First in-human clinical performance of a new non-cavitating handheld lensectomy system in 665 consecutive cataract surgeries.

PURPOSE: To investigate the intraoperative performance and lens fragmentation efficacy of a non-cavitating handheld lensectomy system in mild, moderate, and severe cataract. SETTING: Ambulatory surgical centers. DESIGN: Retrospective consecutive case series. METHODS: 665 consecutive eyes underwent cataract surgery by 12 surgeons using a new handheld non-cavitating lensectomy system for nuclear fragmentations and extraction. Intraoperative measurements included surgical time, miLOOP pretreatment, and irrigation fluid use. RESULTS: Of the 665 eyes, 38 (6%), 468 (70%), 126 (19%), and 33 (5%) were of grade 1, 2, 3, and 4 nuclear densities, respectively, as graded by the surgeon intraoperatively. Successful nuclear fragmentation, lens extraction, and cortical removal were achieved in all eyes. Total nucleus fragmentation and extraction times were 70.1 seconds, 100.3 seconds, 132.6 seconds, and 287.9 seconds for grades 1, 2, 3, and 4, respectively ( P < .001). In addition, irrigation and aspiration cortical removal times were 64.1 seconds, 51.1 seconds, 48.5 seconds, and 59.0 seconds, respectively ( P = .14). There was a low rate of capsular tear (3 cases in 665 surgeries, 0.45%) and no other emergent adverse events. CONCLUSIONS: The miCOR handheld non-cavitating lensectomy system demonstrated nuclear fragmentation and extraction in the absence of intraocular cavitation across all grades of nuclear densities.

Repositioning Rates of Toric IOLs Implanted in Cataract Surgery Patients: A Retrospective Chart Review.

Purpose: To determine the incidence of postoperative repositioning of toric intraocular lenses (IOLs) due to clinically significant rotation. Patients and Methods: This study included consecutive cataract patients with pre-existing astigmatism who had undergone cataract surgery with toric IOL implantation by a single experienced surgeon. Case records of patients who were recommended to undergo toric IOL repositioning surgery due to clinically significant postoperative IOL rotation from the implanted axis were identified. The need for a secondary intervention to manage residual astigmatism was based upon postoperative residual astigmatic error ≥0.75 D, the patient's qualitative dissatisfaction with the level of postoperative distance vision, dilated post-op examination, and confirmation of the significant potential for astigmatism reduction. Results: Case records of 993 eyes implanted with AcrySof toric (N = 362), Tecnis Toric I (N = 53), Tecnis Toric II (N = 308), or enVista Toric IOLs (N = 270) were included. Postoperative toric IOL repositioning was recommended in 16 eyes (1.6%). The repositioning rate was highest in the eyes implanted with Tecnis Toric I (5.7%), followed by AcrySof Toric (2.2%), enVista Toric IOLs (1.1%), and Tecnis Toric II (0.6%). Conclusion: This real-world analysis of eyes implanted with toric IOLs revealed that the rate of surgical IOL repositioning due to clinically significant IOL rotation was lower than 2% for enVista and Tecnis Toric II IOLs. When needed and with appropriate planning, toric IOL repositioning can be very successful.

Comparative Study of Safety Outcomes Following Nucleus Disassembly with and without the miLOOP Lens Fragmentation Device During Cataract Surgery.

Purpose: To determine the effect of a microinterventional lens prefragmentation wire loop device (miLOOP®; Carl Zeiss Meditec AG, Oberkochen, Germany), on adverse events (AEs), cumulative dispersed energy (CDE), and vision outcomes when used before phacoemulsification of high-grade mature cataracts. Setting: Three ambulatory surgical centers in the Peoria, IL region. Design: Retrospective comparative consecutive case series; single-surgeon. Methods: Patient outcomes were compared before and after introduction of miLOOP-assisted lens fragmentation prior to phacoemulsification during cataract surgeries performed 2016‒2020. The primary outcome was intraoperative AE rate/type. Secondary outcomes included ultrasound cumulative dispersed energy (CDE) administered during phacoemulsification, postoperative AEs, and best-corrected visual acuity (BCVA). Results: Data from 765 subjects (mean age 72.9 years; 1025 eyes) comprised 524 conventional lens disassembly (Control) eyes and 501 Device eyes. One hundred percent of the cataracts in both groups were advanced WHO Grade 3+ nuclei. Significantly fewer intraoperative AEs occurred in the Device group versus Controls (2.2% and 6.3% of eyes, respectively; p=0.0011). Postoperative AE rates were comparable between groups (Controls=2.9%, Device=3.5%). Mean CDE from ultrasound was significantly reduced by 21% when the microfilament loop device was used for nuclear disassembly (9.6±5.2 CDE units) versus Controls (11.6±6.4 CDE units; p<0.0001). Median postoperative BCVA was 20/25 Snellen (0.091 logMAR) in both groups. More than 70% of both Control and Device eyes had postoperative BCVA better than 20/30 Snellen. Conclusion: Microinterventional lens fragmentation was associated with lower ultrasound energy use and improved intraoperative safety than traditional unassisted surgery of advanced high-grade cataracts, while maintaining similarly acceptable postoperative complication rates and BCVA functional outcomes.

Potential Financial Effects on Hospitals of the Removal of Common Orthopaedic and Spinal Procedures From Medicare's "Inpatient-Only" List: A Comparison of the Medicare Fee-for-service Payment Model Versus Maryland's Global Budget Revenue Model.

BACKGROUND: When treating Medicare beneficiaries, orthopaedic surgeons must follow Centers for Medicare & Medicaid Services (CMS) policies regarding whether to perform surgical treatments under inpatient or outpatient status. Recently, most orthopaedic and spinal procedures were removed from the CMS's "inpatient-only" list (IPOL). We investigated differences in hospital payments under the Diagnosis Related Group (DRG)/Ambulatory Payment Classification (APC) system when common orthopaedic/spinal procedures are done under outpatient rather than inpatient status. We compared these differences under the DRG/APC model with differences in payments to Maryland hospitals, which are paid under the alternative Global Budget Revenue model. METHODS: We used the CMS Inpatient Pricer and CMS Addendum B to retrieve the mean duration-of-stay data, estimated DRG (inpatient) payment, and APC (outpatient) payment for eight common orthopaedic/spinal procedures for four non-Maryland hospitals (2 urban academic hospitals and 2 neighboring community hospitals). We retrieved Maryland's Health Services Cost Review Commission hospital rates for the same eight procedures done under inpatient or outpatient status to estimate hospital charges for a Maryland urban academic hospital and a neighboring community hospital. RESULTS: Among the four non-Maryland hospitals, estimated differences in payment for hospitalizations under inpatient versus outpatient status for common orthopaedic/spinal procedures with a mean duration of stay of <2 days, whose status would be most subject to change from inpatient to outpatient by its removal from the IPOL, ranged from $19 to $13,042. For the two Maryland hospitals, differences in outpatient versus inpatient payment for these same procedures ranged from $182 to $1,273. DISCUSSION: Non-Maryland hospitals receive widely different CMS payments for common orthopaedic/spinal procedures based on a change in hospitalization status (inpatient to outpatient) prompted by the procedure being removed from the IPOL. The Maryland global budget revenue mitigates most of the effect of hospitalization status on hospital payment and may serve as a guide toward DRG/APC payment reassessment. LEVEL OF EVIDENCE: N/A.

Examining the effects of the histone methyltransferase inhibitor BIX-01294 on histone modifications and gene expression in both a clinical population and mouse models.

Schizophrenia has been consistently characterized by abnormal patterns of gene down-regulation, increased restrictive chromatin assemblies, and reduced transcriptional activity. Histone methyltransferase (HMT) mRNA and H3K9me2 levels are elevated in postmortem brain and peripheral blood cells of persons with schizophrenia. Moreover, this epigenomic state likely contributes to the disease, as HMT levels correlate with clinical symptomatology. This manuscript sought to establish the potential therapeutic value of the HMT inhibitor BIX-01294 (BIX). Human peripheral mononuclear cells (PBMC) from 24 individuals with schizophrenia and 24 healthy individuals were cultured in the presence of BIX (5uM or 10uM). Mice were given once daily intraperitoneal injections of BIX (0.5 or 1mg/kg) for one week. Cultured cells, mouse cortex, or striatum was harvested, RNA extracted and RT-PCR conducted for several schizophrenia candidate genes: IL-6, Gad1, Nanog, KLF4, Reln, and Bdnf9a. Total H3K9me2 levels were measured using western blot while H3K9me2 binding to selected genes of interest was conducted using chromatin immunoprecipitation (ChIP). Neuronal subtype-specific BDNF conditional knockdown was conducted using the cre/lox system of mutant animals. Treatment with BIX decreased H3K9me2 and increased selected mRNA levels in cultured PBMCs from both normal controls and participants with schizophrenia. In mice, peripheral administration of BIX decreased cortical H3K9me2 levels and increased schizophrenia candidate gene expression. In BDNF conditional knockdown animals, BIX administration was able to significantly rescue Bdnf9a mRNA levels in ChAT and D1 Bdnf conditional knockdown mice. The results presented in this manuscript demonstrate a potential for further research into the clinical effectiveness of histone modifying pharmacology in the treatment of schizophrenia.

Obesity Among High School Students in the United States: Risk Factors and Their Population Attributable Fraction.

INTRODUCTION: The prevalence of obesity among children and adolescents in the United States is high. The aim of this study was to assess the association between modifiable risk factors and obesity and to estimate the population attributable fractions (PAFs) of modifiable risk factors among high school students in the United States. METHODS: For this retrospective study, we used a nationally representative sample of 15,624 students who participated in the 2015 Youth Risk Behavior Survey (YRBS). Obesity was defined as body mass index at or above the 95th percentile, based on sex- and age-specific data from the Centers for Disease Control and Prevention. We examined unhealthy dietary behaviors, physical inactivity, and other modifiable risk factors (tobacco use, alcohol consumption, and sleep). We used multivariable logistic regression, accounting for the complex survey design of YRBS, to assess the association between risk factors and obesity and to calculate PAFs. Confidence intervals of PAFs were estimated by using the jackknife repeated replication method. RESULTS: Among all students included in the study, 13.9% were classified as obese. Not being on a sports team (odds ratio [OR], 1.61; 95% confidence interval [CI], 1.31-1.98), current tobacco use (OR, 1.42; 95% CI, 1.14-1.77), and watching television for 3 hours or more per day (OR, 1.38; 95% CI, 1.09-1.76) were significantly correlated with obesity. The combined PAF for all modifiable risk factors was 34.80% (95% CI, 32.09%-37.51%). The single modifiable risk factor with the largest PAF was not participating on a sports team (PAF, 16.57%; 95% CI, 15.30%-17.84%). CONCLUSION: Findings about PAFs help demonstrate the importance of promoting physical activity, healthy diet, and other healthy lifestyles in reducing obesity among high school students in the United States.

Evidence of a sex-dependent restrictive epigenome in schizophrenia.

When compared to women, men have a higher incidence of schizophrenia, with increases in negative and cognitive symptoms, and an overall poorer disease course. Schizophrenia is conceptualized as a disorder of aberrant gene transcription and regulation. Thus, epigenetics, the study of environmentally induced changes in gene regulation, could advance our understanding of the molecular underpinnings of schizophrenia. Peripheral histone methyltransferase (HMT) mRNA levels have been previously shown to be significantly increased in patients with schizophrenia and correlate with symptomology. In this independent study, peripheral lymphocytes were extracted and clinical symptoms were measured on 74 participants, (40 patients with schizophrenia (19 women, 21 men) and 34 healthy individuals (19 women, 15 men)). HMT (G9α, SETDB1 and GLP) mRNA levels and their resulting histone modification H3K9me2 were measured with RT-PCR and ELISA respectively. Plasma estradiol levels were also measured via ELISA and correlated with HMT mRNA. Clinical symptoms were measured utilizing the Positive and Negative Syndrome Scale (PANSS) and the Heinrichs Carpenter Quality of Life Scale (QLS). The results indicate that men with schizophrenia expressed the highest levels of G9α, SETDB1 mRNA and H3K9me2 protein levels. Additionally, higher levels of symptom presentation and an overall poorer quality of life were correlated with higher HMT mRNA and H3K9me2 protein levels in a sex-dependent pattern. These data support the hypothesis of a sex-dependent restrictive epigenome contributing towards the etiology of schizophrenia. The histone methyltransferases measured here could be potential future therapeutic targets for small molecule pharmacology.

Physical function and quality of life in frail and/or elderly patients with metastatic colorectal cancer treated with capecitabine and bevacizumab: an exploratory analysis.

OBJECTIVES: Optimal treatment strategies in frail and/or elderly patients with metastatic colorectal cancer have not been well defined. Using data from a prospective, phase II study of elderly patients with metastatic colorectal cancer treated with bevacizumab and capecitabine, we explored the differences in functional measure and quality of life (QoL) between patients with ECOG performance status (PS) 1 and 2. MATERIALS AND METHODS: Geriatric functional measures included patient reported limitations in ADLs and IADLs, ECOG PS, 3-item recall, hearing acuity, and the "Get up and Go" test. QoL was assessed by means of the FACT-C questionnaire and the EQ-5D questionnaire. The prognostic impact of baseline characteristics on survival was studied using univariate Cox regression analysis. RESULTS: The majority (62%) of the 45 patients had an ECOG PS of 2. The ECOG PS 2 group had more limitations in IADLs, lower baseline QoL, and a lower patient-rated health score. For all participants, QoL significantly improved from baseline to the start of cycle 2 (FACT-C: 99.9 vs. 105.4, p=0.01) and did not deteriorate when baseline scores were compared to when participants went off study (FACT-C: 99.9 vs. 98.6, p=0.59). In the Cox-regression analysis, a positive "Get up and Go" test was prognostic for improved survival (HR=0.31, p=0.01). CONCLUSION: There is significant heterogeneity in functional measures and quality of life among elderly patients with metastatic colorectal cancer with ECOG PS 1 and 2. The "Get up and Go" test may be a useful prognostic indicator for survival in this population.

Quantification of strain and charge co-mediated magnetoelectric coupling on ultra-thin Permalloy/PMN-PT interface.

Strain and charge co-mediated magnetoelectric coupling are expected in ultra-thin ferromagnetic/ferroelectric multiferroic heterostructures, which could lead to significantly enhanced magnetoelectric coupling. It is however challenging to observe the combined strain charge mediated magnetoelectric coupling, and difficult in quantitatively distinguish these two magnetoelectric coupling mechanisms. We demonstrated in this work, the quantification of the coexistence of strain and surface charge mediated magnetoelectric coupling on ultra-thin Ni0.79Fe0.21/PMN-PT interface by using a Ni0.79Fe0.21/Cu/PMN-PT heterostructure with only strain-mediated magnetoelectric coupling as a control. The NiFe/PMN-PT heterostructure exhibited a high voltage induced effective magnetic field change of 375 Oe enhanced by the surface charge at the PMN-PT interface. Without the enhancement of the charge-mediated magnetoelectric effect by inserting a Cu layer at the PMN-PT interface, the electric field modification of effective magnetic field was 202 Oe. By distinguishing the magnetoelectric coupling mechanisms, a pure surface charge modification of magnetism shows a strong correlation to polarization of PMN-PT. A non-volatile effective magnetic field change of 104 Oe was observed at zero electric field originates from the different remnant polarization state of PMN-PT. The strain and charge co-mediated magnetoelectric coupling in ultra-thin magnetic/ferroelectric heterostructures could lead to power efficient and non-volatile magnetoelectric devices with enhanced magnetoelectric coupling.

Repeat oral dose toxicity studies of melamine in rats and monkeys.

Melamine is an important and widely used organic industrial chemical. Recently, clinical findings of renal failure and kidney stones in infants have been associated with ingestion of melamine-contaminated infant formula. To understand the toxicity and clinical outcome of melamine exposure, repeated oral dose studies in rats and monkeys were performed to characterize the subchronic toxicity of melamine. Assessment of toxicity was based on mortality, clinical signs, body weights, ophthalmic findings, clinical pathology, gross pathology, organ weights, and microscopic observations. The first rat study was intended to be a 14-day oral study followed by an 8-day recovery period. The dose levels were 140, 700, and 1,400 mg/kg/day (lowered to 1,000 mg/kg/day subsequently due to mortality). Oral administration of melamine at 700 mg/kg/day for 14 consecutive days in rats produced compound-related clinical signs (red urine), decreased body weights, and changes in clinical pathology (increased serum urea nitrogen and creatinine) and anatomical pathology (renal tubular cell debris, crystal deposition, and hyperactive regeneration of renal tubular epithelium). The kidney was identified as the target organ. Oral administration at 1,400 mg/kg/day (subsequently lowered to 1,000 mg/kg/day) resulted in animal death and moribundity. There were no treatment-related findings in the 140 mg/kg/day group. There were no compound-related findings in the high-dose recovery animals. The second rat study was a 5-day oral toxicity study with genomic biomarkers assayed in the kidney tissues. At the top dose of 1,050 mg/kg/day, similar clinical and anatomical pathology findings as described above were observed. The genes measured, Kim-1, Clu, Spp1, A2m, Lcn2, Tcfrsf12a, Gpnmb, and CD44, were significantly up-regulated (fivefold to 550-fold), while Tff3 was significantly down-regulated (fivefold). These results indicated that genomic markers could sensitively diagnose melamine-induced kidney injury. A 3-month oral study with 4-week recovery in monkeys was also conducted. In this monkey study, the animals were treated with melamine at doses of 60, 200, or 700 mg/kg/day. The administration of 700 mg/kg/day melamine by nasal-gastric gavage to monkeys resulted in test article-related clinical signs including turbid and whitish urine, urine crystals, red blood cell changes, increased serum alanine aminotransferase and kidney and/or liver weights, and microscopic findings including nephrotoxicity, pericarditis, and increased hematopoiesis. Nephrotoxicity was also noted at 200 mg/kg/day. It was concluded that the kidney is the primary target organ and the NOAEL was estimated to be 140 mg/kg/day in rats following a 14-day oral administration and 60 mg/kg/day in the monkey study.

A phase II trial of frontline capecitabine and bevacizumab in poor performance status and/or elderly patients with metastatic colorectal cancer.

OBJECTIVES: This study aims to determine the efficacy and tolerability of capecitabine (CAP) plus bevacizumab (BEV) as treatment for frontline metastatic colorectal cancer (mCRC) in frail and/or elderly patients. MATERIALS AND METHODS: This was an open label, multi-site, single arm, phase II study in frontline mCRC. In this study, patients (pts) who were frail (ECOG 2) or older patients with ECOG 1 performance status (PS) received CAP (1000 mg/m(2) bid, 14 days of every 21 days) plus BEV (7.5mg/kg iv once every 21 days). The primary objective was progression free survival (PFS). Secondary objectives were overall response rate (ORR) and toxicity. RESULTS: In terms of patients: 50 were enrolled; 5 withdrew consent prior to treatment; 45 were treated, and 41 were evaluable. The mean age was 75.9 (range 54-93) and 62% had an ECOG 2 PS. The median PFS was 6.87 months (95% CI, 5.1-11.5 months) and median overall survival was 12.7 months (95% CI, 6.9-12.7 months). The most common grades 3-4 toxicities were: diarrhea (17.8%), fatigue (13.3%), hand-foot syndrome (13.3%), dehydration (8.9%), hypertension (6.7%) and vomiting (6.7%). CONCLUSIONS: The results of this trial support the use of CAP plus BEV as first-line treatment for frail/elderly patients with metastatic CRC. The ORR (40%) is comparable to pooled data in elderly on fluorouracil (5-FU)+BEV. The median PFS (7.2 months) in this study is slightly lower than that seen with 5-FU+BEV but this study had a high percentage of ECOG PS 2 patients. Side effects were manageable with no new safety signals.

Light increases the gap junctional coupling of retinal ganglion cells.

We examined the effect of light adaptation on the gap junctional coupling of α-ganglion cells (α-GCs) in rabbit and mouse retinas. We assayed changes in coupling by measuring parameters of tracer coupling following injection of α-GCs with Neurobiotin and the concerted spike activity of α-GC neighbours under dark- and light-adapted conditions. We found that light adaptation using mesopic or photopic background lights resulted in a dramatic increase in the labelling intensity, number, and spatial extent of ganglion and amacrine cells coupled to OFF α-GCs when compared to levels seen under dark adaptation. While this augmentation of coupling by light did not produce an increase in the concerted spontaneous activity of OFF α-GC neighbours, it did significantly increase correlated light-evoked spiking. This was seen as an increase in the number of correlated spikes for α-GC neighbours and an extension of correlations to second-tier neighbours that was not seen under dark-adapted conditions. Pharmacological studies in the rabbit retina indicated that dopamine mediates the observed changes in coupling by differentially activating D1 and D2 receptors under different adaptation states. In this scheme, activation of dopamine D1 receptors following light exposure triggers cAMP-mediated intracellular pathways resulting in an increase in gap junctional conductance. Overall, our results indicate that as we move from night to day there is an enhanced electrical coupling between α-GCs, thereby increasing the concerted activity believed to strengthen the capacity and efficiency of information flow across the optic nerve.

Prevalence of ceftriaxone-induced red blood cell antibodies in pediatric patients with sickle cell disease and human immunodeficiency virus infection.

Ceftriaxone can be associated with catastrophic immune hemolysis in pediatric patients, particularly those with underlying diseases such as sickle cell disease and human immunodeficiency virus infection. We designed a study to screen for ceftriaxone-induced RBC antibodies in these 2 pediatric populations. The prevalence of anticeftriaxone antibody was 12.5% (8 of 64). Two of these 8 patients with the antibody experienced hemolysis; 1 case was fatal.

Light-induced changes in spike synchronization between coupled ON direction selective ganglion cells in the mammalian retina.

Although electrical coupling via gap junctions is prevalent among ganglion cells in the vertebrate retina, there have been few direct studies of their influence on the light-evoked signaling of these cells. Here, we describe the pattern and function of coupling between the ON direction selective (DS) ganglion cells, a unique subtype whose signals are transmitted to the accessory optic system (AOS) where they initiate the optokinetic response. ON DS cells are coupled indirectly via gap junctions made with a subtype of polyaxonal amacrine cell. This coupling underlies synchronization of the spontaneous and light-evoked spike activity of neighboring ON DS cells. However, we find that ON DS cell pairs show robust synchrony for all directions of stimulus movement, except for the null direction. Null stimulus movement evokes a GABAergic inhibition that temporally shifts firing of ON DS cell neighbors, resulting in a desynchronization of spike activity. Thus, detection of null stimulus movement appears key to the direction selectivity of ON DS cells, evoking both an attenuation of spike frequency and a desynchronization of neighbors. We posit that active desynchronization reduces summation of synaptic potentials at target AOS cells and thus provides a secondary mechanism by which ON DS cell ensembles can signal direction of stimulus motion to the brain.

Gap junctional coupling underlies the short-latency spike synchrony of retinal alpha ganglion cells.

We examined whether coupling between neighboringalpha-type ganglion cells (alpha-GCs) in the rabbit retina underlies their synchronous spike activity. Simultaneous recordings were made from arrays of alpha-GCs to determine the synchrony of both spontaneous and light-evoked spike activity. One cell within each array was then injected with the biotinylated tracer Neurobiotin to determine which of the cells were coupled via gap junctions. Cross-correlation analyses indicated that neighboring off-center alpha-GCs maintain short-latency (approximately 2.5 msec) synchronous spiking, whereas the spontaneous spike activities of on-centeralpha-GC neighbors are not correlated. Without exception, those off-centeralpha-GCs showing synchronous spiking were found to be tracer coupled to both amacrine cells and neighboring off-centeralpha-GCs. In contrast, on-center alpha-GCs were never tracer coupled. Furthermore, whereas spikes initiated in an off-center alpha-GC with extrinsic current injection resulted in short-latency synchronized spiking in neighboring off-center alpha-GCs, this was never seen between on-center alpha-GCs. These results indicate that electrical coupling via gap junctions underlies the short-latency concerted spike activity of neighboring alpha-GCs.

Time and sequence dependence of hydroxyurea in combination with gemcitabine in human KB cells.

Gemcitabine (Gem) is a deoxycytidine analogue whose active metabolite, dFdCTP, blocks DNA elongation and has a cytotoxic effect. Hydroxyurea (HU) is an S-phase specific inhibitor of ribonucleotide reductase (RR) with a broad spectrum of antitumor effects. We report here that low-dose HU enhanced the activity of Gem in a time- and sequence-dependent manner. Exposure of human oropharyngeal carcinoma KB cells to HU followed by the addition of Gem at various times significantly enhanced cytotoxicity when compared to controls. The greatest enhancement of cytotoxicity occurred when Gem was added 8 hours after HU. By treating KB cells with radiolabeled-Gem following HU treatment, we further confirmed that the incorporation of dFdCTP into DNA increased 6-fold over control reactions under these conditions. The mechanism of the time- and sequence-dependent enhancement is associated with a decrease in hRRM2 RNA, protein, and activity between 4 and 8 hours. The subsequent depletion of dNTP pools allows for increased incorporation of dFdCTP into cells arrested in S-phase, resulting in higher levels of cytotoxicity than either treatment alone.