Genome-wide association and multi-omics studies identify MGMT as a novel risk gene for Alzheimer's disease among women.

INTRODUCTION: Variants in the tau gene (MAPT) region are associated with breast cancer in women and Alzheimer's disease (AD) among persons lacking apolipoprotein E ε4 (ε4-). METHODS: To identify novel genes associated with tau-related pathology, we conducted two genome-wide association studies (GWAS) for AD, one among 10,340 ε4- women in the Alzheimer's Disease Genetics Consortium (ADGC) and another in 31 members (22 women) of a consanguineous Hutterite kindred. RESULTS: We identified novel associations of AD with MGMT variants in the ADGC (rs12775171, odds ratio [OR] = 1.4, P = 4.9 × 10-8 ) and Hutterite (rs12256016 and rs2803456, OR = 2.0, P = 1.9 × 10-14 ) datasets. Multi-omics analyses showed that the most significant and largest number of associations among the single nucleotide polymorphisms (SNPs), DNA-methylated CpGs, MGMT expression, and AD-related neuropathological traits were observed among women. Furthermore, promoter capture Hi-C analyses revealed long-range interactions of the MGMT promoter with MGMT SNPs and CpG sites. DISCUSSION: These findings suggest that epigenetically regulated MGMT expression is involved in AD pathogenesis, especially in women.

Curcuminoids Inhibit Angiogenic Behaviors of Human Umbilical Vein Endothelial Cells via Endoglin/Smad1 Signaling.

BACKGROUND: Angiogenesis is primarily attributed to the excessive proliferation and migration of endothelial cells. Targeting the vascular endothelial growth factor (VEGF) is therefore significant in anti-angiogenic therapy. Although these treatments have not reached clinical expectations, the upregulation of alternative angiogenic pathways (endoglin/Smad1) may play a critical role in drug (VEGF-neutralizing agents) resistance. Enhanced endoglin expression following a VEGF-neutralizing therapy (semaxanib®) was noted in patients. Treatment with an endoglin-targeting antibody augmented VEGF expression in human umbilical vein endothelial cells (HUVECs). Therefore, approaches that inhibit both the androgen and VEGF pathways enhance the HUVECs cytotoxicity and reverse semaxanib resistance. The purpose of this study was to find natural-occurring compounds that inhibited the endoglin-targeting pathway. METHODS: Curcuminoids targeting endoglin were recognized from two thousand compounds in the Traditional Chinese Medicine Database@Taiwan (TCM Database@Taiwan) using Discovery Studio 4.5. RESULTS: Our results, obtained using cytotoxicity, migration/invasion, and flow cytometry assays, showed that curcumin (Cur) and demethoxycurcumin (DMC) reduced angiogenesis. In addition, Cur and DMC downregulated endoglin/pSmad1 phosphorylation. CONCLUSIONS: The study first showed that Cur and DMC demonstrated antiangiogenic activity via the inhibition of endoglin/Smad1 signaling. Synergistic effects of curcuminoids (i.e., curcumin and DMC) and semaxanib on HUVECs were found. This might be attributed to endoglin/pSmad1 downregulation in HUVECs. Combination treatment with curcuminoids and a semaxanib is therefore expected to reverse semaxanib resistance.

SparkINFERNO: a scalable high-throughput pipeline for inferring molecular mechanisms of non-coding genetic variants.

SUMMARY: We report Spark-based INFERence of the molecular mechanisms of NOn-coding genetic variants (SparkINFERNO), a scalable bioinformatics pipeline characterizing non-coding genome-wide association study (GWAS) association findings. SparkINFERNO prioritizes causal variants underlying GWAS association signals and reports relevant regulatory elements, tissue contexts and plausible target genes they affect. To achieve this, the SparkINFERNO algorithm integrates GWAS summary statistics with large-scale collection of functional genomics datasets spanning enhancer activity, transcription factor binding, expression quantitative trait loci and other functional datasets across more than 400 tissues and cell types. Scalability is achieved by an underlying API implemented using Apache Spark and Giggle-based genomic indexing. We evaluated SparkINFERNO on large GWASs and show that SparkINFERNO is more than 60 times efficient and scales with data size and amount of computational resources. AVAILABILITY AND IMPLEMENTATION: SparkINFERNO runs on clusters or a single server with Apache Spark environment, and is available at https://bitbucket.org/wanglab-upenn/SparkINFERNO or https://hub.docker.com/r/wanglab/spark-inferno. CONTACT: lswang@pennmedicine.upenn.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

VCPA: genomic variant calling pipeline and data management tool for Alzheimer's Disease Sequencing Project.

SUMMARY: We report VCPA, our SNP/Indel Variant Calling Pipeline and data management tool used for the analysis of whole genome and exome sequencing (WGS/WES) for the Alzheimer's Disease Sequencing Project. VCPA consists of two independent but linkable components: pipeline and tracking database. The pipeline, implemented using the Workflow Description Language and fully optimized for the Amazon elastic compute cloud environment, includes steps from aligning raw sequence reads to variant calling using GATK. The tracking database allows users to view job running status in real time and visualize >100 quality metrics per genome. VCPA is functionally equivalent to the CCDG/TOPMed pipeline. Users can use the pipeline and the dockerized database to process large WGS/WES datasets on Amazon cloud with minimal configuration. AVAILABILITY AND IMPLEMENTATION: VCPA is released under the MIT license and is available for academic and nonprofit use for free. The pipeline source code and step-by-step instructions are available from the National Institute on Aging Genetics of Alzheimer's Disease Data Storage Site (http://www.niagads.org/VCPA). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Evaluation of thirty patients with eustachian tube dysfunction in Taiwan by questionnaire survey.

BACKGROUND: Eustachian tube dysfunction (ETD) is a common otolaryngological disorder. The seven-item Eustachian Tube Dysfunction Questionnaire (ETDQ-7) was used for the assessment of symptoms related to ETD and treatment outcome. Currently, there is no traditional Chinese version of the ETDQ-7 to diagnose ETD in Taiwan. We aim to verify the reliability and validity of the traditional Chinese version of the ETDQ-7 in a clinical setting. METHODS: The traditional Chinese version of the ETDQ-7 was completed by 60 adult subjects composed of 30 healthy controls and 30 subjects diagnosed with ETD. The internal consistency was evaluated using the Cronbach's α coefficient. The discriminant validity was calculated by receiver operating characteristic (ROC) curve as an accuracy measure. RESULTS: The overall Cronbach's α coefficient of the traditional Chinese version ETDQ-7 was 0.717. The mean ETDQ-7 total score was 26.97 in the ETD group and 9.27 in the control group. The area under the ROC curve (AUC) was 99.8%, and the sensitivity and specificity of the traditional Chinese ETDQ-7 was 100% and 99.9%, respectively. CONCLUSION: The traditional Chinese version of the ETDQ-7 is a valid and reliable, disease-specific rating scale that can be used to quantitatively evaluate the severity of subjective symptoms of ETD in adult patients.

A rapid and accurate technique for the identification of the recurrent laryngeal nerve.

OBJECTIVE: We studied the anatomic relationship between the recurrent laryngeal nerve (RLN) and the third tracheal ring, which was very important for rapid identification of RLN in our hands. METHODS: This study was initially performed using 8 fresh cadavers (4 female and 4 male). The transverse nerve location from the third trachea and the depth from its anterior surface were measured. We further observed the topography of RLN in relation to the trachea in 60 patients, between November 2008 and January 2011, at the Tri-Service General Hospital and Buddhist Tzu Chi General Hospital, Taipei Branch, Taiwan, with 46 lobo-isthmectomies and 14 total thyroidectomies. The time spent in identifying the RLN was also recorded. RESULTS: Among cadaver groups, the transverse distance (width) and the vertical distance (depth) averaged 3.3 and 17.6 mm, respectively. Among the clinical cases, the width and depth averaged 4.4 and 14.6 mm, respectively. The depth measured in males was significantly deeper than that in females (22.3 vs 13.2 mm) (P < .05). The time spent in identifying the RLN after starting dissection in the RLN triangle was not statistically significantly different between the cadaver group and the clinical group (10.6 ± 5.7 seconds and 15.5 ± 17.7 seconds, respectively; P > .05). The median time was 9 and 10 seconds, respectively. There was no statistically significant side-to-side difference in terms of the time spent in searching for the RLN. CONCLUSION: Using the third ring as guidance, our inferior-superior technique offers an extra benefit in identifying the RLN safely and quickly, as compared to the conventional inferior approach.

Combined balloon Eustachian tuboplasty/endoscopic sinus surgery for patients with chronic rhinosinusitis and Eustachian tube dysfunction.

BACKGROUND: To elucidate the role of balloon Eustachian tuboplasty (BET) in the management of chronic rhinosinusitis with obstructive Eustachian tube dysfunction (ETD), we evaluated the results of endoscopic sinus surgery (ESS) with and without BET in patients with chronic rhinosinusitis with obstructive ETD. METHODS: This randomized controlled trial conducted in a single-institution tertiary care center setting included 50 patients diagnosed with primary chronic rhinosinusitis and obstructive ETD between July 2018 and June 2022. Twenty-five patients were prospectively enrolled for combined ESS/BET. The control group (25 patients) underwent ESS alone. Outcome measurements of the Sinonasal Outcome Test 22, modified Lund-Kennedy score, Eustachian Tube Dysfunction Questionnaire-7 (ETDQ-7), and serial Eustachian tube function test results were analyzed 3 months postoperatively. RESULTS: The improvement (12.60 ± 6.50) in the ETDQ-7 score in the BET group was significantly higher than that in the control group (6.60 ± 5.58). The ratio of improvement in the ETDQ-7 score was also significantly higher in the BET than in the control group (92% vs. 68%, p = 0.034). Logistic regression analysis showed that performing BET (odds ratio [OR]: 5.41, 95% confidence interval [CI]: 1.02-28.79, p = 0.048) and a low post-modified Lund-Kennedy score (OR: 0.15, 95% CI: 0.04-0.54, p = 0.004) were significantly associated with ETDQ-7 score improvement. CONCLUSION: Combined BET/ESS could decrease otologic symptoms and improve Eustachian tube function. BET may be an appropriate adjunctive procedure for treating chronic rhinosinusitis with obstructive ETD.

Otoprotection against aminoglycoside- and cisplatin-induced ototoxicity focusing on the upstream drug uptake pathway.

Aminoglycoside- and cisplatin-induced ototoxicity, which is a significant issue owing to the widespread use of these drugs in clinical practice, involves the entry of aminoglycosides and cisplatin into the endolymph and hair cells via specific channels or transporters, followed by reactive oxygen species (ROS) generation and hair cells apoptosis. Current strategies focalize primarily on interference with downstream ROS effects; however, recent evidence has demonstrated that inhibiting the uptake of aminoglycosides and cisplatin by hair cells is another promising strategy for tackling the upstream drug uptake pathway. With advances in structural biology, the conformations of certain aminoglycoside and cisplatin channels and transporters, such as the mechanoelectrical transduction channel and organic cation transporter-2, have been largely elucidated. These channels and transporters may become potential targets for the introduction of new otoprotective strategies. This review focuses on the strategies for inhibiting ototoxic drugs uptake by auditory hair cells and provides potential targets for recent developments in the field of otoprotection. Molecular dynamics (MD) simulations of these proteins could help identify the molecules that inhibit the uptake of aminoglycosides and cisplatin by hair cells. Integrating upstream drug uptake pathway targets and MD simulations may help dissect molecular mechanisms and develop novel otoprotective strategies for aminoglycoside- and cisplatin-induced ototoxicity.

Human whole-exome genotype data for Alzheimer's disease.

The heterogeneity of the whole-exome sequencing (WES) data generation methods present a challenge to a joint analysis. Here we present a bioinformatics strategy for joint-calling 20,504 WES samples collected across nine studies and sequenced using ten capture kits in fourteen sequencing centers in the Alzheimer's Disease Sequencing Project. The joint-genotype called variant-called format (VCF) file contains only positions within the union of capture kits. The VCF was then processed specifically to account for the batch effects arising from the use of different capture kits from different studies. We identified 8.2 million autosomal variants. 96.82% of the variants are high-quality, and are located in 28,579 Ensembl transcripts. 41% of the variants are intronic and 1.8% of the variants are with CADD > 30, indicating they are of high predicted pathogenicity. Here we show our new strategy can generate high-quality data from processing these diversely generated WES samples. The improved ability to combine data sequenced in different batches benefits the whole genomics research community.

Using multi-stimulus auditory steady state response to predict hearing thresholds in high-risk infants.

The purpose of this study was to investigate whether multi-stimulus auditory steady-state responses were capable of estimating hearing thresholds in high-risk infants. A retrospective chart review study. Three tertiary referral centers. Infants born between January 2004 and December 2006 who met the criteria for risk factors of congenital hearing loss were enrolled in the study. While under sedation, the multi-stimulus auditory steady-state response was used to determine multi-channel auditory steady-state response thresholds for high-risk infants younger than 13 months. Conditioned play audiometry was then applied to these children at 23-48 months of age to obtain pure tone audiograms. Auditory steady-state response thresholds and pure tone thresholds were then compared. A total of 249 high-risk infants were enrolled in the study. 39 infants were lost during follow-up. The remaining 216 infants completed both examinations. The Pearson correlation coefficients (r) between the ASSR levels and pure tone thresholds were 0.88, 0.94, 0.94 and 0.97 at 500, 1,000, 2,000 and 4,000 Hz, respectively. The strength of the relationship between the auditory steady-state responses and pure tone thresholds increased with more severe degrees of hearing loss and higher frequencies. We conclude that initial multichannel ASSR thresholds measured under sedation are highly correlated with pure tone thresholds obtained 2 or 3 years later. ASSR can be used to predict the frequency-specific hearing thresholds of high-risk infants and can provide information for early hearing intervention.

Copy Number Variation Identification on 3,800 Alzheimer's Disease Whole Genome Sequencing Data from the Alzheimer's Disease Sequencing Project.

Alzheimer's Disease (AD) is a progressive neurologic disease and the most common form of dementia. While the causes of AD are not completely understood, genetics plays a key role in the etiology of AD, and thus finding genetic factors holds the potential to uncover novel AD mechanisms. For this study, we focus on copy number variation (CNV) detection and burden analysis. Leveraging whole-genome sequence (WGS) data released by Alzheimer's Disease Sequencing Project (ADSP), we developed a scalable bioinformatics pipeline to identify CNVs. This pipeline was applied to 1,737 AD cases and 2,063 cognitively normal controls. As a result, we observed 237,306 and 42,767 deletions and duplications, respectively, with an average of 2,255 deletions and 1,820 duplications per subject. The burden tests show that Non-Hispanic-White cases on average have 16 more duplications than controls do (p-value 2e-6), and Hispanic cases have larger deletions than controls do (p-value 6.8e-5).

Effect of Round Window Opening Size on Residual Hearing Preservation in Cochlear Implantation.

OBJECTIVES: This study aims to compare the hearing preservation outcomes in cochlear implant surgery following slit versus full opening of the round window membrane. SETTING: Tertiary referral center. STUDY DESIGN: Comparative study. SUBJECTS AND METHODS: Seventy patients (mean, 26.3 years; range, 2-69 years) who underwent cochlear implantation via the round window approach were included in the study. Thirty-five subjects were prospectively enrolled for cochlear implantation via the open round window technique between August 2018 and January 2019. Thirty-five patients who underwent cochlear implantation from January 2017 to July 2018 via the slit round window opening, frequency matched by sex and age, were retrospectively enrolled. Pre- and postoperative thresholds were obtained. The percentage of hearing preservation was computed with the HEARRING Network formula and classified into complete, partial, and minimal hearing preservation. The results between the groups were compared and analyzed at 6 months postoperatively. RESULTS: The rate of complete hearing preservation in the open group was statistically significant (P = .030) at 71.4% (n = 25) as compared with 45.7% (n = 16) in the slit group. CONCLUSIONS: The widely opened round window may be an optional technique that surgeons can utilize to improve hearing preservation outcomes.

Comparison of Hearing Preservation Outcomes Using Extended Versus Single-Dose Steroid Therapy in Cochlear Implantation.

OBJECTIVES: The purpose of this study was to compare the hearing preservation outcomes of patients who received extended versus single-dose steroid therapy in cochlear implant surgery. DESIGN: Case-control. SETTING: Tertiary referral centers in Taiwan from April 2017 to 2019. PARTICIPANTS: A total of 70 patients aged 1 to 78 years old (mean = 18.04, standard deviation [SD] = 21.51) who received cochlear implantation via the round window approach were included in the study. Prospectively, 35 cases were enrolled for cochlear implantation with single-dose therapy. Thirty-five controls who underwent cochlear implantation with extended therapy were retrospectively enrolled after frequency matching. OUTCOME MEASURES: The main outcome measure was the rate of hearing preservation. This was calculated based on the HEARRING Network formula and results were categorized as complete, partial, and minimal. Impedances served as secondary outcomes. RESULTS: There was no significant difference in the complete hearing preservation rates between the extended and single-dose groups at 6 months postoperatively. Impedances were significantly lower in the extended group after 1 month and 6 months of follow up. When the complete and partial hearing preservation groups were compared, the size of round window opening and speed of insertion were found to be statistically significant. CONCLUSIONS: Both extended and single-dose therapies result in good hearing preservation in patients who undergo cochlear implantation. However, better impedances can be expected from patients who received extended therapy. A slower speed of insertion and a widely opened round window play a role in hearing preservation.

Inferring the Molecular Mechanisms of Noncoding Alzheimer's Disease-Associated Genetic Variants.

Most of the loci identified by genome-wide association studies (GWAS) for late-onset Alzheimer's disease (LOAD) are in strong linkage disequilibrium (LD) with nearby variants all of which could be the actual functional variants, often in non-protein-coding regions and implicating underlying gene regulatory mechanisms. We set out to characterize the causal variants, regulatory mechanisms, tissue contexts, and target genes underlying these associations. We applied our INFERNO algorithm to the top 19 non-APOE loci from the IGAP GWAS study. INFERNO annotated all LD-expanded variants at each locus with tissue-specific regulatory activity. Bayesian co-localization analysis of summary statistics and eQTL data was performed to identify tissue-specific target genes. INFERNO identified enhancer dysregulation in all 19 tag regions analyzed, significant enrichments of enhancer overlaps in the immune-related blood category, and co-localized eQTL signals overlapping enhancers from the matching tissue class in ten regions (ABCA7, BIN1, CASS4, CD2AP, CD33, CELF1, CLU, EPHA1, FERMT2, ZCWPW1). In several cases, we identified dysregulation of long noncoding RNA (lncRNA) transcripts and applied the lncRNA target identification algorithm from INFERNO to characterize their downstream biological effects. We also validated the allele-specific effects of several variants on enhancer function using luciferase expression assays. By integrating functional genomics with GWAS signals, our analysis yielded insights into the regulatory mechanisms, tissue contexts, genes, and biological processes affected by noncoding genetic variation associated with LOAD risk.

Off-Target V(D)J Recombination Drives Lymphomagenesis and Is Escalated by Loss of the Rag2 C Terminus.

Genome-wide analysis of thymic lymphomas from Tp53(-/-) mice with wild-type or C-terminally truncated Rag2 revealed numerous off-target, RAG-mediated DNA rearrangements. A significantly higher fraction of these errors mutated known and suspected oncogenes/tumor suppressor genes than did sporadic rearrangements (p < 0.0001). This tractable mouse model recapitulates recent findings in human pre-B ALL and allows comparison of wild-type and mutant RAG2. Recurrent, RAG-mediated deletions affected Notch1, Pten, Ikzf1, Jak1, Phlda1, Trat1, and Agpat9. Rag2 truncation substantially increased the frequency of off-target V(D)J recombination. The data suggest that interactions between Rag2 and a specific chromatin modification, H3K4me3, support V(D)J recombination fidelity. Oncogenic effects of off-target rearrangements created by this highly regulated recombinase may need to be considered in design of site-specific nucleases engineered for genome modification.

Comparison of intermittent intratympanic steroid injection and near-continual transtympanic steroid perfusion as salvage treatments for sudden sensorineural hearing loss.

OBJECTIVES/HYPOTHESIS: The purpose of this study was to investigate whether near-continual transtympanic steroid perfusion is more effective than intermittent intratympanic steroid injection as a salvage therapy for idiopathic sudden sensorineural hearing loss. STUDY DESIGN: Case control study. METHODS: We designed a case-control study consisting of 60 patients with sudden sensorineural hearing loss who did not respond well to systemic steroid therapy. From November 2008 to October 2010, we prospectively enrolled subjects for the transtympanic steroid perfusion therapy. We retrospectively collected data from age- and sex-matched patients who had undergone intratympanic steroid injection between January 2003 and October 2008. The audiological results of the two groups were compared. RESULTS: The presalvage pure tone threshold was 65.4 ± 13.5 dB in the transtympanic steroid perfusion group. After the therapy, the hearing threshold was improved by an average of 15.0 ± 9.7 dB, and 53.3% of subjects had improved by 10 dB or more. The speech discrimination score was improved from 12.6% ± 7.0% to 54.4 ± 6.4%. In the intratympanic steroid injection group, the presalvage pure tone threshold was 68.8 ± 16.0 dB. After the therapy, the hearing threshold was improved by an average of 10.7 ± 9.8 dB, and 43.3% of subjects had improved by 10 dB or more. The speech discrimination score was improved from 13.3 ± 6.0% to 46.4 ± 12%. The degree of hearing improvement was significantly greater in the transtympanic group. CONCLUSIONS: Both transtympanic steroid perfusion and intratympanic steroid injection can be used as salvage therapies for idiopathic sudden sensorineural hearing loss. Near-continual transtympanic steroid perfusions may provide better audiological results.

Intratympanic steroid injections as a salvage treatment for sudden sensorineural hearing loss: a randomized, double-blind, placebo-controlled study.

OBJECTIVE: The purpose of this study was to determine, through a randomized, double-blind, placebo-controlled trial, whether intratympanic steroid injections (ITSIs) could improve hearing recovery in patients with sudden sensorineural hearing loss (SSHL) who did not respond to initial systemic steroid therapy. STUDY DESIGN: This was a prospective, randomized, double-blind, placebo-controlled study. SETTING: The study was conducted in 2 tertiary referral centers. PATIENTS: A total of 60 patients with idiopathic SSHL who did not respond to an initial round of systemic steroid therapy were included in this study. The subjects were randomized into an ITSI group and an intratympanic normal saline injection (ITNI) group, which were matched by age and sex. A total of 55 subjects completed the study. INTERVENTION: Participants received either ITSIs or ITNIs. Both groups received 4 injections within a 2-week period. MAIN OUTCOME MEASURES: Pure-tone thresholds were compared between the 2 groups 1 month after injection therapy. RESULTS: In the ITNI group, the pure-tone threshold was 69.9 ± 18.5 dB before intratympanic injection therapy. After therapy, the hearing threshold improved by an average of 4.5 ± 6.5 dB, and 10.7% of subjects improved by 10 dB or more. In the ITSI group, the pure-tone threshold was 64.6 ± 17.7 dB before intratympanic injection therapy. After the therapy, the hearing threshold improved by an average of 9.8 ± 8.5 dB, and 44.4% of subjects improved by 10 dB or more. Both the response rate and the level of hearing improvement were significantly greater in the ITSI group than in the ITNI group. CONCLUSION: These results demonstrate that ITSIs are beneficial as a salvage therapy for the treatment of patients with idiopathic SSHL who fail to respond to initial systemic steroid therapy.

Genes and biochemical characterization of three novel chlorophyllase isozymes from Brassica oleracea.

Three full length cDNAs (BoCLH1, 1140 bp; BoCLH2, 1104 bp; BoCLH3, 884 bp) encoding putative chlorophyllases were cloned from the cDNA pools of broccoli (Brassica oleracea) florets and characterized. The amino acid sequence analysis indicated that these three BoCLHs contained a highly conserved lipase motif (GXSXG). However, only BoCLH3 lacked the His residue which is the component of the catalytic triad (Ser-His-Asp). N-terminal sequences of BoCLH1 and BoCLH2 were predicted to have typical signal sequences for the chloroplast, whereas the plasma membrane-targeting sequence was identified in BoCLH3. The predicted molecular masses of BoCLH1, 2, and 3 were 34.7, 35.3, and 23.5 kDa, respectively. The recombinant BoCLHs were successfully expressed in Escherichia coli for the biochemical characterization. The recombinant BoCLH3 showed very low chlorophyllase activity possibly due to its incomplete catalytic triad. BoCLH1 and BoCLH2 showed significant differences in biochemical properties such as pH stability and temperature optimum. Kinetic analysis revealed that BoCLH1 preferably hydrolyzed Mg-free chlorophyll, while BoCLH2 hydrolyzed both chlorophyll and Mg-free chlorophyll at a similar level. Different characteristics between BoCLH1 and BoCLH2 implied that they may have different physiological functions in broccoli. The catalytic triad of recombinant BoCLH2 was identified as Ser141, His247, and Asp170 by site-directed mutagenesis. It suggested that the three broccoli chlorophyllase isozymes were serine hydrolases.

TTK/hMps1 participates in the regulation of DNA damage checkpoint response by phosphorylating CHK2 on threonine 68.

CHK2/hCds1 plays important roles in the DNA damage-induced cell cycle checkpoint by phosphorylating several important targets, such as Cdc25 and p53. To obtain a better understanding of the CHK2 signaling pathway, we have carried out a yeast two-hybrid screen to search for potential CHK2-interacting proteins. Here, we report the identification of the mitotic checkpoint kinase, TTK/hMps1, as a novel CHK2-interacting protein. TTK/hMps1 directly phosphorylates CHK2 on Thr-68 in vitro. Expression of a TTK kinase-dead mutant, TTK(D647A), interferes with the G(2)/M arrest induced by either ionizing radiation or UV light. Interestingly, induction of CHK2 Thr-68 phosphorylation and of several downstream events, such as cyclin B1 accumulation and Cdc2 Tyr-15 phosphorylation, is also affected. Furthermore, ablation of TTK expression using small interfering RNA results not only in reduced CHK2 Thr-68 phosphorylation, but also in impaired growth arrest. Our results are consistent with a model in which TTK functions upstream from CHK2 in response to DNA damage and suggest possible cross-talk between the spindle assembly checkpoint and the DNA damage checkpoint.