Efficacy of adjuvant capecitabine in residual triple negative breast cancer: a multicenter observational Turkish Oncology Group (TOG) study.

BACKGROUND: Triple negative breast cancer (TNBC) is characterized by high rates of recurrence, especially in patients with residual disease after neoadjuvant chemotherapy (NAC). Capecitabine is being used as standard adjuvant treatment in residual TNBC. We aimed to investigate the real-life data regarding the efficacy of capecitabine in residual TNBC. DESIGN AND METHODS: In this retrospective multicenter study, TNBC patients with residual disease were evaluated. Patients, who received standard anthracycline and taxane-based NAC and adjuvant capecitabine were eligible. Overall survival (OS), disease free survival (DFS) and toxicity were analyzed. RESULTS: 170 TNBC patients with residual disease were included. Of these, 62.9% were premenopausal. At the time of analysis, the recurrence rate was 30% and death rate was 18%. The 3-year DFS and OS were 66% and 74%, respectively. In patients treated with adjuvant capecitabine, residual node positive disease stood out as an independent predictor of DFS (p = 0.024) and OS (p = 0.032). Undergoing mastectomy and the presence of T2 residual tumor was independent predictors of DFS (p = 0.016) and OS (p = 0.006), respectively. CONCLUSION: The efficacy of capecitabine was found lower compared to previous studies. Selected patients may have further benefit from addition of capecitabine. The toxicity associated with capecitabine was found lower than anticipated.

Peptide-conjugated Nanoparticle Platforms for Targeted Delivery, Imaging, and Biosensing Applications.

Peptides have become an indispensable tool in engineering of multifunctional nanostructure platforms for biomedical applications such as targeted drug and gene delivery, imaging and biosensing. They can be covalently incorporated into a variety of nanoparticles (NPs) including polymers, metallic nanoparticles, and others. Using different bioconjugation techniques, multifunctional peptide-modified NPs can be formulated to produce therapeutical and diagnostic platforms offering high specificity, lower toxicity, biocompatibility, and stimuli responsive behavior. Targeting peptides can direct the nanoparticles into specific tissues for targeted drug and gene delivery and imaging applications due to their specificity towards certain receptors. Furthermore, due to their stimuli-responsive features, they can offer controlled release of therapeutics into desired sites of disease. In addition, peptide-based biosensors and imaging agents can provide non-invasive detection and monitoring of diseases including cancer, infectious diseases, and neurological disorders. In this review, we covered the design and formulation of recent peptide-based NP platforms, as well as their utilization in in vitro and in vivo applications such as targeted drug and gene delivery, targeting, sensing, and imaging applications. In the end, we provided the future outlook to design new peptide conjugated nanomaterials for biomedical applications.

Computational insights on the hydride and proton transfer mechanisms of L-proline dehydrogenase.

L-Proline dehydrogenase (ProDH) is a flavin-dependent oxidoreductase, which catalyzes the oxidation of L-proline to (S)-1-pyrroline-5-carboxylate. Based on the experimental studies, a stepwise proton and hydride transfer mechanism is supported. According to this mechanism, the amino group of L-proline is deprotonated by a nearby Lys residue, which is followed by the hydride transfer process from C5 position of L-proline to N5 position of isoalloxazine ring of FAD. It was concluded that the hydride transfer step is rate limiting in the reductive half-reaction, however, in the overall reaction, the oxidation of FAD is the rate limiting step. In this study, we performed a computational mechanistic investigation based on ONIOM method to elucidate the mechanism of the reductive half-reaction corresponding to the oxidation of L-proline into iminoproline. Our calculations support the stepwise mechanism in which the deprotonation occurs initially as a fast step as result of a proton transfer from L-proline to the Lys residue. Subsequently, a hydride ion transfers from L-proline to FAD with a higher activation barrier. The enzyme-product complex showed a strong interaction between reduced FAD and iminoproline, which might help to explain why a step in the oxidative half-reaction is rate-limiting.

The safety and efficacy of first-line atezolizumab plus bevacizumab in patients with unresectable hepatocellular carcinoma: A multicenter real-world study from Turkey.

The aim of the study was to evaluate the real-world clinical outcomes of atezolizumab and bevacizumab (Atez/Bev) as the initial therapy for advanced hepatocellular carcinoma (HCC). We retrospectively analyzed 65 patients treated with Atez/Bev for advanced HCC from 22 institutions in Turkey between September 2020 and March 2023. Responses were evaluated by RECIST v1.1 criteria. The median progression-free survival (PFS) and overall survival (OS) were calculated using the Kaplan-Meier method. Cox regression model was employed to conduct multivariate analyses. The median age was 65 (range, 22-89) years, and 83.1% of the patients were male. A total of 1.5% achieved a complete response, 35.4% had a partial response, 36.9% had stable disease, and 26.2% had progressive disease. The disease control rate was 73.8% and associated with alpha-fetoprotein levels at diagnosis and concomitant antibiotic use. The incidence rates of any grade and grade ≥ 3 adverse events were 29.2% and 10.7%, respectively. At a median follow-up of 11.3 (3.4-33.3) months, the median PFS and OS were 5.1 (95% CI: 3-7.3) and 18.1 (95% CI: 6.2-29.9) months, respectively. In univariate analyses, ECOG-PS ≥ 1 (relative to 0), Child-Pugh class B (relative to A), neutrophil-to-lymphocyte ratio (NLR) > 2.9 (relative to ≤ 2.9), and concomitant antibiotic use significantly increased the overall risk of mortality. Multivariate analysis revealed that ECOG-PS ≥ 1 (HR: 2.69, P = .02), NLR > 2.9 (HR: 2.94, P = .017), and concomitant antibiotic use (HR: 4.18, P = .003) were independent predictors of OS. Atez/Bev is an effective and safe first-line therapy for advanced-stage HCC in a real-world setting. The survival benefit was especially promising in patients with a ECOG-PS score of 0, Child-Pugh class A, lower NLR, and patients who were not exposed to antibiotics during the treatment.

Computational Insights on the Hydride and Proton Transfer Mechanisms of D-Arginine Dehydrogenase.

D-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) is an amine oxidase which catalyzes the conversion of D-arginine into iminoarginine. It contains a non-covalent FAD cofactor that is involved in the oxidation mechanism. Based on substrate, solvent, and multiple kinetic isotope effects studies, a stepwise hydride transfer mechanism is proposed. It was shown that D-arginine binds to the active site of enzyme as α-amino group protonated, and it is deprotonated before a hydride ion is transferred from its α-C to FAD. Based on a mutagenesis study, it was concluded that a water molecule is the most likely catalytic base responsible from the deprotonation of α-amino group. In this study, we formulated computational models based on ONIOM method to elucidate the oxidation mechanism of D-arginine into iminoarginine using the crystal structure of enzyme complexed with iminoarginine. The calculations showed that Arg222, Arg305, Tyr249, Glu87, His 48, and two active site water molecules play key roles in binding and catalysis. Model systems showed that the deprotonation step occurs prior to hydride transfer step, and active site water molecule(s) may have participated in the deprotonation process.

Computational Mechanistic Study of l-Aspartate Oxidase by ONIOM Method.

l-Aspartate oxidase (Laspo) is responsible for the oxidation of l-aspartate into iminoaspartate using flavin as a cofactor. During this process flavin is reduced, and it can be reoxidized by either molecular oxygen or fumarate. The overall fold and the catalytic residues of Laspo are similar to succinate dehydrogenase and fumarate reductase. On the basis of deuterium kinetic isotope effects as well as other kinetic and structural data, it is proposed that the enzyme can catalyze the oxidation of l-aspartate through a mechanism similar to amino acid oxidases. It is suggested that a proton is removed from the α-amino group, while a hydride is transferred from C2 to flavin. It is also suggested that the hydride transfer is a rate-limiting step. However, there is still an ambiguity about the stepwise or concerted mechanism of hydride- and proton-transfer steps. In this study, we formulated some computational models to study the hydride-transfer mechanism using the crystal structure of Escherichia colil-aspartate oxidase in complexes with succinate. The calculations involved our own N-layered integrated molecular orbital and molecular mechanics method, and we evaluated the geometry and energetics of the hydride/proton-transfer processes while probing the roles of active site residues. Based on the calculations, it is concluded that proton- and hydride-transfer steps are decoupled, and a stepwise mechanism might be operative as opposed to the concerted one.

The Effect of the COVID-19 Pandemic Process on the Incidence of Intestinal Parasites; Aydin Province Example

OBJECTIVE: The Coronavirus disease-2019 (COVID-19) pandemic, which started in our country in March 2020, has caused a sudden and dramatic change in our habits. As a result of the pandemic measures directly effective in the transmission of parasites, it has become important to investigate the possible effect of the COVID-19 pandemic process on the incidence of intestinal parasites. METHODS: In order to examine the situation before and after the pandemic, all stool and cellophane tape test results examined from March 11, 2018 to March 11, 2022 in Aydin Adnan Menderes University Faculty of Medicine Parasitology Laboratory were evaluated retrospectively. The relationship between the socio-demographic characteristics of the cases, the distribution of the months and years of diagnosis and the prevalence of parasites were also evaluated. RESULTS: Of the 13,036 stool samples examined, 67.42% belong to the pre-pandemic and 32.58% belong to the post-pandemic period. In total, 1.959 stool samples were positive for at least one parasite, of which 71.41% were from the pre-pandemic. Blastocystis spp. was the most frequently detected parasite both before (14.63%) and after the pandemic (12.59%). Of the 3.194 cellophane tape examined, 72.32% belonged to the pre-pandemic and 27.68% post-pandemic period, and Enterobius vermicularis eggs were detected in 246 (7.70%) of all. The majority of E. vermicularis positive samples (82.11%) were belonged to the pre-pandemic period. The number and positivity rates of stool and cellophane tape samples examined in the post-pandemic period showed a significant decrease (p<0.05). CONCLUSION: It was observed that the incidence of intestinal parasites decreased significantly during the COVID-19 pandemic. Important developments in terms of public health, such as measures such as social distance and quarantine during the pandemic process, increased sensitivity to personal hygiene, and informing the public through various tools during the pandemic, are thought to be the reason for the decrease in the prevalence of intestinal parasites.

Discrepancy among microsatellite instability detection methodologies in non-colorectal cancer: Report of 3 cases.

BACKGROUND: Microsatellite instability (MSI) is a predictive biomarker for cancer immunotherapy. The tumor-agnostic nature of MSI makes it a denominator for immunotherapy in several solid tumors. It can be assessed using next-generation sequencing (NGS), fluorescent multiplex PCR, and immunohistochemistry (IHC). CASE SUMMARY: Here, we report 3 cases with discordant MSI results detected using different methods. A cholangiocellular carcinoma case revealed proficient mismatch repair (MMR) by IHC but high MSI (MSI-H) by liquid NGS. A cervical cancer case revealed deficient MMR by IHC, microsatellite stable by PCR, and MSI-H by NGS. Lastly, an endometrial cancer case revealed proficient MMR by IHC but MSI-H by NGS. CONCLUSION: IHC for MMR status is the first choice due to several advantages. However, in cases of indeterminate IHC results, molecular testing by MSI-PCR is preferred. Recently, NGS-based MSI assays are being widely used to detect MSI-H tumors. All three methods have high accuracy; however, the inconsistencies between them may lead to misdiagnosis.

Current trends in perioperative treatment of resectable gastric cancer.

In the last few decades, the treatment strategy for locally advanced resectable gastric cancer (GC) has shifted to a multimodal approach, which potentially decreases recurrence risk and improves survival rates. Perioperative therapy leads to downstaging, increased curative resection rates, and prolonged disease-free and overall survival, by preventing micrometastases in patients with resectable GC. Application of neoadjuvant therapy provides information about tumor biology and in vivo sensitivity. A consensus regarding the therapeutic approach for non-metastatic GC does not exist, and many clinical trials aim to clarify this aspect. Advances in precision medicine and the role of immunotherapy have been the focus of research in GC treatment. Herein, the current status and possible future developments of perioperative therapy for locally advanced resectable GC are reviewed, based on the most recent randomized clinical trials.

Pristine biochar performance investigation to remove metals in primary and secondary treated municipal wastewater for groundwater recharge application.

In this study, pristine biochar derived from date palm at 500°C was used in batch reactors (simulating blending adsorbent in aeration tank) and fixed-bed columns (simulating holding adsorbent in fixed-bed reactors). The removal performance of the biochar was assessed toward single and mixed-metal solutions as well as synthetic primary and secondary treated wastewater for copper (Cu2+), iron (Fe2+), nickel (Ni2+) and zinc (Zn2+). The order of maximum adsorption capacities of the metal ions at pH 7 followed: Fe2+ (2.92/2.94 mg/g)>Cu2+(2.69/2.78 mg/g) >Zn2+(2.03/2.19 mg/g)>Ni2+(1.69/1.02 mg/g) in single/mixed-metal solutions and Zn2+(2.91/11.26 mg/g)>Fe2+(0.60/5.29 mg/g)>Cu2+(0.56/5.05 mg/g)>Ni2+(0.13/2.02 mg/g) in synthetic primary/secondary treated wastewater. Blending biochar in aeration tank reduced metal concentrations. The metal ion concentrations in the final effluent were below the World Health Organization drinking water limits (2, 0.3, 0.1 and 3 mg/L for Cu2+, Fe2+, Ni2+ and Zn2+, respectively) suggesting that treated secondary wastewater can be spread into potable aquifers following disinfection. The Freundlich and the Pseudo-second order models fit best the batch experimental data. Experimental data from column analysis fit well to the Thomas model. The adsorption of metal ions on the surface of biochar was confirmed by Scanning electron microscopy, Energy dispersive X-ray studies, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy and X-ray diffraction. Desorption studies using different eluents demonstrated the reusability potential of the studied biochar.

Intramural Component of Venous, Lymphatic, and Perineural Invasion in Colon Cancer: A Threat or an Illusion?

Background: Extramural venous invasion is an independent predictor of poor outcome in colorectal cancer, whereas the significance of the intramural component of venous and lymphatic and perineural invasion is unclear. Aims: To evaluate the prognostic impact of intramural components for venous, lymphatic, and perineural invasions and the relation of these invasion patterns with clinicopathological features in patients with colon cancer. Study Design: A retrospective cross-sectional study. Methods: The analysis included 626 patients with colon cancer in stages II and III. All patients were divided into four categories (no invasion, intramural invasion only, extramural invasion only, or both intramural and extramural invasions) for vascular invasion, lymphatic invasion and perineural invasion. The primary outcomes were 5-year disease-free and overall survival. Results: Right-sided (for vascular invasion, 24.7% vs. 33.9%, p = 0.007; for perineural invasion, 34.5% vs. 41.5%, p = 0.034) and dMMR tumors (for vascular invasion, 13.5% vs. 33.5, p < 0.001; for perineural invasion, 25% vs. 41.4%, p = 0.004) exhibited less venous and perineural invasion. Compared with no invasion, presence of intramural invasion only, did not exert any effect on disease-free or overall survival for vascular invasion, lymphatic invasion, and perineural invasion. Multivariate analyses revealed that the presence of both intramural and extramural invasion was independently associated with poor disease-free and overall survival for venous (hazard ratios: 2.39, p = 0.001; hazard ratios: 2.46, p = 0.001), lymphatic (hazard ratios: 2.456, p < 0.001; hazard ratios: 2.13, p = 0.02) and perineural invasion (hazard ratios: 2.99, p < 0.001; hazard ratios: 2.68, p < 0.001), respectively. Conclusion: Our data strongly advocates the importance of reporting intramural and extramural components of invasion since the presence of intramural invasion alone may not be considered as a high-risk factor for systemic recurrence.

A Rare Case of Multiple Thrombosis Associated with COVID-19 Pneumonia.

Coronavirus disease 2019 is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 virus. Hypoxic respiratory failure, multiorgan dysfunction, septic shock, thrombosis, and thromboembolic complications have been associated with the severe acute respiratory syndrome coronavirus 2 infection. We report the presentation of the severe acute respiratory syndrome coronavirus 2 infection with acute upper extremity ischemia and mesenteric ischemia clinic. We also report that this patient had an aortic arch mural thrombus as a possible source of thromboembolism, and we emphasize that the aorta should also be carefully evaluated in thromboembolic patients with coronavirus disease 2019.

First detection and molecular characterization of Dientamoeba fragilis in cattle.

Dientamoeba fragilis is a flagellated protozoan with amoeba-like morphology that inhabits the human gastrointestinal tract. It is endemic in a vast geography around the world, including developed countries. There are limited studies on non-human hosts of the parasite, and suitable hosts have not been clarified. The parasite has been detected in non-human primates, pigs, cats, dogs and rats. There is no study in the literature investigating and detecting the presence of this parasite in cattle. In this study, stool samples taken from 163 different cattle and calves from 11 different farms between March 2017 and May 2022 were examined for the detection of D. fragilis via PCR. Trichrome staining was performed on all PCR-positive samples. The isolates with the expected amplicon size were sequenced using the 18S ribosomal RNA region, and their genotypes were determined by BLAST analysis. Sequences were analysed with the most similar and reference sequences in the literature, forming a phylogenetic tree. We detected D. fragilis in 31 (19.01%) of the 163 stool samples. D. fragilis cysts/trophozoites were detected by trichrome staining method in six of 31 samples. All PCR products selected for molecular analysis from positive samples had the same nucleotide sequence. As a result of BLAST analysis, all sequences were determined to belong to D. fragilis genotype 1. This study determined for the first time that cattle are suitable hosts for D. fragilis. Furthermore, the parasite subtype we detected belongs to genotype 1, which is the most common type in humans, suggesting that the parasite may have a zoonotic character. Our result is important in terms of the epidemiology of the parasite, as the mode of transmission is controversial, and available data on its suitable hosts are limited.

Association between RS Time in Electrocardiogram and Right Ventricular Functions in Patients with Chronic Obstructive Pulmonary Disease.

OBJECTIVE: The occurrence of right ventricular (RV) dysfunction in chronic obstructive pulmonary disease (COPD) results in an increased risk of mortality. We aimed to study the diagnostic value of RS time in the recognition of COPD patients with RV dysfunction. METHODS: 120 consecutive COPD patients were divided into two groups, patients with and without RV dysfunction, and compared them in terms of parameters including RS time. RS time was defined as the longest interval from the beginning of the QRS complex to the nadir of the S- or S'-wave in the inferolateral leads on an electrocardiogram. RESULTS: RV dysfunction was observed in 36% of consecutive COPD patients with a mean age of 63.4 ± 9.8 years (83.3% male) and a mean forced expiratory volume in 1 s of 1.51 ± 0.62 lt. The heart rate, right QRS axis deviation frequency, S1S2S3 pattern frequency, and RS time (p < 0.01) were significantly higher in the patients with RV dysfunction than in those without. Body surface area, heart rate, and RS time (p < 0.001) were independent predictors of an RV dysfunction. An ROC analysis showed that the best RS time cutoff value for the prediction of RV dysfunction was 60 ms with a sensitivity of 81.4% and a specificity of 74.0%. CONCLUSION: In patients with COPD, RS time prolongation, which can be easily and quickly determined from the electrocardiogram, may be a marker for RV dysfunction.

Predictors of left ventricular ejection function decline in young patients with ST-segment elevation myocardial infarction.

OBJECTIVE: A decrease in the left ventricular ejection fraction (≤40%) in the setting of ST-segment elevation myocardial infarction is a significant predictor of mortality in the young ST-segment elevation myocardial infarction population. In this study, we aimed to investigate the predictors of left ventricular ejection fraction reduction and evaluate the long-term mortality rates in young ST-segment elevation myocardial infarction patients with or without decreased left ventricular ejection fraction. METHODS: We enrolled retrospectively 411 consecutive ST-segment elevation myocardial infarction patients aged 45 years or below who underwent primary percutaneous coronary intervention. Young ST-segment elevation myocardial infarction patients were divided into two groups according to their left ventricular ejection fraction (≤40%, n=72 and >40%, n=339), which were compared with each other. RESULTS: Statin use, white blood cell count, C-reactive protein, peak creatine kinase-MB, prolonged ischemia time, left anterior descending artery-related infarction, proximally/ostial located lesion, and no-reflow were independently associated with low left ventricular ejection fraction. Additionally, long-term mortality was considerably higher in the left ventricular ejection fraction ≤40% group than those in the left ventricular ejection fraction>40% group (18.1% versus 2.4%; p<0.001). CONCLUSIONS: In young ST-segment elevation myocardial infarction patients, lesion properties (left anterior descending lesion, proximally located lesion), no-reflow, and prolonged ischemia time appeared to be important determinants for the left ventricular ejection fraction decline, rather than coronary disease severity or demographic and hematological parameters. Statin use may be preventive in the development of left ventricular ejection fraction decline in young ST-segment elevation myocardial infarction patients.

Antiprotozoal activity of different Xenorhabdus and Photorhabdus bacterial secondary metabolites and identification of bioactive compounds using the easyPACId approach.

Natural products have been proven to be important starting points for the development of new drugs. Bacteria in the genera Photorhabdus and Xenorhabdus produce antimicrobial compounds as secondary metabolites to compete with other organisms. Our study is the first comprehensive study screening the anti-protozoal activity of supernatants containing secondary metabolites produced by 5 Photorhabdus and 22 Xenorhabdus species against human parasitic protozoa, Acanthamoeba castellanii, Entamoeba histolytica, Trichomonas vaginalis, Leishmania tropica and Trypanosoma cruzi, and the identification of novel bioactive antiprotozoal compounds using the easyPACId approach (easy Promoter Activated Compound Identification) method. Though not in all species, both bacterial genera produce antiprotozoal compounds effective on human pathogenic protozoa. The promoter exchange mutants revealed that antiprotozoal bioactive compounds produced by Xenorhabdus bacteria were fabclavines, xenocoumacins, xenorhabdins and PAX peptides. Among the bacteria assessed, only P. namnaoensis appears to have acquired amoebicidal property which is effective on E. histolytica trophozoites. These discovered antiprotozoal compounds might serve as starting points for the development of alternative and novel pharmaceutical agents against human parasitic protozoa in the future.

Predictors of left ventricular ejection function decline in young patients with ST-segment elevation myocardial infarction

SUMMARY OBJECTIVE: A decrease in the left ventricular ejection fraction (≤40%) in the setting of ST-segment elevation myocardial infarction is a significant predictor of mortality in the young ST-segment elevation myocardial infarction population. In this study, we aimed to investigate the predictors of left ventricular ejection fraction reduction and evaluate the long-term mortality rates in young ST-segment elevation myocardial infarction patients with or without decreased left ventricular ejection fraction. METHODS: We enrolled retrospectively 411 consecutive ST-segment elevation myocardial infarction patients aged 45 years or below who underwent primary percutaneous coronary intervention. Young ST-segment elevation myocardial infarction patients were divided into two groups according to their left ventricular ejection fraction (≤40%, n=72 and >40%, n=339), which were compared with each other. RESULTS: Statin use, white blood cell count, C-reactive protein, peak creatine kinase-MB, prolonged ischemia time, left anterior descending artery-related infarction, proximally/ostial located lesion, and no-reflow were independently associated with low left ventricular ejection fraction. Additionally, long-term mortality was considerably higher in the left ventricular ejection fraction ≤40% group than those in the left ventricular ejection fraction>40% group (18.1% versus 2.4%; p<0.001). CONCLUSIONS: In young ST-segment elevation myocardial infarction patients, lesion properties (left anterior descending lesion, proximally located lesion), no-reflow, and prolonged ischemia time appeared to be important determinants for the left ventricular ejection fraction decline, rather than coronary disease severity or demographic and hematological parameters. Statin use may be preventive in the development of left ventricular ejection fraction decline in young ST-segment elevation myocardial infarction patients.

Computational Analysis of the Inhibition Mechanism of NOTUM by the ONIOM Method.

Notum is a member of serine hydrolyses that cleaves the palmitoleate moiety from Wingless-related integration site (Wnt) ligands. This enzyme plays crucial functions through modulating the Wnt signaling pathway. Inhibition of Notum carries therapeutic effects against a number of maladies including osteoporosis, cancer, and Alzheimer's disease. Recently, a class of irreversible inhibitors based on esters of 4-(indolin-1-yl)-4-oxobutanoic acid have been reported. Using the crystal structures of enzyme-4-(indolin-1-yl)-4-oxobutanoate adduct and 4-(indolin-1-yl)-4-oxobutanoic acid-enzyme complex, we studied computationally the proposed inhibition mechanism using model systems based on the own n-layered integrated molecular orbital and molecular mechanics (ONIOM) method. In the first place, model systems were formulated to investigate the transesterification between the catalytic serine residue, Ser-232, and the methyl ester of 4-(indolin-1-yl)-4-oxobutanoate. In the second place, the hydrolysis mechanism of the resultant enzyme-inhibitor adduct was studied. The energetics of these steps were analyzed using a density functional theory functional in the ONIOM method. In addition, the roles of active-site residues during these steps were highlighted. It was found that the hydrolysis of the covalent adduct is highly endergonic corroborating the irreversible inhibition mechanism. These results will shed light not only on the inhibition mechanism but also on the catalytic mechanism.

Computational Analysis of Histone Deacetylase 10 Mechanism by the ONIOM Method: A Complementary Approach to X-ray and Kinetics Studies.

Histone deacetylase 10 (HDAC 10) catalyzes deacetylation of N8-acetylspermidine into spermidine in the cytosolic region of eukaryotic cells. Inhibition of HDAC 10 has clinical importance in certain types of cancers. Recently, X-ray crystal structures corresponding to the substrate-bound, tetrahedral intermediate-bound, and product-bound enzymes have been resolved using variant forms of humanized HDAC 10. Based on these structures, it was proposed that Y307 residue polarizes the carbonyl of the acetyl group in N8-acetylspermidine together with a zinc atom, which is coordinated by D174, H176, D267, and an H2O molecule. The H2O molecule undergoes nucleophilic addition to the carbonyl carbon of N8-acetylspermidine to form the tetrahedral intermediate. During this process, it is suggested that H136 acts as a general base to deprotonate the H2O molecule. It is further proposed that the protonation of the amide N atom of the tetrahedral intermediate by H137 causes the deacetylation forming the final products, spermidine and acetate ion. In this study, computational models based on the ONIOM method were employed to study the proposed mechanism for the two steps of the deacetylation process based on the crystal structure of the substrate-bound enzyme. The energy profiles of each step as well as the roles of the active site residues were investigated for the catalysis. The calculated activation barrier is in good agreement with the reported kcat value.