Implications of genetic variations, differential gene expression, and allele-specific expression on metformin response in drug-naïve type 2 diabetes.

PURPOSE: Metformin is widely used to treat type 2 diabetes mellitus (T2DM) individuals. Clinically, inter-individual variability of metformin response is of significant concern and is under interrogation. In this study, a targeted exome and whole transcriptome analysis were performed to identify predictive biomarkers of metformin response in drug-naïve T2DM individuals. METHODS: The study followed a prospective study design. Drug-naïve T2DM individuals (n = 192) and controls (n = 223) were enrolled. T2DM individuals were administered with metformin monotherapy and defined as responders and non-responders based on their glycated haemoglobin change over three months. 146 T2DM individuals were used for the final analysis and remaining samples were lost during the follow-up. Target exome sequencing and RNA-seq was performed to analyze genetic and transcriptome profile. The selected SNPs were validated by genotyping and allele specific gene expression using the TaqMan assay. The gene prioritization, enrichment analysis, drug-gene interactions, disease-gene association, and correlation analysis were performed using various tools and databases. RESULTS: rs1050152 and rs272893 in SLC22A4 were associated with improved response to metformin. The copy number loss was observed in PPARGC1A in the non-responders. The expression analysis highlighted potential differentially expressed targets for predicting metformin response (n = 35) and T2DM (n = 14). The expression of GDF15, TWISTNB, and RPL36A genes showed a maximum correlation with the change in HbA1c levels. The disease-gene association analysis highlighted MAGI2 rs113805659 to be linked with T2DM. CONCLUSION: The results provide evidence for the genetic variations, perturbed transcriptome, allele-specific gene expression, and pathways associated with metformin drug response in T2DM.

Proteolysis of the major yolk glycoproteins is regulated by acidification of the yolk platelets in sea urchin embryos.

The precise function of the yolk platelets of sea urchin embryos during early development is unknown. We have shown previously that the chemical composition of the yolk platelets remains unchanged in terms of phospholipid, triglyceride, hexose, sialic acid, RNA, and total protein content after fertilization and early development. However, the platelet is not entirely static because the major 160-kD yolk glycoprotein YP-160 undergoes limited, step-wise proteolytic cleavage during early development. Based on previous studies by us and others, it has been postulated that yolk platelets become acidified during development, leading to the activation of a cathepsin B-like yolk proteinase that is believed to be responsible for the degradation of the major yolk glycoprotein. To investigate this possibility, we studied the effect of addition of chloroquine, which prevents acidification of lysosomes. Consistent with the postulated requirement for acidification, it was found that chloroquine blocked YP-160 breakdown but had no effect on embryonic development. To directly test the possibility that acidification of the yolk platelets over the course of development temporally correlated with YP-160 proteolysis, we added 3-(2,4-dinitroanilo)-3-amino-N-methyldipropylamine (DAMP) to eggs or embryos. This compound localizes to acidic organelles and can be detected in these organelles by EM. The results of these studies revealed that yolk platelets did, in fact, become transiently acidified during development. This acidification occurred at the same time as yolk protein proteolysis, i.e., at 6 h after fertilization (64-cell stage) in Strongylocentrotus purpuratus and at 48 h after fertilization (late gastrula) in L. pictus. Furthermore, the pH value at the point of maximal acidification of the yolk platelets in vivo was equal to the pH optimum of the enzyme measured in vitro, indicating that this acidification is sufficient to activate the enzyme. For both S. purpuratus and Lytechinus pictus, the observed decrease in the pH was approximately 0.8 U, from 7.0 to 6.2. The trypsin inhibitor benzamidine was found to inhibit the yolk proteinase in vivo. By virtue of the fact that this inhibitor was reversible we established that the activity of the yolk proteinase is developmentally regulated even though the enzyme is present throughout the course of development. These findings indicate that acidification of yolk platelets is a developmentally regulated process that is a prerequisite to initiation of the catabolism of the major yolk glycoprotein.

Primary hyperparathyroidism caused by parathyroid-targeted overexpression of cyclin D1 in transgenic mice.

The relationship between abnormal cell proliferation and aberrant control of hormonal secretion is a fundamental and poorly understood issue in endocrine cell neoplasia. Transgenic mice with parathyroid-targeted overexpression of the cyclin D1 oncogene, modeling a gene rearrangement found in human tumors, were created to determine whether a primary defect in this cell-cycle regulator can cause an abnormal relationship between serum calcium and parathyroid hormone response, as is typical of human primary hyperparathyroidism. We also sought to develop an animal model of hyperparathyroidism and to examine directly cyclin D1's role in parathyroid tumorigenesis. Parathyroid hormone gene regulatory region--cyclin D1 (PTH--cyclin D1) mice not only developed abnormal parathyroid cell proliferation, but also developed chronic biochemical hyperparathyroidism with characteristic abnormalities in bone and, notably, a shift in the relationship between serum calcium and PTH. Thus, this animal model of human primary hyperparathyroidism provides direct experimental evidence that overexpression of the cyclin D1 oncogene can drive excessive parathyroid cell proliferation and that this proliferative defect need not occur solely as a downstream consequence of a defect in parathyroid hormone secretory control by serum calcium, as had been hypothesized. Instead, primary deregulation of cell-growth pathways can cause both the hypercellularity and abnormal control of hormonal secretion that are almost inevitably linked together in this common disorder.

Management-related outcomes and radiographic findings of idiopathic condylar resorption: a systematic review.

Idiopathic condylar resorption (ICR) is progressive resorption of the condyle of unknown aetiology. There is no consensus on the approaches for diagnostic imaging and management of this disease. The objective of this systematic review was to examine the best practices for imaging and to appraise the success of surgical and non-surgical therapy of ICR. Eleven search engines were queried via explicit literature searches for studies describing ICR, published until 2012. Two authors independently extracted data using predetermined characteristics. Studies that identified patients as having either ICR or progressive condylar resorption and that described the radiographic findings or treatment options were included. Seventeen studies contributing 178 cases met the eligibility criteria. The major radiographic findings, as assessed mostly by two-dimensional imaging, included decreased ramus height, decreased condylar height, altered volume of the condyle, decreased SNB angle and mandibular plane angle, and a retrognathic profile. Treatments included occlusal splints with orthodontic treatment, condylectomy with costochondral graft, and other surgical approaches. This systematic review was limited by the lack of meta-analysis. Nevertheless, we identified the need for future investigations: characterization of findings on three-dimensional imaging and its contribution to treatment planning, outcomes of non-surgical and pharmacological management of ICR, and randomized trials and comparative studies with longer follow-up periods.

Cyclin D1 in parathyroid disease.

Primary hyperparathyroidism (HPT), most commonly due to parathyroid adenoma, is a disorder characterized by excessive secretion of PTH. So far, abnormalities in two genes, cyclin D1 and MEN1, have been implicated in the development of parathyroid adenomas. Cyclin D1, now an established Oncogene involved in numerous human cancers, was first identified and recognized as an Oncogene in the study of parathyroid tumors. A subset of parathyroid adenomas contains a clonal rearrangement that places the PTH gene's regulatory sequences in proximity to the cyclin D1 Oncogene causing its overexpression, and 20-40% of parathyroid adenomas overexpress the cyclin D1 protein. Transgenic animal models have further confirmed the role of cyclin D1 as a driver of abnormal parathyroid cell proliferation. Future studies on the mechanism of cyclin D1's oncogenicity and its interactions with other parathyroid growth regulators will further our understanding of parathyroid cell biology and may prove useful clinically.

Synthesis and biological activity of a series of potent fluoromethyl ketone inhibitors of recombinant human calpain I.

Calpain I, an intracellular cysteine protease, has been implicated in the neurodegeneration following an episode of stroke. In this paper, we report on a series of potent dipeptide fluoromethyl ketone inhibitors of recombinant human calpain I (rh calpain I). SAR studies revealed that while calpain I tolerates a variety of hydrophobic groups at the P1 site, Leu at P2 is preferred. However, the nature of the N-terminal capping group has a significant effect on the inhibitory activity of this series of compounds. Compound 4e [(1,2,3,4-tetrahydroisoquinolin-2-yl)carbonyl-Leu-D,L-Phe-CH2F+ ++], having a tetrahydroisoquinoline containing urea as the N-terminal capping group, is the most potent dipeptide fluoromethyl ketone inhibitor of calpain I (with a second-order rate constant for inactivation of 276,000 M-1 s-1) yet reported; tripeptide 4k (Cbz-Leu-Leu-D,L-Phe-CH2F) is equipotent. A number of compounds presented in this study displayed excellent selectivity for calpain I over cathepsins B and L, two related cysteine proteases. Compounds which exhibited good inhibitory activity in the assay against isolated rh calpain I also inhibited intracellular calpain I in a human cell line. Thus, in an intact cell assay, compounds 4e and 4k inhibited calpain I with IC50 values of 0.2 and 0.1 microM, respectively. Finally, we also disclose the first example of fluorination of a dipeptide enol silyl ether to generate the corresponding dipeptide fluoromethyl ketone.

Defective modulation of double-strand break repair in ataxia telangiectasia cells by gamma radiation.

Ataxia telangiectasia (AT) cells are defective in responding to damage induced by ionizing radiation. To study the modulation of double-strand break (DSB) repair by ionizing radiation and a defect in such modulation in AT cells, we compared processing of linearized shuttle vector pZ189 (linear DNA) by unirradiated or gamma-irradiated normal and AT lymphoblast hosts. The linear DNA processed in unirradiated AT and normal host cells yielded similar mutation frequencies in the supF-tRNA target gene. Irradiation of normal but not AT host cells decreased plasmid mutation frequency 2-fold if transfection occurred immediately. However, if transfection occurred 2 h after host cell irradiation, mutation frequencies increased 2-fold above those in unirradiated controls in both normal and AT hosts. DSB rejoining capability, based on the ratio of the number of progeny arising from equal amounts of linear DNA and supercoiled, undamaged pZ189, was 25- to 50-fold higher in normal than in AT hosts when both were unirradiated. Irradiation decreased DSB rejoining capability 2- to 5-fold in normal hosts but did not alter that of AT hosts. These findings demonstrate that AT cells normally rejoin DSBs as accurately as normal cells but do so less often, and that AT cells are defective in modulation of DSB rejoining by ionizing radiation.

Evidence of the involvement of p53 in gamma-radiation-induced DNA repair in human lymphoblasts.

PURPOSE: To determine the involvement of p53 in ionizing radiation-induced excision and recombination repair. MATERIALS AND METHODS: Shuttle vector pZ189 containing radiation-induced single strand breaks plus base damage (ocDNA), ultraviolet-radiation damage (uvDNA), or a restriction enzyme-produced double strand break (linDNA) were processed in unirradiated or irradiated p53wt and p53mut lymphoblasts. Mutation frequencies in the supF-tRNA target gene and survival of plasmids processed in p53wt and p53mut hosts were compared. RESULTS: Mutation frequencies of oc-, uv- or linDNA were similar after processing in unirradiated p53wt and p53mut hosts. However, the mutation frequency of ocDNA and uvDNA decreased 50% when processed in irradiated p53wt hosts but was unaltered in irradiated p53mut hosts. In contrast, linDNA mutation frequencies varied similarly whether processed in irradiated p53wt or p53mut hosts: mutation frequency decreased twofold when linDNA was transfected immediately after host irradiation but increased twofold when transfection was delayed by 2h. Double strand break rejoining capacity, determined by the ratio of the number of progenies from linDNA to that from undamaged pZ189, differed both qualitatively and quantitatively in irradiated p53wt and p53mut hosts. CONCLUSIONS: These studies show induction of DNA repair in mammalian cells by ionizing radiation and indicate the involvement of p53 in the modulation of excision repair fidelity and double strand break rejoining capacity.

Heightened induction of c-jun mRNA by PMA, EGF and IL-1 in ataxia telangiectasia lymphoblasts.

PURPOSE: To determine if cells from the cancer-prone autosomal recessive disorder ataxia telangiectasia (A-T) are defective in responding to stimuli other than ionizing-radiation (IR) damage. MATERIALS AND METHODS: The induction of c-jun transcripts by IR, by phorbol 12-myristate 13-acetate (PMA), interleukin 1 (IL-1) and epidermal growth factor (EGF) in normal and A-T lymphoblasts was measured. RESULTS: Treatment with PMA increased c-jun transcripts in a dose- and time-dependent manner two- to three-fold more in A-T than in normal cells. Similarly, treatment with EGF and IL-1 also increased c-jun transcripts more in A-T than in normal lymphoblasts. In contrast, exposure to gamma-radiation increased c-jun transcripts at least twofold more in normal than in A-T lymphoblasts. CONCLUSIONS: These findings indicate that A-T cells are not only defective in responding to IR damage, but also in responding to mitogenic stimuli like IL-1 and EGF. Furthermore, these findings implicate ATM, the gene responsible for the A-T disorder, in the induction of c-jun transcripts by PMA, EGF or IL-1.

Requirement for p53 in ionizing-radiation-inhibition of double-strand-break rejoining by human lymphoblasts.

Ionizing radiation (IR) triggers apoptosis, cell-cycle arrest, and DNA-repair induction in mammalian cells. These responses are mediated by proteins, including p53, which are activated or induced by IR. To determine the role of p53 in double-strand break (DSB) repair following irradiation of mammalian cells, we compared the abilities of unirradiated and irradiated TK6 human lymphoblast line and its derivatives TK6-E6-20C and TK6-E6-5E to repair restriction-enzyme-linearized shuttle pZ189 and the luciferase-reporter plasmid pGL3-control. TK6-E6-20C expresses wild-type p53 like the parental TK6 line, while TK6-E6-5E is p53 null. DSB-rejoining capacity was determined from the ratio of viable progenies arising from DSB-containing plasmids (linDNA) to the number of viable progenies from undamaged, supercoiled plasmids (scDNA). The ratio from the p53wt hosts was two- to three-fold higher than that from the p53null host, using either pZ189 or pGL3-control plasmid. After exposure of both hosts to 0.5 Gy gamma-radiation, DSB-rejoining capacity of p53null increased two-fold compared to unirradiated null controls, if transfection occurred immediately after irradiation. In contrast, the DSB-rejoining capacity of p53wt was unaffected by irradiation. If transfection was delayed for 2 h following irradiation, however, DSB-rejoining declined in both p53wt and p53null hosts. Irradiation also altered DSB-rejoining fidelity, measured from the mutation frequencies, among progenies of pZ189 linDNA. But, unlike rejoining capacity, changes in DSB-rejoining fidelity were similar in p53wt and p53null hosts. Changes in cell-cycle distribution in p53wt and p53null hosts were also similar following irradiation. These findings show that IR increases DSB-rejoining capacity in mammalian cells without functional p53, suggesting that p53 participates in suppressing DSB-rejoining following exposure of mammalian cells to IR.

Calmodulin antagonists and cAMP inhibit ionizing-radiation-enhancement of double-strand-break repair in human cells.

Ionizing radiation (IR) enhances double-strand-break (DSB)-repair fidelity in plasmids processed in normal lymphoblasts but not in lymphoblasts from ataxia telangiectasia (A-T) patients. Putatively, signal-transduction pathways mediate this DNA-repair induction. Because IR inhibition of DNA synthesis is defective in A-T cells and is mediated by a calmodulin (caM)-dependent pathway, we evaluated the involvement of caM-dependent pathways in DSB-repair induction. Human lymphoblasts were gamma-irradiated with or without treatment with caM antagonists and the cells' abilities to repair shuttle pZ189 carrying a single DSB (linDNA) were assessed. In untreated controls, IR enhanced DSB-rejoining fidelity if transfection occurred promptly but diminished fidelity if transfection was delayed. Treatment with two caM antagonists, W-7 and W-13, prior to irradiation blocked this IR-enhancement of DSB-rejoining fidelity. Vinpocetine, a caM-dependent phosphodiesterase inhibitor, and 8-bromo-cAMP also inhibited IR enhancement of repair fidelity, but caM-dependent protein kinase II inhibitor KN62 had no effect. Other protein kinase inhibitors, staurosporine and genistein, also did not inhibit IR enhancement of DSB repair fidelity. However, staurosporine blocked the twofold reduction in DSB-repair fidelity seen if linDNA transfection was delayed 2 h after irradiation. These findings point to the involvement of caM/cAMP-dependent pathway(s) in mediating IR-enhancement of DSB-rejoining fidelity in mammalian cells.

Acoustic effects of lightning.

The effects of lightning on the audiovestibular apparatus vary with the degree of injury. This depends on whether the individual is struck directly or indirectly by lightning. We reported two cases where lightning has caused trauma to audiovestibular apparatus and have reviewed the available literature. One patient was struck directly by lightning leading to rupture of the tympanic membrane and a conductive hearing loss. The other patient was struck indirectly via telephone cable and had a mixed hearing loss with tympanic membrane intact.

Efficacy of the investigational mTOR kinase inhibitor MLN0128/INK128 in models of B-cell acute lymphoblastic leukemia.

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase whose activity contributes to leukemia proliferation and survival. Compounds targeting the mTOR active site inhibit rapamycin-resistant functions and have enhanced anticancer activity in mouse models. MLN0128 (formerly known as INK128) is a novel, orally active mTOR kinase inhibitor currently in clinical development. Here, we evaluated MLN0128 in preclinical models of B-cell acute lymphoblastic leukemia (B-ALL). MLN0128 suppressed proliferation of B-ALL cell lines in vitro and reduced colony formation by primary human leukemia cells from adult and pediatric B-ALL patients. MLN0128 also boosted the efficacy of dasatinib (DA) in Philadelphia Chromosome-positive (Ph+) specimens. In a syngeneic mouse model of lymphoid BCR-ABL+ disease, daily oral dosing of MLN0128 rapidly cleared leukemic outgrowth. In primary xenografts of Ph+ B-ALL specimens, MLN0128 significantly enhanced the efficacy of DA. In non-Ph B-ALL xenografts, single agent MLN0128 had a cytostatic effect that was most pronounced in mice with low disease burden. In all in vivo models, MLN0128 was well tolerated and did not suppress endogenous bone marrow proliferation. These findings support the rationale for clinical testing of MLN0128 in both adult and pediatric B-ALL and provide insight towards optimizing therapeutic efficacy of mTOR kinase inhibitors.

Update on the biological effects of ionizing radiation, relative dose factors and radiation hygiene.

Diagnostic imaging is an indispensable part of contemporary medical and dental practice. Over the last few decades there has been a dramatic increase in the use of ionizing radiation for diagnostic imaging. The carcinogenic effects of high-dose exposure are well known. Does diagnostic radiation rarely cause cancer? We don't know but we should act as if it does. Accordingly, dentists should select patients wisely - only make radiographs when there is patient-specific reason to believe there is a reasonable expectation the radiograph will offer unique information influencing diagnosis or treatment. Low-dose examinations should be made: intraoral imaging - use fast film or digital sensors, thyroid collars, rectangular collimation; panoramic and lateral cephalometric imaging - use digital systems or rare-earth film screen combinations; and cone beam computed tomography - use low-dose machines, restrict field size to region of interest, reduce mA and length of exposure arc as appropriate.

Effect of stress on production of heat labile enterotoxin by Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is an important pathogen responsible for secretory diarrhoea. The production of heat labile enterotoxin (LT), by ETEC, is largely responsible for the pathogenesis of diarrhoea. In the present study we investigated the effect of stress factors such as temperature, pH, osmotic stress and nutritional limitation on the production of LT by ETEC using in-house GMI-ELISA. Four strains of E. coli consisting, one standard strain MTCC 723 and three clinical isolates were used in the study. Maximum amount of LT (OD 3.285) was produced at 37 0 C followed by 40 0 C (OD 3.305). Growth of E. coli in medium with pH 8.6 resulted in maximum amount of LT production (OD 3.489). LT was not detectable when bacteria were grown in medium with pH < or =7.2 and > or = 9.2. Sodium chloride concentration of 0.2 M stimulated maximum amount of LT production. Maximum amount of LT was produced when the bacteria were grown in medium containing 2.5 g/l of glucose. All the stress factors had a significant effect on the LT production by E. coli , though quantitative differences in the various strains were observed.

An unusual soft tissue radiopacity--radiographic appearance of a dermal filler.

We present a case that describes the radiographic findings of Radiesse, a calcium hydroxyapatitie-based dermal filler. This dermal filler was detected during radiographic examination for implant treatment planning. This case illustrates the typical radiographic appearance of this material and the importance of differentiating it from pathological conditions.