Cost-effectiveness analysis of mecapegfilgrastim and recombinant human granulocyte stimulating factor for primary prophylaxis of chemotherapy-induced neutropenia in non-small cell lung cancer.

OBJECTIVE: To evaluate the efficacy, safety, and economics of mecapegfilgrastim and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in the primary prevention of chemotherapy-related neutropenia in non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Data from 181 patients with NSCLC who received intermediate risk chemotherapy were collected from the information system of a tertiary hospital in China. Patients were categorized into two groups: those treated with mecapegfilgrastim (n = 91) and those treated with rhG-CSF (n = 90). The clinical efficacy rates of neutropenia prevention were used as effect indicators, and a cost-effectiveness analysis was conducted from the perspective of the Chinese healthcare system. Logistic regression, generalized linear regression, and bootstrap methods were used for sensitivity analyses. RESULTS: There was no statistical difference between the mecapegfilgrastim and rhG-CSF groups in clinical efficacy rates (98.9 vs. 97.8%). However, the total cost in the mecapegfilgrastim group was significantly higher than that in the rhG-CSF group (16,341.6 CNY vs. 14,371.1 CNY, p = 0.03). The cost-minimization analysis shows that mecapegfilgrastim is not cost-effective. The sensitivity analyses confirm that these results are robust. CONCLUSION: Compared with rhG-CSF, mecapegfilgrastim is not a cost-effective strategy for NSCLC patients in neutropenia prevention in China.

The association between blood eosinophils and clinical outcome of acute exacerbations of chronic obstructive pulmonary disease: A systematic review and meta-analysis.

OBJECTIVES: Studies have shown an association between eosinophilia and clinical outcomes in acute exacerbation of chronic obstructive pulmonary disease (AECOPD). However, contradictory findings exist. Our study aims to systematically evaluate the association between elevated peripheral blood eosinophils and clinical outcome of patients with AECOPD. METHODS: An electronic search was conducted for relevant studies published from database inception to February 28, 2023, on PubMed, EMBASE, Cochrane Library, and Web of Science. The analysis covered studies on the correlation between EOS AECOPD and mortality, hospital stay duration, readmission and hospitalization rates, and invasive mechanical ventilation. Where applicable, relative risk (RR) and weighted mean difference (WMD) were extracted, pooled, and assessed using meta-analysis. Sensitivity analysis was performed to explore the source of heterogeneity. RESULTS: Fifteen high-quality studies including 14 cohort studies and one case-control study were included in the meta-analysis. Compared with non-eosinophilic AECOPD patients, those with eosinophilic AECOPD had a lower risk of mortality (RR = 0.65, 95 % confidence interval [CI] 0.54, 0.77, P < 0.001), shorter length of hospital stay (WMD = -1.56, 95%CI -2.16, -0.96, P < 0.001), and higher readmission rate (RR = 1.07, 95%CI 1.01,1.13, P = 0.029). No difference was found concerning the rate of hospitalization and invasive mechanical ventilation between the two groups. CONCLUSION: Individuals diagnosed with eosinophilic AECOPD had a reduced mortality rate, a truncated period of hospitalization, and an insubstantial increase in the probability of readmission relative to their non-eosinophilic AECOPD counterparts. The level of eosinophils in blood has been shown to serve as a potential predictive biomarker for AECOPD patients.

Analysis of 12 cases of antineoplastic agents-induced interstitial lung disease.

Objective: To summarize the situation of antineoplastic agents-induced interstitial lung diseases (ILD), provide reference for strengthening clinical management of druginduced interstitial lung diseases (DILD). Methods: We retrospectively investigated the medical records of 12 patients with antineoplastic agents-induced ILD in a hospital between January and December 2020. Data collected included patients' characteristic (gender, age, ECOG PS score, smoking history, primary tumor, concurrent diseases or complications.) and treatment conditions (DILD-causing drugs, clinical symptoms, chest CT, DILD treatment drugs, onset cycle, onset time, severity of DILD, DILD course and prognosis.). Results: The median age of 12 DILD cases was 68%, 66.67% of the patients were male, lung cancer accounted for 58.33% (7/12). DILD was induced by cytotoxicity drugs, targeted drugs and immune checkpoint inhibitors (ICIs), of which ICIs accounted for 66.67% (8/12). Scattered patchy, cord-like, grid-like or flocculent shadows were observed on chest CT, mainly under the pleura of lungs. Once DILD occurs, the suspected antineoplastic agents were stopped and glucocorticoid was given, among which 83.33% (10/12) patients were treated with antibiotics. Finally, 16.67% (2/12) were cured, 33.33% (4/12) were improved, 16.67% (2/12) were not cured and 33.33% (4/12) were dead. Conclusion: Antineoplastic agents-induced ILD is mostly found in elderly male lung cancer patients with smoking history. The clinical symptoms of DILD are diverse and lack of specificity. ICIs-ILD has the characteristic of high incidence and poor prognosis compared with other antineoplastic agents. Comprehensive evaluation before medication, regular review, early and adequate glucocorticoid shock therapy after onset can improve the prognosis of DILD patients.

Impact of pharmaceutical care pathway established in hepatobiliary surgery.

OBJECTIVES: Clinical pharmacists play a pivotal role in ensuring medication safety due to their detailed understanding of the medication-use process. This study aimed to propose the concept of pharmaceutical care pathway (PCP) in surgical care and design the work pattern and workflow in the healthcare systems of China. SETTING: Data were collected from patients in the Department of Hepatobiliary Surgery of the First People's Hospital of Lianyungang in China between January 2019 and December 2019. MATERIALS AND METHODS: The study was conducted using 346 patients in the control group and 363 in the intervention group. The control group was managed only by the clinical pathway (CP), while the intervention group was managed by the CP and PCP. MAIN OUTCOME MEASURE: Adverse drug reactions (ADRs), patient satisfaction, hospital expense, drug cost, length of stay, and prescription situations were documented. RESULTS: Using PCP, the rational use of drugs increased from 56% in the control group to 94.2% in the intervention group. Further, 124 (35.8%) ADRs in the control group and 44 (12.1%) ADRs in the intervention group were assessed using the Karch and -Lasagna scale. The mean hospital expense was 21,949.12 ± 2,311.25 yuan in the control group and 17,566.25 ± 1,082.56 yuan in the intervention group. The mean drug cost was 6,250.69 ± 589.35 yuan and 4,894.22 ± 356.14 yuan (1 US$ = 6.37 yuan). The mean length of stay was 12.23 ± 2.51 days and 8.35 ± 1.32 days in the control and intervention groups, respectively. Patient satisfaction increased significantly. CONCLUSION: PCP reduced the length of stay for patients and drug-related adverse events, increased the rational use of drugs, cost-effectiveness, patient satisfaction, and consequently, improved the quality of service in surgery medicine.

A real-world cost-effectiveness analysis of nebulized budesonide and intravenous methylprednisolone in acute exacerbation of chronic obstructive pulmonary disease.

Objective: To assess the cost-effectiveness of nebulized budesonide and intravenous methylprednisolone in the treatment of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) in a real-world setting. Materials and methods: Data from 291 patients with AECOPD were collected from the information system of a tertiary hospital in China. Patients were categorized into two groups: those treated with nebulized budesonide (n = 148) and those treated with intravenous methylprednisolone (n = 143). Clinical efficacy and the rate of no readmission within 1 year after discharge were used as effect indicators, and a cost-effectiveness analysis was conducted from the perspective of the Chinese healthcare system. Logistic regression, generalized linear regression, and bootstrap methods were used for sensitivity analyses. Results: There was no statistical difference between the budesonide and methylprednisolone groups in clinical efficacy rates (94.6% vs. 93.7%). The cost-minimization analysis shows that budesonide is not cost-effective owing to higher total cost. In terms of readmission rates, budesonide was again not cost-effective, with an incremental cost-effectiveness ratio (ICER) of 22276.62 CNY, which is higher than the willingness to pay (WTP) of 20206.20 CNY, the mean per admission expenditure in China. The sensitivity analyses confirm that these results are robust. Conclusion: Compared with intravenous methylprednisolone, nebulized budesonide is not a cost-effective strategy for AECOPD patients in China.

Characterization and identification of the metabolites of dihydromethysticin by ultra-high-performance liquid chromatography orbitrap high-resolution mass spectrometry.

Dihydromethysticin, a natural component from Piper methysticum Forst, has been reported to display pharmacological effects in mental disorders and some malignant tumors. However, the metabolism of this component remained unknown. The goal of this work was conducted to discover the metabolic profiles of dihydromethysticin. The in vitro incubation was performed by incubating dihydromethysticin with rat, monkey, and human liver microsomes and hepatocytes. An analytical assay of ultra-high performance liquid chromatography combined with Orbitrap high-resolution mass spectrometry was utilized to detect and identify the metabolites. With high resolution mass spectrometric determination, the accurate mass, elemental composition, and product ions of the metabolites were determined, which enabled structural characterization to become easy. Under the present conditions, four phase-I metabolites, as well as six phase-II metabolites, were detected and their tentative structures were characterized by mass spectra. M4 was found as the most abundant metabolite both in liver microsomes and hepatocytes. Cytochrome P450 1A2, 2C9, and 3A4 contributed to the formation of this metabolite by using human recombinant P450 enzymes. M4 can be oxidized into reactive ortho-quinone intermediate followed by conjugating with glutathione. M4 was also subject to glucuronidation (M1 and M2) and methylation (M5). Demethylenation, oxidation, hydroxylation, glucuronidation, glutathionylation, and methylation were the primary metabolic pathways of dihydromethysticin. This study provides in vitro metabolism data of dihydromethysticin, which is indispensable for understanding the disposition of this compound.

The Nonclinical Disposition and Pharmacokinetic/Pharmacodynamic Properties of N-Acetylgalactosamine-Conjugated Small Interfering RNA Are Highly Predictable and Build Confidence in Translation to Human.

Conjugation of oligonucleotide therapeutics, including small interfering RNAs (siRNAs) or antisense oligonucleotides, to N-acetylgalactosamine (GalNAc) ligands has become the primary strategy for hepatocyte-targeted delivery, and with the recent approvals of GIVLAARI (givosiran) for the treatment of acute hepatic porphyria, OXLUMO (lumasiran) for the treatment of primary hyperoxaluria, and Leqvio (inclisiran) for the treatment of hypercholesterolemia, the technology has been well validated clinically. Although much knowledge has been gained over decades of development, there is a paucity of published literature on the drug metabolism and pharmacokinetic properties of GalNAc-siRNA. With this in mind, the goals of this minireview are to provide an aggregate analysis of these nonclinical absorption, distribution, metabolism, and excretion (ADME) data to build confidence on the translation of these properties to human. Upon subcutaneous administration, GalNAc-conjugated siRNAs are quickly distributed to the liver, resulting in plasma pharmacokinetic (PK) properties that reflect rapid elimination through asialoglycoprotein receptor-mediated uptake from circulation into hepatocytes. These studies confirm that liver PK, including half-life and, most importantly, siRNA levels in RNA-induced silencing complex in hepatocytes, are better predictors of pharmacodynamics (PD) than plasma PK. Several in vitro and in vivo nonclinical studies were conducted to characterize the ADME properties of GalNAc-conjugated siRNAs. These studies demonstrate that the PK/PD and ADME properties of GalNAc-conjugated siRNAs are highly conserved across species, are largely predictable, and can be accurately scaled to human, allowing us to identify efficacious and safe clinical dosing regimens in the absence of human liver PK profiles. SIGNIFICANCE STATEMENT: Several nonclinical ADME studies have been conducted in order to provide a comprehensive overview of the disposition and elimination of GalNAc-conjugated siRNAs and the pharmacokinetic/pharmacodynamic translation between species. These studies demonstrate that the ADME properties of GalNAc-conjugated siRNAs are well correlated and predictable across species, building confidence in the ability to extrapolate to human.

Impact of Serum Proteins on the Uptake and RNAi Activity of GalNAc-Conjugated siRNAs.

Serum protein interactions are evaluated during the drug development process since they determine the free drug concentration in blood and thereby can influence the drug's pharmacokinetic and pharmacodynamic properties. While the impact of serum proteins on the disposition of small molecules is well understood, it is not yet well characterized for a new modality, RNA interference therapeutics. When administered systemically, small interfering RNAs (siRNAs) conjugated to the N-acetylgalactosamine (GalNAc) ligand bind to proteins present in circulation. However, it is not known if these protein interactions may impact the GalNAc-conjugated siRNA uptake into hepatocytes mediated through the asialoglycoprotein receptor (ASGPR) and thereby influence the activity of GalNAc-conjugated siRNAs. In this study, we assess the impact of serum proteins on the uptake and activity of GalNAc-conjugated siRNAs in primary human hepatocytes. We found that a significant portion of the GalNAc-conjugated siRNAs is bound to serum proteins. However, ASGPR-mediated uptake and activity of GalNAc-conjugated siRNAs were minimally impacted by the presence of serum relative to their uptake and activity in the absence of serum. Therefore, in contrast to small molecules, serum proteins are expected to have minimal impact on pharmacokinetic and pharmacodynamic properties of GalNAc-conjugated siRNAs.

RT-qPCR Methods to Support Pharmacokinetics and Drug Mechanism of Action to Advance Development of RNAi Therapeutics.

The goal of this study was to develop a reverse transcription quantitative polymerase chain reaction (RT-qPCR) method for the accurate quantification of chemically modified small interfering RNA (siRNA) including but not restricted to thermally destabilizing modifications such as glycol nucleic acid (GNA). RT-qPCR was found to be superior to mass spectrometry-based siRNA detection in terms of sensitivity and throughput. However, mass spectrometry is still the preferred method when specific metabolite detection is required and is also insensitive to siRNA chemical modifications such as GNA. The RT-qPCR approach can be optimized to take chemical modifications into account and works robustly in different matrices without optimization, unlike mass spectrometry. RT-qPCR and mass spectrometry both have their strengths and weaknesses for the detection of siRNA and must be used appropriately depending on the questions at hand. Considerations such as desired throughput, assay sensitivity, and metabolite identification must be weighed when choosing which methodology to apply.

Comparative efficacies of nebulized budesonide and systemic corticosteroids in the treatment of exacerbations of chronic obstructive pulmonary disease: A systematic review and meta-analysis.

WHAT IS KNOWN AND OBJECTIVE: Corticosteroids are recommended by almost all international guidelines for the management of exacerbations of chronic obstructive pulmonary disease (COPD). Nevertheless, due to their side effects, there are still concerns regarding the use of systemic corticosteroids (SCs). The Global Initiative for Chronic Obstructive Lung Disease guideline states nebulized budesonide (NB) may be a suitable alternative to SCs for treating COPD exacerbations. We conducted this study to systematically compare the efficacies of NB and SCs by using a meta-analysis. METHODS: PubMed, EMBASE and Cochrane Library databases were searched from database inception to 10 October 2019. Our main end points were change in pulmonary function and blood gas analysis. Secondary end points were numbers of exacerbations and hyperglycaemia. RESULTS AND DISCUSSION: Of 645 identified studies, 6 were eligible and were included in our analysis (N = 867 participants). Compared with SCs, NB was non-inferior on the change in FEV1 %predicted at 24 hours, 48-72 hours and 5-7 days; FEV1 at 5-7 days; FEV1 /FVC at 7 days. For blood gas analysis, our meta-analysis indicated that PaO2 , PaCO2 at 24 hours, 48-72 hours and 7-10 days and SaO2 at 24 hours and 7-10 days showed a non-significant difference in both groups, whereas the SaO2 was significant higher in NB group at 48-72 hours after treatment. Hyperglycaemia was less frequent with NB (odds ratio, 0.1; 95% CI, 0.01-0.85; P = .04). WHAT IS NEW AND CONCLUSION: Based on our meta-analysis, NB was not inferior to SCs when used in the treatment of COPD exacerbations. However, additional well-designed prospective studies are needed to identify the optimal dose of nebulized budesonide and the effects of nebulized budesonide in outpatients, or patients in ICU settings.

Evaluation of electrophoretic mobility shift assay as a method to determine plasma protein binding of siRNA.

Aim: The electrophoretic mobility shift assay (EMSA) was evaluated as an alternative to ultrafiltration (UF) to assess plasma protein binding (PPB) of small interfering RNAs (siRNA) and antisense oligonucleotides (ASO). Results & methodology: EMSA analysis showed that PPB depended on siRNA and plasma concentration. Conversely, when analyzed by ultrafiltration, siRNA bound the filtration device nonspecifically and PPB remained >98% across physiologically relevant siRNA concentrations. Using EMSA, siRNA exhibited charge-based interactions with plasma proteins, while ASO remained highly bound to plasma proteins or albumin in the presence of 500 mM salt. Conclusion: PPB characteristics of siRNA and ASO can be distinguished using EMSA. Characterization of siRNA PPB by EMSA enhances our knowledge of siRNA absorption, distribution, metabolism and excretion and advanced development of RNA interference therapeutics.

In Vitro Drug-Drug Interaction Evaluation of GalNAc Conjugated siRNAs Against CYP450 Enzymes and Transporters.

Small interfering RNAs (siRNAs) represent a new class of medicines that are smaller (∼16,000 Da) than biologic therapeutics (>150,000 Da) but much larger than small molecules (<900 Da). Current regulatory guidance on drug-drug interactions (DDIs) from the European Medicines Agency, Food and Drug Administration, and Pharmaceutical and Medical Devices Agency provides no recommendations for oligonucleotide therapeutics including siRNAs; therefore, small molecule guidance documents have historically been applied. Over ∼10 years, in vitro DDI investigations with siRNAs conjugated to a triantennary N-acetylgalactosamine [(GalNAc)-siRNA] ligand have been conducted during nonclinical drug development to elucidate the potential clinical DDI liability. GalNAc siRNAs were evaluated as substrates, inhibitors, or inducers of major cytochrome P450s (P450s) and as substrates and inhibitors of transporters. Aggregate analysis of these data demonstrates a low potential for DDI against P450s. Zero of five, 10, and seven are inducers, time-dependent inhibitors, or substrates, respectively, and nine of 12 do not inhibit any P450 isoform evaluated. Three GalNAc siRNAs inhibited CYP2C8 at supratherapeutic concentrations, and one mildly inhibited CYP2B6. The lowest K i value of 28 µM is >3000-fold above the therapeutic clinical C max at steady state, and importantly no clinical inhibition was projected. Of four GalNAc siRNAs tested none were substrates for transporters and one caused inhibition of P-glycoprotein, calculated not to be clinically relevant. The pharmacological basis for DDIs, including consideration of the target and/or off-target profiles for GalNAc siRNAs, should be made as part of the overall DDI risk assessment. If modulation of the target protein does not interfere with P450s or transporters, then in vitro or clinical investigations into the DDI potential of the GalNAc siRNAs are not warranted. SIGNIFICANCE STATEMENT: Recommendations for evaluating DDI potential of small molecule drugs are well established; however, guidance for novel modalities, particularly oligonucleotide-based therapeutics are lacking. Given the paucity of published data in this field, in vitro DDI investigations are often conducted. The aggregate analysis of GalNAc-siRNA data reviewed herein demonstrates that, like new biological entities, these oligonucleotide-based therapeutic drugs are unlikely to result in DDIs; therefore, it is recommended that the need for in vitro or clinical investigations similarly be determined on a case-by-case basis. Given the mechanism of siRNA action, special consideration should be made in cases where there may be a pharmacological basis for DDIs.

Part 3: Notch-sparing γ-secretase inhibitors: SAR studies of 2-substituted aminopyridopyrimidinones.

In search for novel lead compounds as γ-secretase inhibitors, analogs of aminopyrido[2,3-d]pyrimidin-7-ones (I) were synthesized and evaluated for inhibitory effects on amyloid-ß-peptide production and cleavage of the Notch1 receptor mediated by γ-secretase. Selected pyridopyrimidines, such as 1, 8, 9, 10, 11 and 16 are γ-secretase inhibitors that did not have an effect on Notch1 receptor processing.

Part 1: Notch-sparing γ-secretase inhibitors: The identification of novel naphthyl and benzofuranyl amide analogs.

γ-Secretase is one of two proteases directly involved in the production of the amyloid ß-peptide (Aß), which is pathogenic in Alzheimer's disease. Inhibition of γ-secretase to suppress the production of Aß should not block processing of one of its alternative substrates, Notch1 receptors, as interference with Notch1 signaling leads to severe toxic effects. In the course of our studies to identify γ-secretase inhibitors with selectivity for APP over Notch, 1 [3-(benzyl(isopropyl)amino)-1-(naphthalen-2-yl)propan-1-one] was found to inhibit γ-secretase-mediated Aß production without interfering with γ-secretase-mediated Notch processing in purified enzyme assays. As 1 is chemically unstable, efforts to increase the stability of this compound led to the identification of 2 [naphthalene-2-carboxylic acid benzyl-isopropyl-amide] which showed similar biological activity to compound 1. Synthesis and evaluation of a series of amide analogs resulted in benzofuranyl amide analogs that showed promising Notch-sparing γ-secretase inhibitory effects. This class of compounds may serve as a novel lead series for further study in the development of γ-secretase inhibitors.

Part 2. Notch-sparing γ-secretase inhibitors: The study of novel γ-amino naphthyl alcohols.

One therapeutic approach for Alzheimer's disease is to inhibit the cleavage of the amyloid precursor protein (APP) by γ-secretase. At the beginning of a series of studies from our laboratories, a series of novel γ-amino alcohols (1) were found to possess γ-secretase inhibitory activity and Notch-sparing effects. A new one-pot synthesis of γ-amino alcohols and the structure-activity relationship (SAR) of these analogs will be discussed.

Nicastrin functions to sterically hinder γ-secretase-substrate interactions driven by substrate transmembrane domain.

γ-Secretase is an intramembrane-cleaving protease that processes many type-I integral membrane proteins within the lipid bilayer, an event preceded by shedding of most of the substrate's ectodomain by α- or ß-secretases. The mechanism by which γ-secretase selectively recognizes and recruits ectodomain-shed substrates for catalysis remains unclear. In contrast to previous reports that substrate is actively recruited for catalysis when its remaining short ectodomain interacts with the nicastrin component of γ-secretase, we find that substrate ectodomain is entirely dispensable for cleavage. Instead, γ-secretase-substrate binding is driven by an apparent tight-binding interaction derived from substrate transmembrane domain, a mechanism in stark contrast to rhomboid--another family of intramembrane-cleaving proteases. Disruption of the nicastrin fold allows for more efficient cleavage of substrates retaining longer ectodomains, indicating that nicastrin actively excludes larger substrates through steric hindrance, thus serving as a molecular gatekeeper for substrate binding and catalysis.

Quantification of gamma-secretase modulation differentiates inhibitor compound selectivity between two substrates Notch and amyloid precursor protein.

BACKGROUND: Deposition of amyloid-ß protein (Aß) is a major pathological hallmark of Alzheimer's disease (AD). Aß is generated from γ-secretase cleavage of amyloid precursor protein (APP). In addition to APP, γ-secretase also cleaves other type I integral membrane proteins, including the Notch receptor, a key molecule involved in embryonic development. RESULTS: To explore selective γ-secretase inhibitors, a combination of five methods was used to systematically determine these inhibitors' profiles on the γ-secretase cleavage of APP and Notch. When two potent γ-secretase inhibitors, compound E (cpd E) and DAPT, were used in a conventional in vitro γ-secretase activity assay, cpd E completely blocked Aß generation from the cleavage of substrate APP C100, but only had a minor effect on Notch cleavage and NICD generation. Next, cpd E and DAPT were applied to HEK293 cells expressing a truncated Notch substrate NotchDeltaE. Both cpd E and DAPT were more potent in blocking Aß generation than NICD generation. Third, a reporter construct was created that carried the NICD targeting promoter with three Su(H) binding sequences followed by the luciferase gene. We found that the inhibition of NICD generation by cpd E and DAPT was consistent with the reduced expression of luciferase gene driven by this Notch targeting promoter. Fourth, levels of "Notch-Aß-like" (Nß*) peptide derived from two previously reported chimeric APP with its transmembrane domain or the juxtamembrane portion replaced by the Notch sequence were quantified. Measurement of Nß* peptides by ELISA confirmed that EC50's of cpd E were much higher for Nß* than Aß. Finally, the expression levels of Notch target gene her6 in cpd E or DAPT-treated zebrafish were correlated with the degree of tail curvature due to defective somitogenesis, a well characterized Notch phenotype in zebrafish. CONCLUSION: Our ELISA-based quantification of Aß and Nß* in combination with the test in zebrafish provides a novel approach for efficient cell-based screening and in vivo validation of APP selective γ-secretase inhibitors.

Properties of the type B histone acetyltransferase Hat1: H4 tail interaction, site preference, and involvement in DNA repair.

The Hat1 histone acetyltransferase catalyzes the acetylation of H4 at lysines 5 and 12, the same sites that are acetylated in newly synthesized histone H4. By performing histone acetyltransferase (HAT) assays on various synthetic H4 N-terminal peptides, we have examined the interactions between Hat1 and the H4 tail domain. It was found that acetylation requires the presence of positively charged amino acids at positions 8 and 16 of H4, positions that are normally occupied by lysine; however, lysine per se is not essential and can be replaced by arginine. In contrast, replacing Lys-8 and -16 of H4 with glutamines reduces acetylation to background levels. Similarly, phosphorylation of Ser-1 of the H4 tail depresses acetylation by both yeast Hat1p and the human HAT-B complex. These results strongly support the model proposed by Ramakrishnan and colleagues for the interaction between Hat1 and the H4 tail (Dutnall, R. N., Tafrov, S. T., Sternglanz, R., and Ramakrishnan, V. (1998) Cell 94, 427-438) and may have implications for the genetic analysis of histone acetylation. It was also found that Lys-12 of H4 is preferentially acetylated by human HAT-B, in further agreement with the proposed model of H4 tail binding. Finally, we have demonstrated that deletion of the hat1 gene from the fission yeast Schizosaccharomyces pombe causes increased sensitivity to the DNA-damaging agent methyl methanesulfonate in the absence of any additional mutations. This is in contrast to results obtained with a Saccharomyces cerevisiae hat1Delta strain, which must also carry mutations of the acetylatable lysines of H3 for heightened methyl methanesulfonate sensitivity to be observed. Thus, although the role of Hat1 in DNA damage repair is evolutionarily conserved, the ability of H3 acetylation to compensate for Hat1 deletion appears to be more variable.

Modifications of H3 and H4 during chromatin replication, nucleosome assembly, and histone exchange.

Histone posttranslational modifications that accompany DNA replication, nucleosome assembly, and H2A/H2B exchange were examined in human tissue culture cells. Through microsequencing analysis and chromatin immunoprecipitation, it was found that a subset of newly synthesized H3.2/H3.3 is modified by acetylation and methylation at sites that correlate with transcriptional competence. Immunoprecipitation experiments suggest that cytosolic predeposition complexes purified from cells expressing FLAG-H4 contain H3/H4 dimers, not tetramers. Studies of the deposition of newly synthesized H2A/H2B onto replicating and nonreplicating chromatin demonstrated that H2A/H2B exchange takes place in chromatin regions that contain acetylated H4; however, there is no single pattern of H4 acetylation that accompanies exchange. H2A/H2B exchange is also largely independent of the deposition of replacement histone variant, H3.3. Finally, immunoprecipitation of nucleosomes replicated in the absence of de novo nucleosome assembly showed that histone modifications do not prevent the transfer of parental histones to newly replicated DNA and thus have the potential to serve as means of epigenetic inheritance. Our experiments provide an in-depth analysis of the "histone code" associated with chromatin replication and dynamic histone exchange in human cells.