Cerebellar control of visceral responses-possible mechanisms involved

It seems reasonable to assume that cerebellar autonomic control operates according to similar principles as those utilized in the somatomotor coordination. The unique and very uniform neuronal architecture throughout the cerebellum speaks in favour of such a view.     

CTGF-mediated autophagy-senescence transition in tumor stroma promotes anabolic tumor growth and metastasis

Comment on: Capparelli C, et al. Cell Cycle 2012; 11:2272-84 and Capparelli C, et al. Cell Cycle 2012; 11:2285-302.Otto Warburg first observed that cancer cells preferentially undergo glycolysis instead of the mitochondrial TCA cycle even under oxygen-rich conditions. This form of energy metabolism in cancer cells is called “aerobic glycolysis” or the “Warburg effect.”¹ Lisanti and colleagues have previously proposed an alternative model called the “the reverse Warburg effect,” in which aerobic glycolysis predominantly occurs in stromal fibroblasts.² During this process, cancer cells secrete oxidative stress factors, such as hydrogen peroxide, into the tumor microenvironment, which induces autophagy. This leads to degradation of mitochondria (mitophagy) and elevated glycolysis in cancer-associated fibroblasts.³ Aerobic glycolysis results in the elevated production of pyruvate, ketone bodies and L-lactate, which can be utilized by cancer cells for anabolic growth and metastasis. At the molecular level, stromal fibroblasts lose expression of caveolin-1 and activate HIF-1a (Fig. 1), TGFβ and NFκB signaling.⁴ Stromal caveolin-1 expression predicts clinical outcome in breast cancer patients.⁵Open in a separate windowFigure 1. CTGF-mediated autophagy-senescence transition in tumor stroma promotes anabolic tumor growth and metastasis. Cancer cells secrete oxidative stress factors (H₂O₂) that induce autophagy in cancer-associated fibroblasts. Additionally, caveolin-1 (cav-1) loss leads to activation of connective tissue growth factor (CTGF) and HIF-1α that mediate autophagy and senescence in these stromal cells. This is called the autophagy-senescence transition (AST). AST leads to mitophagy and elevated glycolysis in cancer-associated fibroblasts. Aerobic glycolysis results in the elevated production of several nutrients (pyruvate, ketone bodies and L-lactate), which can be utilized by cancer cells for tumor growth and metastasis.In the June 15, 2012 issue of Cell Cycle, two studies by Capparelli et al. further validate the “autophagic tumor stroma model of cancer” described above, as well as identify novel mechanisms involved in this process.^6,7 Autophagy and senescence are induced by the same stimuli and are known to occur simultaneously in cells. In the first study, the authors hypothesize that the onset of senescence in the tumor stroma in response to autophagy/mitophagy contributes to mitochondrial dysfunction and aerobic glycolysis. In order to genetically validate this process of autophagy-senescence transition (AST) (Fig. 1), Capparelli et al. overexpressed several autophagy-promoting factors (BNIP3, cathepsin B, Beclin-1 and ATG16L1) in hTERT fibroblasts to constitutively induce autophagy. Autophagic fibroblasts lost caveolin-1 expression and displayed enhanced tumor growth and metastasis when co-injected with breast cancer cells in mice, without an increase in angiogenesis. In contrast, constitutive activation of autophagy in breast cancer cells inhibited in vivo tumor growth. Autophagic fibroblasts also showed mitochondrial dysfunction, increased production of nutrients (L-lactate and ketone bodies) and features of senescence (β-galactosidase activity and p21 activation). AST was also demonstrated in human breast cancer patient samples.⁷ In the second study, using a similar experimental approach, the authors evaluated the role of the TGFβ target gene, connective tissue growth factor (CTGF), in the induction of AST and aerobic glycolysis in cancer-associated fibroblasts. CTGF would be activated in the tumor stroma upon loss of caveolin-1. CTGF overexpression in fibroblasts induced autophagy/mitophagy, glycolysis and L-lactate production in a HIF-1α-dependent manner along with features of senescence and oxidative stress. CTGF overexpression in fibroblasts also promoted tumor growth when co-injected with breast cancer cells in mice (Fig. 1), independent of angiogenesis. As expected, CTGF overexpression in breast cancer cells inhibited tumor growth. CTGF is known to be involved in extracellular matrix synthesis; however, the effects of CTGF overexpression in fibroblasts and tumor cells were found to be independent of this function.⁶Overall, the authors have identified a novel mechanism by which CTGF promotes AST and aerobic glycolysis in cancer-associated fibroblasts. In turn, the stromal cells stimulate anabolic tumor growth and metastasis. The authors also genetically validate the two-compartment model of cancer metabolism, whereby autophagy genes and CTGF have differential effects in stromal cells and tumor cells. The current studies have several implications for cancer therapy. The finding that HIF-1 activation is necessary for the induction of autophagy and senescence downstream of caveolin-1 loss and CTGF activation in stromal fibroblasts is intriguing. Activation of HIF-1 in the hypoxic tumor microenvironment is known to promote tumor cell growth, survival and therapeutic resistance.⁸ Therefore, targeting HIF-1 has the potential to block tumor progression through dual inhibitory effects on hypoxic cancer cell growth and survival as well as the induction of autophagy in stromal fibroblasts. CTGF and AST in the tumor stroma could serve as biomarkers for predicting clinical outcome, therapy response and metastasis. The two-compartment model of tumor metabolism raises further questions regarding the use of antioxidants and autophagy inhibitors/inducers for cancer therapy. The use of these agents in the clinic should be carefully evaluated considering their differential effects on stromal cells and cancer cells.     

Prenatal antiepileptic exposure associates with neonatal DNA methylation differences

Antiepileptic drugs (AEDs) are used to treat a variety of neuropsychiatric illnesses commonly encountered in women during their reproductive years, including epilepsy and bipolar disorder. Despite their widespread use, the impact of prenatal exposure on fetal development remains obscure. To evaluate whether AEDs taken by pregnant mothers influence DNA methylation patterns in their neonates, DNA was extracted from the umbilical cord blood of 201 neonates whose mothers were treated for neuropsychiatric illness during pregnancy and interrogated across 27,578 CpG sites using the Illumina HumanMethylation27 BeadChip. The association of each methylation value with the cumulative duration of prenatal AED exposure was examined using a linear mixed model. The average methylation level across all CpG sites was calculated for each subject, and this global methylation measure was evaluated similarly. Neonates with a longer duration of AED exposure in pregnancy showed a decrease in average global methylation (p = 0.0045). Further, DNA methylation of CpG sites in 14 genes significantly decreased with the duration of prenatal AED exposure even after adjusting for multiple comparisons (FDR < 0.05). For a small subset (n = 19) of these neonates, a second tissue, placenta, was available in addition to cord blood. Methylation of 3 of these 14 CpG sites was also significantly decreased in placental tissue. These novel data suggest decreased DNA methylation in neonates of mothers who took AEDs during pregnancy. The long-term stability and potential impact of these changes warrant further attention, and caution may be warranted before prescribing AEDs to pregnant women.     

Deep Rooting In-Situ Expansion of mtDNA Haplogroup R8 in South Asia

Background

The phylogeny of the indigenous Indian-specific mitochondrial DNA (mtDNA) haplogroups have been determined and refined in previous reports. Similar to mtDNA superhaplogroups M and N, a profusion of reports are also available for superhaplogroup R. However, there is a dearth of information on South Asian subhaplogroups in particular, including R8. Therefore, we ought to access the genealogy and pre-historic expansion of haplogroup R8 which is considered one of the autochthonous lineages of South Asia.

Methodology/Principal Findings

Upon screening the mtDNA of 5,836 individuals belonging to 104 distinct ethnic populations of the Indian subcontinent, we found 54 individuals with the HVS-I motif that defines the R8 haplogroup. Complete mtDNA sequencing of these 54 individuals revealed two deep-rooted subclades: R8a and R8b. Furthermore, these subclades split into several fine subclades. An isofrequency contour map detected the highest frequency of R8 in the state of Orissa. Spearman''s rank correlation analysis suggests significant correlation of R8 occurrence with geography.

Conclusions/Significance

The coalescent age of newly-characterized subclades of R8, R8a (15.4±7.2 Kya) and R8b (25.7±10.2 Kya) indicates that the initial maternal colonization of this haplogroup occurred during the middle and upper Paleolithic period, roughly around 40 to 45 Kya. These results signify that the southern part of Orissa currently inhabited by Munda speakers is likely the origin of these autochthonous maternal deep-rooted haplogroups. Our high-resolution study on the genesis of R8 haplogroup provides ample evidence of its deep-rooted ancestry among the Orissa (Austro-Asiatic) tribes.     

Genetics in public health: Rarely explored

The availability and the integration of genetic information into our understanding of normal and abnormal growth and development are driving important changes in health care. These changes have fostered the hope that the availability of genetic information will promote a better understanding of disease etiology and permit early, even pre-symptomatic diagnosis and preventive intervention to avoid disease onset. Hence, our aim was to review and provide the insight into the role of genetics in public health and its scope as well as barriers. The use of genetics along with their goals and essential public health functions are discussed. From the era of eugenics to the present era, this area has seen many turns in which geneticists have put through their effort to tie together the strings of both molecular genetics and public health. Though still the dark clouds of eugenics, the predictive power of genes, genetic reductionism, non-modifiable risk factors, individuals or populations, resource allocation, commercial imperative, discrimination and understanding and education are hanging above. The technological and scientific advances that have fundamentally changed our perception of human diseases fuel the expectations for this proactive health.     

Molecular Basis for the Role of Staphylococcus aureus Penicillin Binding Protein 4 in Antimicrobial Resistance

Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the d-Ala-d-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-l-Ala-d-Glu-l-Lys-d-Ala-d-Ala. β-Lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a β-lactamase and is not trapped as an acyl intermediate with β-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.Penicillin binding proteins (PBPs) are critical components of the cell wall synthesis machinery in bacteria. These membrane-associated proteins are broadly classified as low-molecular-mass (LMM) PBPs that are monofunctional d,d-carboxypeptidase enzymes or multimodular high-molecular-mass (HMM) PBPs with multiple functional roles. PBPs, in general, are anchored to the cytoplasmic membrane by a noncleavable pseudo-signal peptide. In the case of the HMM PBPs, the cytoplasmic C-terminal domain binds penicillin and catalyzes peptidoglycan cross-linking, whereas the juxtamembrane N-terminal domain participates in transglycosylation (12). The catalytic penicillin-binding (PB) module also occurs as part of penicillin sensor transducers, such as Staphylococcus aureus MecR and Bacillus licheniformis BlaR (15). The transpeptidase activity in HMM PBPs is based on a conserved lysine residue located in the so-called catalytic S-X-X-K motif, whereas the other conserved S-X-N and K(H)-T(S)-G motifs govern carboxypeptidase activity and bind penicillin (20). The carboxypeptidase domain of PBPs is the target for β-lactam antibiotics in susceptible staphylococci (with penicillin MICs as low as 1 μg/ml).The transpeptidase activity of the PBPs occurs at the d-Ala-d-Ala terminus of a precursor disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-l-Ala-d-Ala-l-Lys-d-Ala-d-Ala. This reaction is initiated by acylation involving a nucleophilic attack by the active-site serine on the penultimate d-Ala residue to form an acyl-enzyme complex. The C-terminal d-Ala is subsequently released from the peptide chain, followed by deacylation. In the case of HMM PBPs, deacylation occurs when an amino group on a second peptide substrate acts as an acceptor, resulting in a peptide cross-link between two adjacent peptidoglycan strands. The carboxypeptidase activity of LMM PBPs follows a similar reaction scheme, except that the acceptor in this case is a water molecule. β-Lactam antibiotics mimic the substrates of the PBPs. However, unlike the natural substrate, the β-lactam-PBP acyl adduct is stable and results in irreversible inhibition of PBP function. The β-lactam-PBP acyl adduct has been characterized extensively, with over 50 protein-antibiotic complexes reported to date (37). Thus, in contrast to the nonessential LMM PBPs, HMM PBPs constitute lethal targets for β-lactam antibiotics (6).Staphylococcus aureus is a gram-positive coccus and is one of the leading causes of high morbidity and mortality associated with both community- and hospital-associated infections (42, 46). This coccus shows extensive genomic variation, with over 22% of the genome dedicated to dispensable regions. A genome-scale analysis of a clinical strain of S. aureus is of particular interest in this context, wherein the conversion of a susceptible strain of S. aureus to a multidrug-resistant phenotype was shown to involve just 35 mutations in 13 loci, achieved within 3 months (36). Of the five PBPs in S. aureus, an acquired PBP, PBP2a, is the most extensively examined, as it was noted to be a specific marker for methicillin-resistant S. aureus (MRSA) strains. Among the intrinsic PBPs, PBP1 has been shown to play a key role in cell growth and division (2). PBP2 is a dual-function enzyme with both transglycosylase and transpeptidase activities, and inhibition of this protein leads to restrained peptidoglycan elongation and subsequent leakage of cytoplasmic contents due to cell lysis (34, 40). Inactivation of PBP3 neither changes the muropeptide composition of the cell wall nor significantly decreases the rate of autolysis. However, cells of abnormal size and shape and with disoriented septa are produced when bacteria with inactivated PBP3 are grown with sub-MIC levels of methicillin (29).S. aureus PBP4 is a carboxypeptidase and is needed for the secondary cross-linking of peptidoglycan (19). However, it is not essential for cell growth under laboratory conditions, because mutants of S. aureus defective in PBP4 are viable (48). Overexpression of PBP4 was noted to result in an increase in β-lactam resistance and in greater cross-linking of the peptidoglycan (18). S. aureus PBP4 is similar to other LMM PBPs and is grouped within the superfamily of penicillin-susceptible and penicillin-interacting enzymes. However, homologues of PBP4 have a different phenotype in other species (1, 15). For example, a mutation of PBP4 in Pseudomonas aeruginosa triggers an AmpR-dependent overproduction of the chromosomal β-lactamase AmpC. The P. aeruginosa PBP4 mutant also activates CreBC, a two-component regulator, thereby mediating β-lactam resistance (33). Indeed, S. aureus PBP4 has been suggested to have different functions in strains with different genetic backgrounds (26). However, based on in vitro and genetic data, S. aureus PBP4 is primarily a transpeptidase and has little d,d-carboxypeptidase activity. This is also supported by the observation that increased carboxypeptidase activity decreases cell wall cross-linking due to loss of the free d-Ala-d-Ala termini necessary for transpeptidation (10). In this context, it is pertinent that pbp4 gene knockout strains of S. aureus were more resistant to the glycopeptide antibiotic vancomycin (46).Here we present the biochemical and structural characteristics of PBP4 from S. aureus strain COL. S. aureus PBP4 is a β-lactamase. A comparison of the crystal structure of S. aureus PBP4 in complex with antibiotic with that of its Escherichia coli homologue, PBP5, provides a conformational and biochemical rationale for the β-lactamase activity of PBP4. Monitoring the expression of PBP4 in the MRSA strain COL and representative clinical strains of S. aureus suggested that the expression level of PBP4 does not fluctuate substantially across these strains. Together, these data on the structure, expression, activity, and regulation of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.     

Thiadiazolopiperazinyl ureas as inhibitors of fatty acid amide hydrolase

A series of thiadiazolopiperazinyl aryl urea fatty acid amide hydrolase (FAAH) inhibitors is described. The molecules were found to inhibit the enzyme by acting as mechanism-based substrates, forming a covalent bond with Ser241. SAR and PK properties are presented.     

Computational modelling of local calcium ions release from calcium phosphate-based scaffolds

A variety of natural or synthetic calcium phosphate (CaP)-based scaffolds are currently produced for dental and orthopaedic applications. These scaffolds have been shown to stimulate bone formation due to their biocompatibility, osteoconductivity and osteoinductivity. The release of the \(\hbox {Ca}^{2+}\) ions from these scaffolds is of great interest in light of the aforementioned properties. It can depend on a number of biophysicochemical phenomena such as dissolution, diffusion and degradation, which in turn depend on specific scaffold characteristics such as composition and morphology. Achieving an optimal release profile can be challenging when relying on traditional experimental work alone. Mathematical modelling can complement experimentation. In this study, the in vitro dissolution behaviour of four CaP-based scaffold types was investigated experimentally. Subsequently, a mechanistic finite element method model based on biophysicochemical phenomena and specific scaffold characteristics was developed to predict the experimentally observed behaviour. Before the model could be used for local \(\hbox {Ca}^{2+}\) ions release predictions, certain parameters such as dissolution constant (\(k_{\mathrm{dc}}\)) and degradation constant (\(k_\mathrm{sc}\)) for each type of scaffold were determined by calibrating the model to the in vitro dissolution data. The resulting model showed to yield release characteristics in satisfactory agreement with those observed experimentally. This suggests that the mathematical model can be used to investigate the local \(\hbox {Ca}^{2+}\) ions release from CaP-based scaffolds.     

Helospectin I and II evoke vasodilation in the intact peripheral microcirculation

Helospectin I and II, two closely related mammalian neuropeptides of the secretin/glucagons/vasoactive intestinal peptide (VIP) superfamily of peptides, are co-localized with VIP in nerve fibers surrounding vascular smooth muscle. However, the role if any, VIP receptors play in transducing the vasorelaxant effects of helospectin I and II in the intact peripheral microcirculation is uncertain. The purpose of this study was to determine whether helospectin I and II elicit vasodilation in the intact peripheral microcirculation and, if so, whether this response is mediated, in part, by VIP or pituitary adenylate cyclase activating peptide (PACAP) receptor engagement, and through local elaboration of cyclooxygenase products of arachidonic acid metabolism. Using intravital microscopy, we found that suffusion of helospectin I and II (each, 1.0 nmol) evoked potent vasodilation and of similar magnitude in the intact hamster cheek pouch microcirculation (P < 0.05). Suffusion of 0.1 nmol helospectin I and II had no significant effects on arteriolar diameter. Pretreatment with VIP(10-28), a VPAC1/VPAC2 receptor antagonist, or PACAP(6-38), a PAC1/VPAC2 receptor antagonist, had no significant effects on helospectin I- and II-induced responses. In addition, pretreatment with indomethacin had no significant effects on helospectin I- and II-induced vasodilation. Collectively, these data indicate that helospectin I and II evoke potent vasodilation in the intact peripheral microcirculation that is not transduced by VIP or PACAP receptors nor through cyclooxygenase products of arachidonic acid metabolism.