Optimizing Optical Flow Cytometry for Cell Volume-Based Sorting and Analysis

Cell size is a defining characteristic central to cell function and ultimately to tissue architecture. The ability to sort cell subpopulations of different sizes would facilitate investigation at genomic and proteomic levels of mechanisms by which cells attain and maintain their size. Currently available cell sorters, however, cannot directly measure cell volume electronically, and it would therefore be desirable to know which of the optical measurements that can be made in such instruments provide the best estimate of volume. We investigated several different light scattering and fluorescence measurements in several different cell lines, sorting cell fractions from the high and low end of distributions, and measuring volume electronically to determine which sorting strategy yielded the best separated volume distributions. Since we found that different optical measurements were optimal for different cell lines, we suggest that following this procedure will enable other investigators to optimize their own cell sorters for volume-based separation of the cell types with which they work

A mathematical and computational review of Hartree-Fock SCF methods in Quantum Chemistry

We present here a review of the fundamental topics of Hartree-Fock theory in Quantum Chemistry. From the molecular Hamiltonian, using and discussing the Born-Oppenheimer approximation, we arrive to the Hartree and Hartree-Fock equations for the electronic problem. Special emphasis is placed in the most relevant mathematical aspects of the theoretical derivation of the final equations, as well as in the results regarding the existence and uniqueness of their solutions. All Hartree-Fock versions with different spin restrictions are systematically extracted from the general case, thus providing a unifying framework. Then, the discretization of the one-electron orbitals space is reviewed and the Roothaan-Hall formalism introduced. This leads to a exposition of the basic underlying concepts related to the construction and selection of Gaussian basis sets, focusing in algorithmic efficiency issues. Finally, we close the review with a section in which the most relevant modern developments (specially those related to the design of linear-scaling methods) are commented and linked to the issues discussed. The whole work is intentionally introductory and rather self-contained, so that it may be useful for non experts that aim to use quantum chemical methods in interdisciplinary applications. Moreover, much material that is found scattered in the literature has been put together here to facilitate comprehension and to serve as a handy reference.Comment: 64 pages, 3 figures, tMPH2e.cls style file, doublesp, mathbbol and subeqn package

Folding of the apolipoprotein A1 driven by the salt concentration as a possible mechanism to improve cholesterol trapping

The folding of the cholesterol trapping apolipoprotein A1 in aqueous solution at increasing ionic strength is studied using atomically detailed molecular dynamics simulations. We calculate various structural properties to characterize the conformation of the protein, such as the radius of gyration, the radial distribution function and the end to end distance. Additionally we report information using tools specifically tailored for the characterization of proteins, such as the mean smallest distance matrix and the Ramachandran plot. We find that two qualitatively different configurations of this protein are preferred, one where the protein is extended, and one where it forms loops or closed structures. It is argued that the latter promote the association of the protein with cholesterol and other fatty acids.Comment: 14 pages, 6 figures. To appear in "Selected Topics of Computational and Experimental Fluid Mechanics", Springer, J. Klapp, G. Ru\'iz, A. Medina, A. L\'opez & L. Di G. Sigalotti (eds.), 201

Design Novel Dual Agonists for Treating Type-2 Diabetes by Targeting Peroxisome Proliferator-Activated Receptors with Core Hopping Approach

Owing to their unique functions in regulating glucose, lipid and cholesterol metabolism, PPARs (peroxisome proliferator-activated receptors) have drawn special attention for developing drugs to treat type-2 diabetes. By combining the lipid benefit of PPAR-alpha agonists (such as fibrates) with the glycemic advantages of the PPAR-gamma agonists (such as thiazolidinediones), the dual PPAR agonists approach can both improve the metabolic effects and minimize the side effects caused by either agent alone, and hence has become a promising strategy for designing effective drugs against type-2 diabetes. In this study, by means of the powerful “core hopping” and “glide docking” techniques, a novel class of PPAR dual agonists was discovered based on the compound GW409544, a well-known dual agonist for both PPAR-alpha and PPAR-gamma modified from the farglitazar structure. It was observed by molecular dynamics simulations that these novel agonists not only possessed the same function as GW409544 did in activating PPAR-alpha and PPAR-gamma, but also had more favorable conformation for binding to the two receptors. It was further validated by the outcomes of their ADME (absorption, distribution, metabolism, and excretion) predictions that the new agonists hold high potential to become drug candidates. Or at the very least, the findings reported here may stimulate new strategy or provide useful insights for discovering more effective dual agonists for treating type-2 diabetes. Since the “core hopping” technique allows for rapidly screening novel cores to help overcome unwanted properties by generating new lead compounds with improved core properties, it has not escaped our notice that the current strategy along with the corresponding computational procedures can also be utilized to find novel and more effective drugs for treating other illnesses

QM/MM MD and Free Energy Simulations of G9a-Like Protein (GLP) and Its Mutants: Understanding the Factors that Determine the Product Specificity

Certain lysine residues on histone tails could be methylated by protein lysine methyltransferases (PKMTs) using S-adenosyl-L-methionine (AdoMet) as the methyl donor. Since the methylation states of the target lysines play a fundamental role in the regulation of chromatin structure and gene expression, it is important to study the property of PKMTs that allows a specific number of methyl groups (one, two or three) to be added (termed as product specificity). It has been shown that the product specificity of PKMTs may be controlled in part by the existence of specific residues at the active site. One of the best examples is a Phe/Tyr switch found in many PKMTs. Here quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations are performed on wild type G9a-like protein (GLP) and its F1209Y and Y1124F mutants for understanding the energetic origin of the product specificity and the reasons for the change of product specificity as a result of single-residue mutations at the Phe/Tyr switch as well as other positions. The free energy barriers of the methyl transfer processes calculated from our simulations are consistent with experimental data, supporting the suggestion that the relative free energy barriers may determine, at least in part, the product specificity of PKMTs. The changes of the free energy barriers as a result of the mutations are also discussed based on the structural information obtained from the simulations. The results suggest that the space and active-site interactions around the ε-amino group of the target lysine available for methyl addition appear to among the key structural factors in controlling the product specificity and activity of PKMTs

Modeling a New Water Channel That Allows SET9 to Dimethylate p53

SET9, a protein lysine methyltransferase, has been thought to be capable of transferring only one methyl group to target lysine residues. However, some reports have pointed out that SET9 can dimethylate Lys372 of p53 (p53-K372) and Lys4 of histone H3 (H3-K4). In order to understand how p53 can be dimethylated by SET9, we measured the radius of the channel that surrounds p53-K372, first on the basis of the crystal structure of SET9, and we show that the channel is not suitable for water movement. Second, molecular dynamic (MD) simulations were carried out for 204 ns on the crystal structure of SET9. The results show that water leaves the active site of SET9 through a new channel, which is made of G292, A295, Y305 and Y335. In addition, the results of molecular docking and MD simulations indicate that the new water channel continues to remain open when S-adenosyl-L-methionine (AdoMet) or S-adenosyl-L-homocysteine (AdoHcy) is bound to SET9. The changes in the radii of these two channels were measured in the equilibrium phase at the constant temperature of 300 K. The results indicate that the first channel still does not allow water to get into or out of the active site, but the new channel is large enough to allow this water to circulate. Our results indicate that water can be removed from the active site, an essential process for allowing the dimethylation reaction to occur

Distribution and diel vertical movements of mesopelagic scattering layers in the Red Sea

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Marine Biology 159 (2012): 1833-1841, doi:10.1007/s00227-012-1973-y.The mesopelagic zone of the Red Sea represents an extreme environment due to low food concentrations, high temperatures and low oxygen waters. Nevertheless, a 38 kHz echosounder identified at least four distinct scattering layers during the daytime, of which the 2 deepest layers resided entirely within the mesopelagic zone. Two of the acoustic layers were found above a mesopelagic oxygen minimum zone (OMZ), one layer overlapped with the OMZ, and one layer was found below the OMZ. Almost all organisms in the deep layers migrated to the near-surface waters during the night. Backscatter from a 300 kHz lowered Acoustic Doppler Current Profiler indicated a layer of zooplankton within the OMZ. They carried out DVM, yet a portion remained at mesopelagic depths during the night. Our acoustic measurements showed that the bulk of the acoustic backscatter was restricted to waters shallower than 800 m, suggesting that most of the biomass in the Red Sea resides above this depth.This research is based in part on work supported by Award Nos. USA 00002, KSA 00011 and KSA 00011/02 made by KAUST to the Woods Hole Oceanographic Institution

A Molecular Study on the Prevalence and Virulence Potential of Aeromonas spp. Recovered from Patients Suffering from Diarrhea in Israel

Background: Species of the genus Aeromonas are native inhabitants of aquatic environments and have recently been considered emerging human pathogens. Although the gastrointestinal tract is by far the most common anatomic site from which aeromonads are recovered, their role as etiologic agents of bacterial diarrhea is still disputed. Aeromonas-associated diarrhea is a phenomenon occurring worldwide; however, the exact prevalence of Aeromonas infections on a global scale is unknown. Methodology/Principal Findings: The prevalence and virulence potential of Aeromonas in patients suffering from diarrhea in Israel was studied using molecular methods. 1,033 diarrheal stools were sampled between April and September 2010 and Aeromonas species were identified in 17 (,2%) patients by sequencing the rpoD gene. Aeromonas species identity and abundance was: A. caviae (65%), A. veronii (29%) and Aeromonas taiwanensis (6%). This is the first clinical record of A. taiwanensis as a diarrheal causative since its recent discovery from a wound infection in a patient in Taiwan. Most of the patients (77%) from which Aeromonas species were isolated were negative for any other pathogens. The patients ranged from 1 to 92 years in age. Aeromonas isolates were found to possess different virulence-associated genes: ahpB (88%), pla/ lip/lipH3/apl-1 (71%), act/hlyA/aerA (35%), alt (18%), ast (6%), fla (65%), lafA (41%), TTSS ascV (12%), TTSS ascF-ascG (12%), TTSS-dependent ADP-ribosylating toxins aexU (41%) and aexT (6%) in various combinations. Most of the identified strain

Falls, Depression and Antidepressants in Later Life: A Large Primary Care Appraisal

BACKGROUND: Depression and falls are common and co-exist for older people. Safe management of each of these conditions is important to quality of life. METHODS: A cross-sectional survey was used to examine medication use associated with injurious and non-injurious falls in 21,900 community-dwelling adults, aged 60 years or over from 383 Australian general practices recruited for the DEPS-GP Project. Falls and injury from falls, medication use, depressive symptoms (Primary Health Questionnaire (PHQ-9)), clinical morbidity, suicidal ideation and intent, health status (SF-12 Health Survey), demographic and lifestyle information was reported in a standardised survey. FINDINGS: Respondents were 71.8 years (sd 7.7) of age and 58.4% were women. 24% 11% and 8% reported falls, fall related injury, and sought medical attention respectively. Antidepressant use (odds ratio, OR: 1.46; 95% confidence interval, 95%CI: 1.25, 1.70), questionable depression (5-14 on PHQ OR: 1.32, 95%CI: 1.13, 1.53) and clinically significant symptoms of depression (15 or more on PHQ OR: 1.70, 95%CI: 1.14, 1.50) were independently associated with multiple falls. SSRI use was associated with the highest risk of multiple falls (OR: 1.66, 95%CI: 1.36, 2.02) amongst all psychotropic medications. Similar associations were observed for injurious falls. Over 60% of those with four accumulated risk factors had multiple falls in the previous year (OR: 3.40, 95%CI: 1.79, 6.45); adjusted for other demographic and health factors. INTERPRETATION: Antidepressant use (particularly SSRIs) was strongly associated with falls regardless of presence of depressive symptoms. Strategies to prevent falls should become a routine part of the management of older people with depression

Precise Regulation of Gene Expression Dynamics Favors Complex Promoter Architectures

Promoters process signals through recruitment of transcription factors and RNA polymerase, and dynamic changes in promoter activity constitute a major noise source in gene expression. However, it is barely understood how complex promoter architectures determine key features of promoter dynamics. Here, we employ prototypical promoters of yeast ribosomal protein genes as well as simplified versions thereof to analyze the relations among promoter design, complexity, and function. These promoters combine the action of a general regulatory factor with that of specific transcription factors, a common motif of many eukaryotic promoters. By comprehensively analyzing stationary and dynamic promoter properties, this model-based approach enables us to pinpoint the structural characteristics underlying the observed behavior. Functional tradeoffs impose constraints on the promoter architecture of ribosomal protein genes. We find that a stable scaffold in the natural design results in low transcriptional noise and strong co-regulation of target genes in the presence of gene silencing. This configuration also exhibits superior shut-off properties, and it can serve as a tunable switch in living cells. Model validation with independent experimental data suggests that the models are sufficiently realistic. When combined, our results offer a mechanistic explanation for why specific factors are associated with low protein noise in vivo. Many of these findings hold for a broad range of model parameters and likely apply to other eukaryotic promoters of similar structure