated in progression from impaired glucose tolerance to type 2 diabetes.

Making the best of mixed-field orientation of polar molecules: a recipe for achieving adiabatic dynamics in an electrostatic field combined with laser pulses.

We have experimentally and theoretically investigated the mixed-field orientation of rotational-state-selected OCS molecules and achieved strong degrees of alignment and orientation. The applied moderately intense nanosecond laser pulses are long enough to adiabatically align molecules. However, in combination with a weak dc electric field, the same laser pulses result in nonadiabatic dynamics of the mixed-field orientation. These observations are fully explained by calculations employing both adiabatic and nonadiabatic (time-dependent) models.

Control and femtosecond time-resolved imaging of torsion in a chiral molecule.

We study how the combination of long and short laser pulses can be used to induce torsion in an axially chiral biphenyl derivative (3,5-difluoro-3',5'-dibromo-4'-cyanobiphenyl). A long, with respect to the molecular rotational periods, elliptically polarized laser pulse produces 3D alignment of the molecules, and a linearly polarized short pulse initiates torsion about the stereogenic axis. The torsional motion is monitored in real-time by measuring the dihedral angle using femtosecond time-resolved Coulomb explosion imaging. Within the first 4 picoseconds (ps), torsion occurs with a period of 1.25 ps and an amplitude of 3° in excellent agreement with theoretical calculations. At larger times, the quantum states of the molecules describing the torsional motion dephase and an almost isotropic distribution of the dihedral angle is measured. We demonstrate an original application of covariance analysis of two-dimensional ion images to reveal strong correlations between specific ejected ionic fragments from Coulomb explosion. This technique strengthens our interpretation of the experimental data.

Expression and localization of microRNAs in perinatal rat pancreas: role of miR-21 in regulation of cholesterol metabolism.

OBJECTIVE: To investigate the expression of pancreatic microRNAs (miRNAs) during the period of perinatal beta-cell expansion and maturation in rats, determine the localization of these miRNAs and perform a pathway analysis with predicted target mRNAs expressed in perinatal pancreas. RESEARCH DESIGN AND METHODS: RNA was extracted from whole pancreas at embryonic day 20 (E20), on the day of birth (P0) and two days after birth (P2) and hybridized to miRNA microarrays. Differentially expressed miRNAs were verified by northern blotting and their pancreatic localization determined by in situ hybridization. Pathway analysis was done using regulated sets of mRNAs predicted as targets of the miRNAs. Possible target genes were tested using reporter-gene analysis in INS-1E cells. RESULTS: Nine miRNAs were differentially expressed perinatally, seven were confirmed to be regulated at the level of the mature miRNA. The localization studies showed endocrine localization of six of these miRNAs (miR-21, -23a, -29a, -125b-5p, -376b-3p and -451), and all were expressed in exocrine cells at one time point at least. Pathways involving metabolic processes, terpenoid and sterol metabolism were selectively affected by concomitant regulation by miRNAs and mRNAs, and Srebf1 was validated as a target of miR-21. CONCLUSIONS: The findings suggest that miRNAs are involved in the functional maturation of pancreatic exocrine and endocrine tissue following birth. Pathway analysis of target genes identify changes in sterol metabolism around birth as being selectively affected by differential miRNA expression during this period.

Stark-selected beam of ground-state OCS molecules characterized by revivals of impulsive alignment.

We make use of an inhomogeneous electrostatic dipole field to impart a quantum-state-dependent deflection to a pulsed beam of OCS molecules, and show that those molecules residing in the absolute ground state, X(1)Σ(+), |00(0)0>, J = 0, can be separated out by selecting the most deflected part of the molecular beam. Past the deflector, we irradiate the molecular beam by a linearly polarized pulsed nonresonant laser beam that impulsively aligns the OCS molecules. Their alignment, monitored via velocity-map imaging, is measured as a function of time, and the time dependence of the alignment is used to determine the quantum state composition of the beam. We find significant enhancements of the alignment ( = 0.84) and of state purity (>92%) for a state-selected, deflected beam compared with an undeflected beam.