Determining Candidate Single Nucleotide Polymorphisms in Acquired Laryngotracheal Stenosis.

OBJECTIVES/HYPOTHESIS: Despite wide adoption of strategies to prevent injury from prolonged intubation and tracheotomy, acquired laryngotracheal stenosis (ALTS) has not disappeared. ALTS' persistence may be due to patient factors that confer unique susceptibility for some. We sought to identify genetic markers in genes associated with wound healing that could be associated with ALTS. STUDY DESIGN: Case-control study. METHODS: One hundred thirty-eight patients were recruited, 53 patients with ALTS and 85 control patients who underwent intubation or tracheotomy without evidence of ALTS. The patients' DNA was isolated from whole blood. Custom primers were designed, and the TaqMan assay employing allele-specific polymerase chain reaction was used to interrogate single nucleotide polymorphisms (SNPs) rs1799750, rs522616, rs2276109, rs2569190, rs1800469, and rs1024611 of candidate wound healing genes MMP1, MMP3, MMP12, CD14, TGFß1, and MCP1, respectively. A logistic regression model was used to examine the association of candidate gene polymorphisms with the presence or absence of ALTS. RESULTS: All 138 patients were successfully genotyped. No significant association was found between candidate SNPs and development of ALTS in the overall group. However, subgroup analysis within each ethnicity identified SNPs that are associated with ALTS depending upon the ethnic background. CONCLUSIONS: Patient factors such as variations in wound healing due to functional SNPs may shed light on the development of ALTS. There may be a difference in susceptibility to developing ALTS in different ethnic backgrounds. These preliminary findings need to be corroborated in larger population studies. LEVEL OF EVIDENCE: 3b. Laryngoscope, 128:E111-E116, 2018.

Genetic findings in sport-related concussions: potential for individualized medicine?

Concussion is a traumatic transient disturbance of the brain. In sport, the initial time and severity of concussion is known giving an opportunity for subsequent analysis. Variability in susceptibility and recovery between individual athletes depends, among other parameters, on genetic factors. The genes-encoding polypeptides that determine incidence, severity and prognosis for concussion are the primary candidates for genetic analysis. Genetic polymorphisms in the genes contributing to plasticity and repair (APOE), synaptic connectivity (GRIN2A), calcium influx (CACNA1E), uptake and deposit of glutamate (SLC17A7) are potential biomarkers of concussion incidence and recovery rate. With catalogued genetic variants, prospective genotyping of athletes at the beginning of their career will allow medical professionals to improve concussion management and return-to-play decisions.

Genetic variation in SLC17A7 promoter associated with response to sport-related concussions.

OBJECTIVE: To determine the association of the single nucleotide polymorphism (SNP) rs74174284 within SLC17A7 promoter with concussion severity or duration. DESIGN: A between-subjects design was utilized. METHODS: Saliva samples and concussion severity and duration data were collected from 40 athletes diagnosed with a sport-related concussion by a physician, utilizing a standardized concussion assessment protocol. DNA was extracted, estimated and genotyped. RESULTS: An association was found between the dominant genetic model (CC vs GG + GC; p = 0.0179) and recovery, where those carrying the minor allele were 6.33-times more likely to experience prolonged recovery rates. Within the ImPACT assessment, those carrying the CC genotype (33.38 ± 10.15, p = 0.01) had worse motor speed scores upon initial assessment compared to both heterozygous (CG) and homozygous (GG) genotypes (41.59 ± 7.39). CONCLUSIONS: This study was the first to demonstrate an association between genetic polymorphism at rs7417284 SNP in the promoter region of the SLC17A7 gene and concussion severity and duration. Based upon these findings, rs74174284 is a potential predictive genetic marker for identifying athletes who are more susceptible for altered recovery times and worse motor speed ImPACT scores after sport-related concussion.

Disruption of NAD(+) binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions.

AIM: To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS: We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters (diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching (FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD(+) cofactor binding. RESULTS: Using MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD(+) binding center of GAPDH at Y94, S98, and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH, we demonstrated accumulation of phospho-T99-GAPDH in the nuclear fractions of A549, HCT116, and SW48 cancer cells after cytotoxic stress. We performed site-mutagenesis, and estimated enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD(+) binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD(+) (Km = 741 ± 257 µmol/L in T99I vs 57 ± 11.1 µmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD(+) binding with GAPDH. FRAP (fluorescence recovery after photo bleaching) analysis showed that mutations in NAD(+) binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION: Our results suggest an important functional role of phosphorylated amino acids in the NAD(+) binding center in GAPDH interactions with its intranuclear partners.

Association between GRIN2A promoter polymorphism and recovery from concussion.

OBJECTIVE: To determine genetic variability within the N-methyl-D-aspartate receptor 2A sub-unit (GRIN2A) gene promoter and its association with concussion recovery time. The hypothesis tested was that there would be a difference in allele and/or genotype distribution between two groups of athletes with normal and prolonged recovery. METHODS: DNA was extracted from saliva collected from a total of 87 athletes with a physician-diagnosed concussion. The (GT) variable number tandem repeats (VNTR) within the promoter region of GRIN2A was genotyped. The long (L) allele was an allele with ≥25 repeats and the short (S) allele was an allele with <25 repeats in the GT tract. Participants' recovery time was followed prospectively and was categorized as normal (≤60 days) or prolonged (>60 days). RESULTS: LL carriers were 6-times more likely to recover longer than 60 days following the concussive event (p = 0.0433) when compared to SS carriers. Additionally, L allele carriers were found more frequently in the prolonged recovery group (p = 0.048). CONCLUSION: Determining genetic influence on concussion recovery will aid in future development of genetic counselling. The clinical relevance of genotyping athletes could improve management of athletes who experience concussion injuries.

Effects of prostaglandin E1 on callus formation in rabbits.

BACKGROUND: Recent research has focused on identifying chemical modulators of osteogenesis. We present initial findings on the osteoinductive properties of prostaglandin Е1 (Vasaprostan), using a rabbit model. METHODS: Data were collected on callus formation in 14 male rabbits. These were divided into two groups (control and treatment) with 7 animals in each group. In all animals, the right tibia was fractured using a standardized protocol and stabilized by an intramedullary nail. Treatment group received a 5 µg/kg subcutaneous injection of PGE1/day during 10 postoperative days. Visual and radiological evaluation of callus formation was prospectively collected. After 30 days, all animals were killed and the tibia specimens were examined histologically. RESULTS: In all the treatment group animals, fractures were consolidated radiologically by day 30. No treatment group animals and two control group animals were excluded form the experiment. In the control group, 4 animals demonstrated slower callus formation than the main group. Two control group animals were excluded from the experiment on the 20th day due to wound infection; one developed a nonunion. The mean coefficient of bone callus thickening in the main group was 2.08 (±0, 16) and 1.77 (±0.05) (p < 0.05) in the control group. Calculation of mean quantity of neogenic vessels in 10 random visual fields of the bone callus revealed 78 (±9.82) in the main group and 40 (±4.68) in the control group (p < 0.05). CONCLUSIONS: Our study demonstrates an increased rate and amount of bone callus formation in the group treated with prostaglandin E1 compared to the control group. Prospective radiological analysis was corroborated by histologic evaluation.

Synthesis and evaluation of Strychnos alkaloids as MDR reversal agents for cancer cell eradication.

Natural products represent the fourth generation of multidrug resistance (MDR) reversal agents that resensitize MDR cancer cells overexpressing P-glycoprotein (Pgp) to cytotoxic agents. We have developed an effective synthetic route to prepare various Strychnos alkaloids and their derivatives. Molecular modeling of these alkaloids docked to a homology model of Pgp was employed to optimize ligand-protein interactions and design analogues with increased affinity to Pgp. Moreover, the compounds were evaluated for their (1) binding affinity to Pgp by fluorescence quenching, and (2) MDR reversal activity using a panel of in vitro and cell-based assays and compared to verapamil, a known inhibitor of Pgp activity. Compound 7 revealed the highest affinity to Pgp of all Strychnos congeners (Kd=4.4µM), the strongest inhibition of Pgp ATPase activity, and the strongest MDR reversal effect in two Pgp-expressing cell lines. Altogether, our findings suggest the clinical potential of these synthesized compounds as viable Pgp modulators justifies further investigation.

Translational genomics of acquired laryngotracheal stenosis.

OBJECTIVES/HYPOTHESIS: Acquired laryngotracheal stenosis (ALTS) results from abnormal mucosal wound healing after laryngeal and/or tracheal injury. Patients with ALTS often present late after significant reduction of the airway lumen and onset of symptoms. Motivated by the need for earlier detection of affected patients, we sought to investigate genetic markers for ALTS that would identify susceptible patients. STUDY DESIGN: Pilot Case-Control Study. METHODS: Seventy-six patients were recruited, 40 patients with ALTS and 36 control patients with airway injury but without ALTS. DNA was isolated from whole blood and formalin-fixed paraffin-embedded specimens from patients. Custom primers were designed and the TaqMan assay employing allele-specific polymerase chain reaction was used to interrogate single nucleotide polymorphisms (SNPs): rs2569190, rs1799750, and rs1800469 located in candidate genes CD14, matrix metalloproteinase-1 (MMP-1), and transforming growth factor-ß1 (TGF-ß1), respectively. A logistic regression model was used to examine the association of candidate gene polymorphisms with the presence or absence of ALTS. RESULTS: All 76 patients were successfully genotyped at the three loci of interest by optimizing the genotyping protocol. MMP-1 SNP rs1799750 was most significantly associated with development of ALTS (P = 0.005). CONCLUSION: Identification of SNPs associated with development of ALTS will provide new experimental targets to study wound healing in human subjects. The association found in the current study between ALTS and SNP rs1799750 is being validated in a larger population examining an expanded set of relevant SNPs. Identifying patients with genetic susceptibility to ALTS and poor wound healing in the upper airway will be useful for management of patients after upper-airway injury.

The letrozole phase 1 metabolite carbinol as a novel probe drug for UGT2B7.

Carbinol [4,4'-(hydroxymethylene)dibenzonitrile] is the main phase 1 metabolite of letrozole, a nonsteroidal aromatase inhibitor used for endocrine therapy in postmenopausal breast cancer. We elucidated the contribution of UDP-glucuronosyltransferase (UGT) isoforms on the glucuronidation of carbinol. Identification of UGT isoforms was performed using a panel of recombinant human UGT enzymes. Kinetic studies were done in recombinant human UGT2B7 and pooled human liver microsomes (HLMs). A liquid chromatography-tandem mass spectrometry method was used for detection of metabolites. To assess the impact of UGT2B7*2, we determined the carbinol glucuronidation activity using HLM as well as UGT2B7 protein expression in 148 human livers. Moreover, we analyzed the plasma concentrations of 60 letrozole-treated breast cancer patients. We identified UGT2B7 as the predominant UGT isoform involved in carbinol glucuronidation. In HLMs and recombinant UGT2B7, we determined K(m) values (9.99 and 9.56 µM) and V(max) values (3430 and 2399 pmol/min per milligram of protein), respectively. In the set of 148 human livers, carbinol glucuronidation activity significantly correlated with UGT2B7 protein as determined by Western blotting (r(s) = 0.5088, P < 0.0001). Neither carbinol glucuronidation activity (*1/*1: n = 25, 2434 ± 1018; *1/*2: n = 80, 2356 ± 1372; *2/*2: n = 43, 2251 ± 1421 pmol/min per milligram of protein) nor UGT2B7 protein expression was altered by the UGT2B7*2 genotype. No impact of UGT2B7*2 on plasma levels of carbinol and carbinol-gluc [bis(4-cyanophenyl)methyl hexopyranosiduronic acid] in 60 letrozole-treated patients was found. Taken together, these findings suggest carbinol as a novel in vitro probe substrate for UGT2B7. In vitro and in vivo data suggest a lack of influence of the UGT2B7*2 polymorphism on carbinol glucuronidation.

The total ankle arthroplasty learning curve with third-generation implants: a single surgeon's experience.

BACKGROUND: Renewed interest in total ankle arthroplasty (TAA) has developed globally as a result of recent literature supporting new-generation implants as a viable alternative to arthrodesis. The literature also demonstrates a learning curve among surgeons adopting TAA. The purpose of this study is to better define this learning curve for surgeons using third-generation implants. METHODS: Charts and radiographs were reviewed for the initial 26 TAA procedures performed by the senior author. Three third-generation implants were used: SBi (Small Bone Innovations) STAR, Salto Talaris, and Wright Medical INBONE. We report perioperative and early postoperative complications. RESULTS: Two perioperative fractures occurred in the first 9 cases, and the incidence subsequently dropped to 0 (P = .0431). Two cases of component malalignment occurred in the first 3 patients receiving the STAR implant, and the incidence then dropped to 0 (P = .0034). Five wound complications (4 minor and 1 major) occurred, all in the final 14 patients. No cases of nerve injury, tendon laceration, or deep vein thrombosis occurred. Two patients returned to the operating room as a result of complications, and the total perioperative and early postoperative complication rate was 27%. CONCLUSION: The observed rate of perioperative and early postoperative complications in this case series was low relative to other similar-sized studies, suggesting that third-generation implants can reduce adverse events. Our results demonstrate that some common complications could be avoided altogether (nerve/tendon injuries), some decreased quickly with experience (intraoperative fractures and component malpositioning), and some persisted unchanged throughout this study (wound complications). These findings should influence surgical training, surgeon willingness to adopt this procedure, and patient counseling. LEVELS OF EVIDENCE: Therapeutic, Level IV, Retrospective Case Series.

Establishing a model for assessing DNA damage in murine brain cells as a molecular marker of chemotherapy-associated cognitive impairment.

AIMS: Chemotherapy-associated cognitive impairment often follows cancer chemotherapy. We explored chemotherapy-induced DNA damage in the brain cells of mice treated with 5-fluorouracil (5FU), an antineoplastic agent, to correlate the extent of DNA damage to behavioral functioning in an autoshaping-operant mouse model of chemotherapy-induced learning and memory deficits (Foley et al., 2008). MAIN METHODS: Male, Swiss-Webster mice were injected once with saline or 75 mg/kg 5FU at 0, 12, and 24h and weighed every 24h. Twenty-four h after the last injection, the mice were tested in a two-day acquisition and the retention of a novel response task for food reinforcement. Murine brain cells were analyzed for the presence of single- and double-strand DNA breaks by the single cell gel electrophoresis assay (the Comet assay). KEY FINDINGS: We detected significant differences (p<0.0001) for all DNA damage characteristics (DNA "comet" tail shape, migration pattern, tail moment and olive moments) between control mice cohort and 5FU-treated mice cohort: tail length - 119 vs. 153; tail moment - 101 vs. 136; olive moment - 60 vs. 82, correspondingly. We found a positive correlation between increased response rates (r=0.52, p<0.05) and increased rate of errors (r=0.51, p<0.05), and DNA damage on day 1. For all 15 mice (saline-treated and 5FU-treated mice), we found negative correlations between DNA damage and weight (r=-0.75, p<0.02). SIGNIFICANCE: Our results indicate that chemotherapy-induced DNA damage changes the physiological status of the brain cells and may provide insights to the mechanisms for cognitive impairment after cancer chemotherapy.

Institutional Profile: Jayne Haines Center for Pharmacogenomics and Drug Safety: educating future generations of healthcare professionals.

The Jayne Haines Center for Pharmacogenomics and Drug Safety operates in the Temple University School of Pharmacy and serves as an educational and research facility for professional pharmacy students, graduate students, residents, postdoctoral fellows and faculties. The Center is involved in educational and investigational projects in a setting that includes an inner city research/teaching hospital and the Temple University Schools of Pharmacy, Medicine, Dentistry and Health Professions. The mission of the Haines Center is to facilitate the basic science approach to the problems of pharmacotherapy, and to provide education for future healthcare professionals in the genetics of drug response. With the expanding role of genetic analysis in preventing adverse effects of pharmacotherapy, we are working towards truly personalized pharmacotherapy that fully exploits the advances of modern biomedical science.

Cytotoxicity of chemotherapeutic agents in glyceraldehyde-3-phosphate dehydrogenase-depleted human lung carcinoma A549 cells with the accelerated senescence phenotype.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays a central role in glycolysis. Because cancer cells rely on aerobic glycolysis rather than oxidative phosphorylation, GAPDH-depleting agents have a therapeutic potential to impede cancer cell proliferation. Knockdown of GAPDH by RNA interference induced the accelerated senescent phenotype in A549 cells, suggesting that GAPDH is a potential molecular target for combination chemotherapy. The cytotoxic effects of a panel of anticancer drugs, 5-fluorouracil, 5-fluorouridine, 5-fluorodeoxyuridine, 6-thioguanine, cytarabine, fludarabine, cladribine, clofarabine, 2-chloroadenosine, and doxorubicin, were assessed in GAPDH-depleted A549 cells using a cell proliferation assay. GAPDH-depleted A549 cells, when compared with control cells, exhibited increased chemoresistance to several antimetabolite agents including cytarabine [inhibitory concentration 50 (IC50) 1.7±0.3 vs. 0.03±0.02 µmol/l], 2-chloroadenosine (IC50 7.1±1.8 vs. 1.5±0.6 µmol/l), 6-thioguanine (IC50 7.5±1.6 vs. 1.4±0.5 µmol/l), 5-fluorouracil (IC50 13.2±2.5 vs. 3.0±0.7 µmol/l), and 5-fluorodeoxyuridine (IC50 >100 vs. 3.7±0.9 µmol/l), which we designated as group A agents. In contrast, GAPDH-deficient and GAPDH-proficient cells were equally sensitive to group B agents including doxorubicin (IC50 0.05±0.02 vs. 0.04±0.02 µmol/l), fludarabine (IC50 18.5±2.3 vs. 15.7±2.8 µmol/l), 5-fluorouridine (IC50 0.1±0.03 vs. 0.1±0.03 µmol/l), clofarabine (IC50 0.7±0.3 vs. 0.5±0.3 µmol/l), and cladribine (IC50 0.5±0.1 vs. 0.5±0.2 µmol/l). After treatment with group B agents at concentrations equivalent to 7-10-fold the IC50 value, the fraction of apoptotic cells in GAPDH-depleted, senescent A549 cells was similar to that in GAPDH-proficient cells. Our study identified the antimetabolite drugs active in senescent cells that can be used in combination with GAPDH inhibitors in cancer treatment. GAPDH-targeted combination therapy is a novel strategy to control the proliferation of tumor cells.

Clinical response and side effects of metoclopramide: associations with clinical, demographic, and pharmacogenetic parameters.

OBJECTIVES: Metoclopramide is associated with variable efficacy and side effects when used in the treatment of gastroparesis. AIM: To determine associations of clinical and pharmacogenetic parameters with response and side effects to metoclopramide in patients with upper gastrointestinal symptoms suggestive of gastroparesis. METHODS: Gastroparetic patients treated with metoclopramide were enrolled. Clinical parameters recorded were age, sex, weight, diabetic status, gastric emptying result, daily dose, effectiveness, and side effects. DNA was isolated from salivary samples; 20 single nucleotide polymorphisms were genotyped in 8 candidate genes (ABCB1, ADRA1D, CYP1A2, CYP2D6, DRD2, DRD3, HTR4, KCNH2). RESULTS: One hundred gastroparetic patients treated with metoclopramide participated. Dose averaged 33±16 mg/d for 1.1±1.7 years. Responders (53 of 100 patients) were older (48±15 vs. 38±11 y; P=0.0004) and heavier (body mass index of 28±7 vs. 25±7; P=0.0125). Efficacy was associated with polymorphisms in KCNH2 (rs1805123, P=0.020) and ADRA1D (rs2236554, P=0.035) genes. Side effects, occurred in 64 patients, were more common in females (83% vs. 64%; P=0.037), nondiabetics (77% vs. 47%; P=0.004), and patients with normal gastric emptying (41% vs. 17%; P=0.015). Side effects were associated with polymorphisms in CYP2D6 (rs1080985, P=0.045; rs16947, P=0.008; rs3892097, P=0.049), KCNH2 (rs3815459, P=0.015), and serotonin 5-HT4 receptor HTR4 gene (rs9325104, P=0.026). CONCLUSIONS: Side effects to metoclopramide were more common in nondiabetic patients with normal gastric emptying. Polymorphisms in CYP2D6, KCNH2, and 5-HT4 receptor HTR4 genes were associated with side effects, whereas polymorphisms in KCNH2 and ADRA1D genes were associated with clinical response. Clinical parameters and pharmacogenetic testing may be useful in identifying patients before treatment with metoclopramide to enhance efficacy and minimize side effects.

DNA microarray SNP associations with clinical efficacy and side effects of domperidone treatment for gastroparesis.

BACKGROUND: Domperidone treatment for gastroparesis is associated with variable efficacy as well as the potential for side effects. DNA microarray single nucleotide polymorphism (SNP) analysis may help to elucidate the role of genetic variability on the therapeutic effectiveness and toxicity of domperidone. AIM: The aim of this study was to identify SNPs that are associated with clinical efficacy and side effects of domperidone treatment for gastroparesis from DNA microarray experiments. This will help develop a strategy for rational selection of patients for domperidone therapy. METHODS: DNA samples extracted from the saliva of 46 patients treated with domperidone were analyzed using Affymetrix 6.0 SNP microarrays. Then least angle regression (LARS) was used to select SNPs that are related to domperidone efficacy and side effects. Decision tree based prediction models were constructed with the most correlated features selected by LARS. RESULTS: Using the most stable SNP selected by LARS a prediction model for side effects of domperidone achieved (95 ± 0)% true negative rate (TN) and (78 ± 11)% true positive rate (TP) in nested leave-one-out tests. For domperidone efficacy, the prediction based on five most stable SNPs achieved (85 ± 7)% TP and (61±4)% TN. Five identified SNPs are related to ubiquitin mediated proteolysis, epithelial cell signaling, leukocyte, cell adhesion, and tight junction signaling pathways. Genetic polymorphisms in three genes that are related to cancer and hedgehog signaling were found to significantly correlate with efficacy of domperidone. CONCLUSION: LARS was found to be a useful tool for statistical analysis of domperidone-related DNA microarray data generated from a small number of patients.

Neuronal structural protein polymorphism and concussion in college athletes.

OBJECTIVE: To examine the association between a neuronal structural protein polymorphism and the frequency and severity of concussions in college athletes. METHODS: Forty-eight athletes with previous self-reported history of a concussion were matched with 48 controls that did not report a history of concussion. Each group was genotyped for neurofilament heavy (NEFH) polymorphism rs#165602 in this retrospective case-control study. RESULTS: There was no significant association (χ(2 )= 0.487, p = 0.485) between carrying the NEFH rare allele and a history of one or more concussions due to small effect sizes. A Fisher's exact test revealed no significant association (p = 1.00, ϕ = -0.03) between the presence of NEFH rare allele and a history of multiple concussions. The independent t-tests revealed no significant differences in duration of signs and symptoms (t = 1.41, p = 0.17, d = 0.48) or return to play (t = 0.23, p = 0.82, d = 0.08) between NEFH rare allele carrier and non-carriers. CONCLUSIONS: Among college athletes, carrying the rare allele assessed may not influence an athlete's susceptibility to sustaining a concussion or return to play duration following a concussion.

Somatic deletions of genes regulating MSH2 protein stability cause DNA mismatch repair deficiency and drug resistance in human leukemia cells.

DNA mismatch repair enzymes (for example, MSH2) maintain genomic integrity, and their deficiency predisposes to several human cancers and to drug resistance. We found that leukemia cells from a substantial proportion of children (∼11%) with newly diagnosed acute lymphoblastic leukemia have low or undetectable MSH2 protein levels, despite abundant wild-type MSH2 mRNA. Leukemia cells with low levels of MSH2 contained partial or complete somatic deletions of one to four genes that regulate MSH2 degradation (FRAP1 (also known as MTOR), HERC1, PRKCZ and PIK3C2B); we also found these deletions in individuals with adult acute lymphoblastic leukemia (16%) and sporadic colorectal cancer (13.5%). Knockdown of these genes in human leukemia cells recapitulated the MSH2 protein deficiency by enhancing MSH2 degradation, leading to substantial reduction in DNA mismatch repair and increased resistance to thiopurines. These findings reveal a previously unrecognized mechanism whereby somatic deletions of genes regulating MSH2 degradation result in undetectable levels of MSH2 protein in leukemia cells, DNA mismatch repair deficiency and drug resistance.

Accelerated cellular senescence phenotype of GAPDH-depleted human lung carcinoma cells.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a pivotal glycolytic enzyme, and a signaling molecule which acts at the interface between stress factors and the cellular apoptotic machinery. Earlier, we found that knockdown of GAPDH in human carcinoma cell lines resulted in cell proliferation arrest and chemoresistance to S phase-specific cytotoxic agents. To elucidate the mechanism by which GAPDH depletion arrests cell proliferation, we examined the effect of GAPDH knockdown on human carcinoma cells A549. Our results show that GAPDH-depleted cells establish senescence phenotype, as revealed by proliferation arrest, changes in morphology, SA-ß-galactosidase staining, and more than 2-fold up-regulation of senescence-associated genes DEC1 and GLB1. Accelerated senescence following GAPDH depletion results from compromised glycolysis and energy crisis leading to the sustained AMPK activation via phosphorylation of α subunit at Thr172. Our findings demonstrate that GAPDH depletion switches human tumor cells to senescent phenotype via AMPK network, in the absence of DNA damage. Rescue experiments using metabolic and genetic models confirmed that GAPDH has important regulatory functions linking the energy metabolism and the cell cycle networks. Induction of senescence in LKB1-deficient non-small cell lung cancer cells via GAPDH depletion suggests a novel strategy to control tumor cell proliferation.

Motor vehicle transportation in hip spica casts: are our patients safely restrained?

BACKGROUND: Federal guidelines and state laws mandate that all children must be appropriately restrained while traveling in motor vehicles to reduce the risk of injury and death secondary to motor vehicle accidents. The purpose of this study is to identify the methods of restraint in motor vehicles for children in hip spica casts. METHODS: Children placed in hip spica casts between August 1, 2006 and August 1, 2008 were recruited. Demographic data, type of spica cast placed, and reason for cast placement were recorded. Before discharge, all children were evaluated by a physical therapist to determine adequate restraint with the least cost. At each follow-up visit and at the time of cast removal, parents filled out standardized nonvalidated questionnaires to determine the method of restraint, mode of transportation, the approximate number of trips taken per week, and the occurrence of traffic violations or accidents. RESULTS: Thirty-one children, average age of 5 years (range, 1.3 to 13 y), in a total of 35 spica casts were enrolled in the study. After evaluation by the physical therapist, none of the children were recommended to be transported in their personal car seat, 12 children were advised to travel by ambulance and 23 were advised to use a specially manufactured car seat. Overall, 8 of 35 children (23%) followed the initial recommendation of the physical therapist. On the basis of our discharge protocol's recommendations, children in 24 spica casts (69%) were suboptimally transported after discharge, 6 children who should have had ambulance transportation and 18 who should have been transported by a specially manufactured car seat. CONCLUSIONS: The majority of children in hip spica casts are not safely restrained when traveling in motor vehicles. Pediatric hospitals must develop better strategies to improve adherence to prescribed safe transportation protocols for patients in hip spica casts. Improved parental education, expansion of insurance coverage for restraints, hospital-based loaner programs and financial assistance to families are potential solutions to explore.

Domperidone treatment for gastroparesis: demographic and pharmacogenetic characterization of clinical efficacy and side-effects.

BACKGROUND: Domperidone is a useful alternative to metoclopramide for treatment of gastroparesis due to better tolerability. Effectiveness and side-effects from domperidone may be influenced by patient-related factors including polymorphisms in genes encoding drug-metabolizing enzymes, drug transporters, and domperidone targets. AIMS: The aim of this study was to determine if demographic and pharmacogenetic parameters of patients receiving domperidone are associated with response to treatment or side-effects. METHODS: Patients treated with domperidone for gastroparesis provided saliva samples from which DNA was extracted. Fourteen single-nucleotide polymorphisms (SNPs) in seven candidate genes (ABCB1, CYP2D6, DRD2, KCNE1, KCNE2, KCNH2, KCNQ1) were used for genotyping. SNP microarrays were used to assess single-nucleotide polymorphisms in the ADRA1A, ADRA1B, and ADRA1D loci. RESULTS: Forty-eight patients treated with domperidone participated in the study. DNA was successfully obtained from each patient. Age was associated with effectiveness of domperidone (p=0.0088). Genetic polymorphism in KCNH2 was associated with effectiveness of domperidone (p=0.041). The efficacious dose was associated with polymorphism in ABCB1 gene (p=0.0277). The side-effects of domperidone were significantly associated with the SNPs in the promoter region of ADRA1D gene. CONCLUSIONS: Genetic characteristics associated with response to domperidone therapy included polymorphisms in the drug transporter gene ABCB1, the potassium channel KCNH2 gene, and α1D--adrenoceptor ADRA1D gene. Age was associated with a beneficial response to domperidone. If verified in a larger population, this information might be used to help determine which patients with gastroparesis might respond to domperidone and avoid treatment in those who might develop side-effects.