Bactericidal/permeability increasing protein: A multifaceted protein with functions beyond LPS neutralization

Bactericidal permeability increasing protein (BPI), a 55-60kDa protein, first reported in 1975, has gone a long way as a protein with multifunctional roles. Its classical role in neutralizing endotoxin (LPS) raised high hopes among septic shock patients. Today, BPI is not just a LPS-neutralizing protein, but a protein with diverse functions. These functions can be as varied as inhibition of endothelial cell growth and inhibition of dendritic cell maturation, or as an anti-angiogenic, chemoattractant or opsonization agent. Though the literature available is extremely limited, it is fascinating to look into how BPI is gaining major importance as a signalling molecule. In this review, we briefly summarize the recent research focused on the multiple roles of BPI and its use as a therapeutic.

Healing time for the growth of thin films on patterned substrates

The healing times for the growth of thin films on patterned substrates are studied using simulations of two discrete models of surface growth: the Family model and the Das Sarma-Tamborenea (DT) model. The healing time, defined as the time at which the characteristics of the growing interface are ``healed'' to those obtained in growth on a flat substrate, is determined via the study of the nearest-neighbor height difference correlation function. Two different initial patterns are considered in this work: a relatively smooth tent-shaped triangular substrate and an atomically rough substrate with singlesite pillars or grooves. We find that the healing time of the Family and DT models on aL x L triangular substrate is proportional to L-z, where z is the dynamical exponent of the models. For the Family model, we also analyze theoretically, using a continuum description based on the linear Edwards-Wilkinson equation, the time evolution of the nearest-neighbor height difference correlation function in this system. The correlation functions obtained from continuum theory and simulation are found to be consistent with each other for the relatively smooth triangular substrate. For substrates with periodic and random distributions of pillars or grooves of varying size, the healing time is found to increase linearly with the height (depth) of pillars (grooves). We show explicitly that the simulation data for the Family model grown on a substrate with pillars or grooves do not agree with results of a calculation based on the continuum Edwards-Wilkinson equation. This result implies that a continuum description does not work when the initial pattern is atomically rough. The observed dependence of the healing time on the substrate size and the initial height (depth) of pillars (grooves) can be understood from the details of the diffusion rule of the atomistic model. The healing time of both models for pillars is larger than that for grooves with depth equal to the height of the pillars. The calculated healing time for both Family and DT models is found to depend on how the pillars and grooves are distributed over the substrate. (C) 2014 Elsevier B.V. All rights reserved.

Identification of a novel AMPK-PEA15 axis in the anoikis-resistant growth of mammary cells

Introduction: Matrix detachment triggers anoikis, a form of apoptosis, in most normal epithelial cells, while acquisition of anoikis resistance is a prime requisite for solid tumor growth. Of note, recent studies have revealed that a small population of normal human mammary epithelial cells (HMECs) survive in suspension and generate multicellular spheroids termed `mammospheres'. Therefore, understanding how normal HMECs overcome anoikis may provide insights into breast cancer initiation and progression. Methods: Primary breast tissue-derived normal HMECs were grown as adherent monolayers or mammospheres. The status of AMP-activated protein kinase (AMPK) and PEA15 signaling was investigated by immunoblotting. Pharmacological agents and an RNA interference (RNAi) approach were employed to gauge their roles in mammosphere formation. Immunoprecipitation and in vitro kinase assays were undertaken to evaluate interactions between AMPK and PEA15. In vitro sphere formation and tumor xenograft assays were performed to understand their roles in tumorigenicity. Results: In this study, we show that mammosphere formation by normal HMECs is accompanied with an increase in AMPK activity. Inhibition or knockdown of AMPK impaired mammosphere formation. Concomitant with AMPK activation, we detected increased Ser(116) phosphorylation of PEA15, which promotes its anti-apoptotic functions. Inhibition or knockdown of AMPK impaired PEA15 Ser(116) phosphorylation and increased apoptosis. Knockdown of PEA15, or overexpression of the nonphosphorylatable S116A mutant of PEA15, also abrogated mammosphere formation. We further demonstrate that AMPK directly interacts with and phosphorylates PEA15 at Ser(116) residue, thus identifying PEA15 as a novel AMPK substrate. Together, these data revealed that AMPK activation facilitates mammosphere formation by inhibition of apoptosis, at least in part, through Ser(116) phosphorylation of PEA15. Since anoikis resistance plays a critical role in solid tumor growth, we investigated the relevance of these findings in the context of breast cancer. Significantly, we show that the AMPK-PEA15 axis plays an important role in the anchorage-independent growth of breast cancer cells both in vitro and in vivo. Conclusions: Our study identifies a novel AMPK-PEA15 signaling axis in the anchorage-independent growth of both normal and cancerous mammary epithelial cells, suggesting that breast cancer cells may employ mechanisms of anoikis resistance already inherent within a subset of normal HMECs. Thus, targeting the AMPK-PEA15 axis might prevent breast cancer dissemination and metastasis.

Novel Functions of (p)ppGpp and Cyclic di-GMP in Mycobacterial Physiology Revealed by Phenotype Microarray Analysis of Wild-Type and Isogenic Strains of Mycobacterium smegmatis

The bacterial second messengers (p)ppGpp and bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulate important functions, such as transcription, virulence, biofilm formation, and quorum sensing. In mycobacteria, they regulate long-term survival during starvation, pathogenicity, and dormancy. Recently, a Pseudomonas aeruginosa strain lacking (p) ppGpp was shown to be sensitive to multiple classes of antibiotics and defective in biofilm formation. We were interested to find out whether Mycobacterium smegmatis strains lacking the gene for either (p)ppGpp synthesis (Delta rel(Msm)) or c-di-GMP synthesis (Delta dcpA) would display similar phenotypes. We used phenotype microarray technology to compare the growth of the wild-type and the knockout strains in the presence of several antibiotics. Surprisingly, the Delta rel(Msm) and Delta dcpA strains showed enhanced survival in the presence of many antibiotics, but they were defective in biofilm formation. These strains also displayed altered surface properties, like impaired sliding motility, rough colony morphology, and increased aggregation in liquid cultures. Biofilm formation and surface properties are associated with the presence of glycopeptidolipids (GPLs) in the cell walls of M. smegmatis. Thin-layer chromatography analysis of various cell wall fractions revealed that the levels of GPLs and polar lipids were reduced in the knockout strains. As a result, the cell walls of the knockout strains were significantly more hydrophobic than those of the wild type and the complemented strains. We hypothesize that reduced levels of GPLs and polar lipids may contribute to the antibiotic resistance shown by the knockout strains. Altogether, our data suggest that (p)ppGpp and c-di-GMP may be involved in the metabolism of glycopeptidolipids and polar lipids in M. smegmatis.

Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis

Glioblastoma (grade IV glioma/GBM) is the most common primary adult malignant brain tumor with poor prognosis. To characterize molecular determinants of tumor-stroma interaction in GBM, we profiled 48 serum cytokines and identified macrophage colony-stimulating factor (MCSF) as one of the elevated cytokines in sera from GBM patients. Both MCSF transcript and protein were up-regulated in GBM tissue samples through a spleen tyrosine kinase (SYK)-dependent activation of the PI3K-NF kappa B pathway. Ectopic overexpression and silencing experiments revealed that glioma-secreted MCSF has no role in autocrine functions and M2 polarization of macrophages. In contrast, silencing expression of MCSF in glioma cells prevented tube formation of human umbilical vein endothelial cells elicited by the supernatant from monocytes/microglial cells treated with conditioned medium from glioma cells. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture showed that glioma-derived MCSF induces changes in microglial secretome and identified insulin-like growth factor-binding protein 1 (IGFBP1) as one of the MCSF-regulated proteins secreted by microglia. Silencing IGFBP1 expression in microglial cells or its neutralization by an antibody reduced the ability of supernatants derived from microglial cells treated with glioma cell-conditioned medium to induce angiogenesis. In conclusion, this study shows up-regulation of MCSF in GBM via a SYK-PI3K-NF kappa B-dependent mechanism and identifies IGFBP1 released by microglial cells as a novel mediator of MCSF-induced angiogenesis, of potential interest for developing targeted therapy to prevent GBM progression.

Regulation of Growth, Cell Shape, Cell Division, and Gene Expression by Second Messengers (p)ppGpp and Cyclic Di-GMP in Mycobacterium smegmatis

The alarmone (p)ppGpp regulates transcription, translation, replication, virulence, lipid synthesis, antibiotic sensitivity, biofilm formation, and other functions in bacteria. Signaling nucleotide cyclic di-GMP (c-di-GMP) regulates biofilm formation, motility, virulence, the cell cycle, and other functions. In Mycobacterium smegmatis, both (p) ppGpp and c-di-GMP are synthesized and degraded by bifunctional proteins Rel(Msm) and DcpA, encoded by rel(Msm) and dcpA genes, respectively. We have previously shown that the Delta rel(Msm) and Delta dcpA knockout strains are antibiotic resistant and defective in biofilm formation, show altered cell surface properties, and have reduced levels of glycopeptidolipids and polar lipids in their cell wall (K. R. Gupta, S. Kasetty, and D. Chatterji, Appl Environ Microbiol 81:2571-2578, 2015, http://dx.doi.org/10.1128/AEM.03999-14). In this work, we have explored the phenotypes that are affected by both (p) ppGpp and c-di-GMP in mycobacteria. We have shown that both (p) ppGpp and c-di-GMP are needed to maintain the proper growth rate under stress conditions such as carbon deprivation and cold shock. Scanning electron microscopy showed that low levels of these second messengers result in elongated cells, while high levels reduce the cell length and embed the cells in a biofilm-like matrix. Fluorescence microscopy revealed that the elongated Delta rel(Msm) and Delta dcpA cells are multinucleate, while transmission electron microscopy showed that the elongated cells are multiseptate. Gene expression analysis also showed that genes belonging to functional categories such as virulence, detoxification, lipid metabolism, and cell-wall-related processes were differentially expressed. Our results suggests that both (p) ppGpp and c-di-GMP affect some common phenotypes in M. smegmatis, thus raising a possibility of cross talk between these two second messengers in mycobacteria. IMPORTANCE Our work has expanded the horizon of (p) ppGpp and c-di-GMP signaling in Gram-positive bacteria. We have come across a novel observation that M. smegmatis needs (p) ppGpp and c-di-GMP for cold tolerance. We had previously shown that the Delta rel(Msm) and Delta dcpA strains are defective in biofilm formation. In this work, the overproduction of (p) ppGpp and c-di-GMP encased M. smegmatis in a biofilm-like matrix, which shows that both (p) ppGpp and c-di-GMP are needed for biofilm formation. The regulation of cell length and cell division by (p) ppGpp was known in mycobacteria, but our work shows that c-di-GMP also affects the cell size and cell division in mycobacteria. This is perhaps the first report of c-di-GMP regulating cell division in mycobacteria.

Negative regulators of a growth factor-mediated signaling pathway in the nematode Caenorhabditis elegans

Vulval differentiation in C. elegans is mediated by an Epidermal growth factor (EGF)- EGF receptor (EGFR) signaling pathway. I have cloned unc-101, a negative regulator of vulval differentiation of the nematode C. elegans. unc-101 encodes a homolog of AP47, the medium chain of the trans-Golgi clathrin-associated protein complex. This identity was confirmed by cloning and comparing sequence of a C. elegans homolog of AP50, the medium chain of the plasma membrane clathrin-associated protein complex. I provided the first genetic evidence that the trans-Golgi clathrin-coated vesicles are involved in regulation of an EGF signaling pathway. Most of the unc-101 alleles are deletions or nonsense mutations, suggesting that these alleles severely reduce the unc-101 activity. A hybrid gene that contains parts of unc-101 and mouse AP4 7 rescued at least two phenotypes of unc-101 mutations, the Unc and the suppression of vulvaless phenotype of let-23(sy1) mutation. Therefore, the functions of AP47 are conserved between nematodes and mammals.

unc-101 mutations can cause a greater than wild-type vulval differentiation in combination with certain mutations in sli-1, another negative regulator of the vulval induction pathway. A mutation in a new gene, rok-1, causes no defect by itself, but causes a greater than wild-type vulval differentiation in the presence of a sli-1 mutation. The unc-101; rok-1; sli-1 triple mutants display a greater extent of vulval differentiation than any double mutant combinations of unc-101, rok-1 and sli-1. Therefore, rok-1 locus defines another negative regulator of the vulval induction pathway.

I analyzed a second gene encoding an AP47 homolog in C. elegans. This gene, CEAP47, encodes a protein 72% identical to both unc-101 and mammalian AP47. A hybrid gene containing parts of unc-101 and CEAP47 sequences can rescue phenotypes of unc-101 mutants, indicating that UNC- 101 and CEAP47 proteins can be redundant if expressed in the same set of cells.

A simple approach for the estimation of food consumption from growth rates at different environmental conditions and its application to juvenile cod (Gadus morhua L.) of a fjordic sea loch on the west coast of Scotland

Abstract Growth and condition of fish are functions of available food and environmental conditions. This led to the idea of using fish as a “consumption sensor” for the measurement of food intake over a defined period of time. A bio-physical model for the estimation of food consumption was developed based on the von Bertalanffy model. Whereas some of the input variables of the model, the initial and final lengths and masses of a fish and the temperature within the time period considered can easily be measured, internal characteristics of the species have to be determined indirectly. Three internal parameters are used in the model: the maintenance consumption at 0°C, the temperature dependence of this consumption and the food efficiency, the percentage of the ingested food utilized. Estimates of the parameters for a given species can be determined by feeding experiments. Here, data from published feeding experiments on juvenile cod, Gadus morhua L., were used to validate the model. The average of the relative error for the food intake predicted by the model for individual fish was about 24 %, indicating that fish used the food with different efficiencies. However, grouping the fish according to size classes and temperature lowered the relative error of the predicted food intake for the group to typically 5 %. For a group containing all fish of the feeding experiment the relative prediction error was about 2 %. Zusammenfassung Wachstum und Kondition der Fische sind von der verfügbaren Nahrung und von Umweltbedingungen abhängig. Dies führte zur Idee, Fisch als „Konsum-Sensor“ für die Messung der Nahrungsaufnahme über einen definierten Zeitraum zu verwenden. Auf Grundlage des von Bertalanffy-Modells wurde ein bio-physikalisches Modell zur Schätzung der Futteraufnahme entwickelt. Während einige der Eingangsgrößen des Modells leicht gemessen werden können (Anfangs- und Endlänge und -körpermasse der Fische und die Temperatur innerhalb des betrachteten Zeitraum), können interne Parameter der betrachteten Art nur indirekt bestimmt werden. Drei interne Parameter werden in dem Modell verwendet: Die Erhaltungskonsumtion bei 0° C, die Temperaturabhängigkeit dieser Rate und der Wirkungsgrad der Nahrung (der Anteil der Nahrung ,der aufgenommen und verwendet und nicht ungenutzt wieder ausgeschieden wird). Die Modellparameter für eine bestimmte Art können durch Fütterungsversuche bestimmt werden. Um das Modell zu validieren wurden Daten aus veröffentlichten Fütterungsversuchen mit juvenilen Kabeljau (Gadus morhua L.) verwendet. Modell und Wirklichkeit weichen in der Regel voneinander ab. Der durchschnittliche relative Fehler der durch das Modell vorhergesagten Nahrungsaufnahme betrug für Einzelfische etwa 24%, was darauf hinweist, dass einzelne Fisch die Nahrung mit unterschiedlichen Wirkungsgraden verwerten. Allerdings senkte die Gruppierung der Fische nach Größenklassen und Temperatur den relativen Vorhersagefehler für die Nahrungsaufnahme der Gruppe auf etwa 5%. Für alle Fische im Fütterungsversuch ist der relative Vorhersagefehler etwa 2%.

Estimation of the growth curve parameters in Macrobrachium rosenbergii

Growth is one of the most important characteristics of cultured species. The objective of this study was to determine the fitness of linear, log linear, polynomial, exponential and Logistic functions to the growth curves of Macrobrachium rosenbergii obtained by using weekly records of live weight, total length, head length, claw length, and last segment length from 20 to 192 days of age. The models were evaluated according to the coefficient of determination (R2), and error sum off square (ESS) and helps in formulating breeders in selective breeding programs. Twenty full-sib families consisting 400 PLs each were stocked in 20 different hapas and reared till 8 weeks after which a total of 1200 animals were transferred to earthen ponds and reared up to 192 days. The R2 values of the models ranged from 56 – 96 in case of overall body weight with logistic model being the highest. The R2 value for total length ranged from 62 to 90 with logistic model being the highest. In case of head length, the R2 value ranged between 55 and 95 with logistic model being the highest. The R2 value for claw length ranged from 44 to 94 with logistic model being the highest. For last segment length, R2 value ranged from 55 – 80 with polynomial model being the highest. However, the log linear model registered low ESS value followed by linear model for overall body weight while exponential model showed low ESS value followed by log linear model in case of head length. For total length the low ESS value was given by log linear model followed by logistic model and for claw length exponential model showed low ESS value followed by log linear model. In case of last segment length, linear model showed lowest ESS value followed by log linear model. Since, the model that shows highest R2 value with low ESS value is generally considered as the best fit model. Among the five models tested, logistic model, log linear model and linear models were found to be the best models for overall body weight, total length and head length respectively. For claw length and last segment length, log linear model was found to be the best model. These models can be used to predict growth rates in M. rosenbergii. However, further studies need to be conducted with more growth traits taken into consideration

Simulation of growth process of thin film on non-planar substrate

In (2 + 1) dimension, growth process of thin film on non-planar substrate in Kuramoto-Sivashinsky model is studied with numerical simulation approach. 15 x 15 semi-ellipsoids arranged orderly on the surface of substrate are used to represent initial rough surface. The results show that at the initial stage of growth process, the surface morphology of thin film appears to be grid-structure, and the interface width constantly decreases with the growth time, then reaches minimum. However, the grid-structure becomes ambiguous, and granules of different sizes distribute evenly on the surface of thin film with the increase of growth time. Thereafter, the average size of granules and the interface width gradually increase, and the surface morphology of thin film presents fractal properties. The numerical results of height-height correlation functions of thin film verify the surface morphology of thin film to be fractal for a longer growth time. By fitting of the height-height correlation functions of thin film with different growth times, the growth process is described quantitatively. (c) 2004 Elsevier B.V. All rights reserved.

An allometric smoothing function to describe the relation between otolith and somatic growth over the lifespan of walleye pollock (Theragra chalcogramma)

We propose a new equation to describe the relation between otolith length (OL) and somatic length (fork length [FL]) of fish for the entire lifespan of the fish. The equation was developed by applying a mathematical smoothing method based on an allometric equation with a constant term for walleye pollock (Theragra chalcogramma) —a species that shows an extended longevity (>20 years). The most appropriate equation for defining the relation between OL and FL was a four-phase allometric smoothing function with three inflection points. The inflection points correspond to the timing of settlement of walleye pollock, changes in sexual maturity, and direction of otolith growth. Allometric smoothing functions describing the relation between short otolith radius and FL, long otolith radius and FL, and FL and body weight were also developed. The proposed allometric smoothing functions cover the entire lifespan of walleye pollock. We term these equations “allometric smoothing functions for otolith and somatic growth over the lifespan of walleye pollock.”

An environmentally based growth model that uses finite difference calculus with maximum likelihood method: its application to the brackish water bivalve Corbicula japonica in Lake Abashiri, Japan

We present a growth analysis model that combines large amounts of environmental data with limited amounts of biological data and apply it to Corbicula japonica. The model uses the maximum-likelihood method with the Akaike information criterion, which provides an objective criterion for model selection. An adequate distribution for describing a single cohort is selected from available probability density functions, which are expressed by location and scale parameters. Daily relative increase rates of the location parameter are expressed by a multivariate logistic function with environmental factors for each day and categorical variables indicating animal ages as independent variables. Daily relative increase rates of the scale parameter are expressed by an equation describing the relationship with the daily relative increase rate of the location parameter. Corbicula japonica grows to a modal shell length of 0.7 mm during the first year in Lake Abashiri. Compared with the attain-able maximum size of about 30 mm, the growth of juveniles is extremely slow because their growth is less susceptible to environmental factors until the second winter. The extremely slow growth in Lake Abashiri could be a geographical genetic variation within C. japonica.

Individual growth rates and movement of juvenile white shrimp (Litopenaeus setiferus) in a tidal marsh nursery

We measured growth and movements of individually marked free-ranging juvenile white shrimp (Litopenaeus setiferus) in tidal creek subsystems of the Duplin River, Sapelo Island, Georgia. Over a period of two years, 15,974 juvenile shrimp (40−80 mm TL) were marked internally with uniquely coded microwire tags and released in the shallow upper reaches of four salt marsh tidal creeks. Subsequent samples were taken every 3−6 days from channel segments arranged at 200-m intervals along transects extending from the upper to lower reach of each tidal creek. These collections included 201,384 juvenile shrimp, of which 184 were marked recaptures. Recaptured shrimp were at large an average of 3−4 weeks (range: 2−99 days) and were recovered a mean distance of <0.4 km from where they were initially marked. Mean residence times in the creek subsystems ranged from 15.2 to 25.5 days and were estimated from exponential decay functions describing the proportions of marked individuals recaptured with increasing days at large. Residence time was not significantly correlated with creek length (Pearson=−0.316, P=0.684 ), but there was suggestive evidence of positive associations with either intertidal (Pearson r=0.867, P=0.133) or subtidal (Pearson r=0.946, P=0.054) drainage area. Daily mean specific growth rates averaged 0.009 to 0.013 among creeks; mean absolute growth rates ranged from 0.56−0.84 mm/d, and were lower than those previously reported for juvenile penaeids in estuaries of the southeastern United States. Mean individual growth rates were not significantly different between years (t-test, P>0.30) but varied significantly during the season, tending to be greater in July than November. Growth rates were size-dependent, and temporal changes in size distributions rather than temporal variation in physical environmental factors may have accounted for seasonal differences in growth. Growth rates differed between creeks in 1999 (t-test, P<0.015), but not in 1998 (t-test, P>0.5). We suggest that spatial variation in landscape structure associated with access to intertidal resources may have accounted for this apparent interannual difference in growth response.

Melanoma cell growth inhibition by beta gamma-CAT, which is a novel non-lens betagamma-crystallin and trefoil factor complex from frog Bombina maxima skin

In vertebrates, non-lens beta gamma-crystallins are widely expressed in various tissues but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained