Wavelet-based selection-and-recalibration network for Parkinson's disease screening in OCT images.

BACKGROUND AND OBJECTIVE: Parkinson's disease (PD) is one of the most prevalent neurodegenerative brain diseases worldwide. Therefore, accurate PD screening is crucial for early clinical intervention and treatment. Recent clinical research indicates that changes in pathology, such as the texture and thickness of the retinal layers, can serve as biomarkers for clinical PD diagnosis based on optical coherence tomography (OCT) images. However, the pathological manifestations of PD in the retinal layers are subtle compared to the more salient lesions associated with retinal diseases. METHODS: Inspired by textural edge feature extraction in frequency domain learning, we aim to explore a potential approach to enhance the distinction between the feature distributions in retinal layers of PD cases and healthy controls. In this paper, we introduce a simple yet novel wavelet-based selection and recalibration module to effectively enhance the feature representations of the deep neural network by aggregating the unique clinical properties, such as the retinal layers in each frequency band. We combine this module with the residual block to form a deep network named Wavelet-based Selection and Recalibration Network (WaveSRNet) for automatic PD screening. RESULTS: The extensive experiments on a clinical PD-OCT dataset and two publicly available datasets demonstrate that our approach outperforms state-of-the-art methods. Visualization analysis and ablation studies are conducted to enhance the explainability of WaveSRNet in the decision-making process. CONCLUSIONS: Our results suggest the potential role of the retina as an assessment tool for PD. Visual analysis shows that PD-related elements include not only certain retinal layers but also the location of the fovea in OCT images.

Copper overload exacerbates testicular aging mediated by lncRNA:CR43306 deficiency through ferroptosis in Drosophila.

Testicular aging manifests as impaired spermatogenesis and morphological alterations in Drosophila. Nonetheless, the comprehensive molecular regulatory framework remains largely undisclosed. This investigation illustrates the impact of copper overload on testicular aging and underscores the interplay between copper overload and lncRNA. Copper overload triggers Cuproptosis through the mitochondrial TCA cycle, facilitating intracellular interactions with Ferroptosis, thereby governing testicular aging. Dysfunction of lncRNA:CR43306 also contributes to testicular aging in Drosophila, emphasizing the significance of lncRNA:CR43306 as a novel aging-associated lncRNA. Moreover, copper overload exacerbates spermatid differentiation defects mediated by lncRNA:CR43306 deficiency through oxidative stress, copper, and iron transport. Therapeutically, Ferrostatin-1 and Resveratrol emerge as potential remedies for addressing testicular aging. This study offers perspectives on the regulatory mechanisms involving copper overload and lncRNA:CR43306 deficiency in the context of testicular aging.

Layer-by-layer growth of Cu3(HHTP)2 films on Cu(OH)2 nanowire arrays for high performance ascorbic acid sensing.

Growing three-dimensional (3D) metal organic frameworks (MOFs) via heterogeneous epitaxial growth on metal hydroxide arrays are effective for constructing electrochemical sensor. However, the growth of MOFs is difficult to control, resulting in thick and irregular morphologies and even damage the metal hydroxide template. In this work, Cu3(HHTP)2 (HHTP = 2, 3, 6, 7, 10, 11-hexahydroxytriphenylene) films with controllable thickness and morphology were successfully prepared on Cu(OH)2 nanowire arrays (NWAs) through layer-by-layer (LBL) growth method. We have discovered that the LBL cycle and the reaction solvent composition are crucial for growing homogenous MOF thin films. The Cu3(HHTP)2 based ascorbic acid (AA) sensor, fabricated in ethanol within 10 LBL cycles, generated an ultrahigh sensitivity of 821.64 µA mM-1 cm-2 in the range of 6-981.41 µM, a low detection limit of 60 nM as well as the great selectivity, stability and reproducibility. Moreover, the relative deviation for AA detection in two fruit juices were 3.22 % and 3.71 %, and the test result for human sweat fall within the normal AA concentration range, verifying the feasibility of as-prepared sensor for practical application.

Lanosterol Synthase Prevents EMT During Lens Epithelial Fibrosis Via Regulating SREBP1.

Purpose: Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is a predominant pathological process underlying fibrotic cataracts. Here we investigated the role and mechanism of lanosterol synthase (LSS), a key rate-limiting enzyme in sterol biosynthesis, in EMT of LECs. Methods: Human lens epithelial explants, primary rabbit LECs, and whole rat lenses were treated with TGFß2. RNA-sequencing was conducted to explore genetic changes during fibrosis of human lens epithelial explants. Loss- and gain-of-function studies were performed in primary LECs to investigate roles and mechanisms of LSS, lanosterol and sterol regulatory element binding transcription protein 1 (SREBP1) in EMT. Rat lenses were applied to evaluate the potential effect of lanosterol on lens fibrosis. Expression of LSS, SREBP1, EMT-related regulators, and markers were analyzed by Western blot, qRT-PCR, or immunofluorescent staining. Results: LSS and steroid biosynthesis were downregulated in TGFß2-induced lens fibrosis. LSS inhibition directly triggered EMT by inducing Smad2/3 phosphorylation and nucleus translocation, an overexpression of LSS protected LECs from EMT by inhibiting Smad2/3 activation. Moreover, LSS inhibition decreased the expression of SREBP1, which regulated EMT via intervening TGFß2/Smad2/3 transduction. Furthermore, lanosterol protected LECs from EMT caused by both TGFß2 treatment and LSS inhibition via suppressing Smad2/3 activation and maintained lens transparency by preventing fibrotic plaques formation. Conclusions: We first identified that LSS protected LECs from EMT and played an antifibrotic role to maintain lens transparency. Additionally, lanosterol and sterol biosynthesis regulation might be promising strategies for preventing and treating fibrotic cataracts.

Graphene/metal-organic framework nano-sandwiches derived N, P-codoped porous carbon nanosheets as robust material for electrochemical analysis.

Construction of novel two-dimensional porous carbon nanosheets with superior electrochemical activity is of great challenge. Here, graphene/ZIF-8 nano-sandwiches derived N, P-codoped porous carbon nanosheets (N, P-codoped PCN) was easily obtained by sequential room temperature self-assembly and high-temperature carbonization method. Relative to the widely used physically exfoliated graphene nanosheets (GN) and graphene/ZIF-8 derived N-doped porous carbon nanosheets (N-doped PCN), N, P-codoped PCN displayed larger active surface, faster electron transport ability and stronger physical adsorption ability, which can be ascribed to the dual doping effect of heteroatoms N and P. As a result, N, P-codoped PCN exhibited remarkable oxidation signal enhancement for tumor marker (8-hydroxy-2'-deoxyguanosine), analgesic and antipyretic drug (acetaminophen) and organic pesticide (benomyl). Besides, the limits of detection were measured as low as 1.58 nM, 7.50 nM and 2.10 nM with sensitivity of 270.00 µA µM-1 cm-2, 757.14 µA µM-1 cm-2 and 272.86 µA µM-1 cm-2 for 8-hydroxy-2'-deoxyguanosine, acetaminophen and benomyl, respectively. Basing on this, a novel and highly sensitive electrochemical sensing platform was developed. It is believed that the reported two-dimensional N, P-codoped PCN with unique structure and composition is highly valuable for the development of carbon-based electrochemical sensors.

Facile synthesis of core-shell Co-MOF with hierarchical porosity for enhanced electrochemical detection of furaltadone in aquaculture water.

Metal-organic frameworks (MOFs) exhibited huge application potential in electrochemical analysis field, how to facilely and effectively boost the electrochemical sensing activity of MOFs materials still face enormous challenges. In this work, core-shell Co-MOF (Co-TCA@ZIF-67) polyhedrons with hierarchical porosity was easily synthesized via simple chemical etching reaction by selecting thiocyanuric acid as the etching reagent. Benefiting from the introduction of mesopores and thiocyanuric acid/Co2+ complex on the surface of ZIF-67 frameworks, the property and functions of the pristine ZIF-67 was seriously tailored. Compared with the pristine ZIF-67, the as-resulted Co-TCA@ZIF-67 nanoparticles displayed greatly enhanced physical adsorption capacity and electrochemical reduction activity toward the antibiotic drug furaltadone. As a result, a novel furaltadone electrochemical sensor with high sensitivity was fabricated. The linear detection range was from 50 nM to 5 µM with sensitivity of 110.40 µA-1 µM-1 cm-2 and detection limit of 12 nM. This work demonstrated chemical etching strategy is truly a facile and effective way to modify the electrochemical sensing performance of MOFs-based materials, and we believed the chemically etched MOFs materials will play a stronger role in terms of food safety and environmental conservation.

Long Non-Coding RNA H19 Prevents Lens Fibrosis through Maintaining Lens Epithelial Cell Phenotypes.

The integrity of lens epithelial cells (LECs) lays the foundation for lens function and transparency. By contrast, epithelial-mesenchymal transition (EMT) of LECs leads to lens fibrosis, such as anterior subcapsular cataracts (ASC) and fibrotic forms of posterior capsule opacification (PCO). However, the underlying mechanisms remain unclear. Here, we aimed to explore the role of long non-coding RNA (lncRNA) H19 in regulating TGF-ß2-induced EMT during lens fibrosis, revealing a novel lncRNA-based regulatory mechanism. In this work, we identified that lncRNA H19 was highly expressed in LECs, but downregulated by exposure to TGF-ß2. In both human lens epithelial explants and SRA01/04 cells, knockdown of H19 aggravated TGF-ß2-induced EMT, while overexpressing H19 partially reversed EMT and restored lens epithelial phenotypes. Semi-in vivo whole lens culture and H19 knockout mice demonstrated the indispensable role of H19 in sustaining lens clarity through maintaining LEC features. Bioinformatic analyses further implied a potential H19-centered regulatory mechanism via Smad-dependent pathways, confirmed by in vitro experiments. In conclusion, we uncovered a novel role of H19 in inhibiting TGF-ß2-induced EMT of the lens by suppressing Smad-dependent signaling, providing potential therapeutic targets for treating lens fibrosis.

A high-performance electrochemical sensor for sensitive detection of tetracycline based on a Zr-UiO-66/MWCNTs/AuNPs composite electrode.

Herein, a high-performance electrochemical sensor was constructed based on a metal-organic framework (Zr-UiO-66)/multi-carbon nanotubes/gold nanoparticles (Zr-UiO-66/MWCNTs/AuNPs) composite modified glassy carbon electrode for sensitive determination of tetracycline. The morphology, structure, and performance of Zr-UiO-66/MWCNTs/AuNPs were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), energy dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy (FT-IR), and electrochemical techniques. The Zr-UiO-66/MWCNTs/AuNPs nanohybrids exhibited excellent electrocatalytic activity towards the oxidation of tetracycline, mainly because of the synergistic effect of the MOFs, MWCNTs, and gold nanoparticles. The electrochemical kinetics and catalytical mechanism of tetracycline were demonstrated, proving that tetracycline's electrocatalytic oxidation reaction was an absorption-controlled two-step process involving the transfer of two protons and two electrons, respectively. Furthermore, a simple and facile method was used to achieve the regeneration of the absorbed saturated electrode. A low concentration of tetracycline was detected by amperometry with the linear ranges of 5 × 10-7 to 2.25 × 10-4 mol L-1 (R2 = 0.9941), the sensitivity was 45.4 mA L mol-1, and the limit of detection was as low as 1.67 × 10-7 mol L-1 (S/N = 3). In addition, the composite electrode demonstrated high selectivity (interference deviation of ± 5%), satisfactory reproductivity (RSD of 5.31%), and long-term stability (easy regeneration) and was successfully applied in the analysis of tetracycline antibiotics in actual samples. Thus, the proposed electrode provides a promising and prospective MOFs-based sensing platform for the detection of tetracyclines in the environment.

Simultaneous and efficient removal of fluoride and phosphate in phosphogypsum leachate by acid-modified sulfoaluminate cement.

The high concentration of fluoride and phosphate in phosphogypsum leachate is harmful to the environment and ecosystem. Thus, there is a need to develop feasible materials or technologies to remove both fluoride and phosphate in acidic phosphogypsum leachate. In this study, sulfoaluminate cement (SC) was used to simultaneously remove fluoride and phosphate in wastewater based on its moderate alkalinity and rich content of metal elements (Ca, Al and Fe, etc). The acidized sulfoaluminate cement (ASC) composite was prepared through modifying SC with hydrochloric acid, which can increase the specific surface areas of the raw SC, as well as the activity of the metal elements in SC. Compared with other coagulants, ASC showed excellent removal performance for fluoride and phosphate, such as higher removal efficiency, better effluent quality, and accelerated settling rate. The fluoride and phosphate removal performances of ASC herein were investigated at different dosages, pH values, coexisting substances, and initial concentrations. As a result, ASC exhibited wide pH adaptability and satisfactory selectivity for fluoride and phosphate. The possible removal mechanisms of fluoride and phosphate by ASC included chemisorption, ion exchange, and precipitation. The main end products associated with fluoride were fluorite (CaF2), aluminum fluoride (AlF3), and iron trifluoride (FeF3). The main final products amid phosphate removal, on the other hand, were brushite (CaHPO4·2H2O), aluminophosphate ((H3O)·AlP2O6(OH)2), silicocarnotite (Ca2SiO4·Ca3(PO4)2) and iron phosphate (Fe(H2PO4)3). More importantly, ASC can effectively treat the phosphogypsum leachate at a wide range of concentrations, and the concentrations of phosphate and fluoride in the effluents were lower than 0.5 mg P L-1 and 4 mg L-1, respectively. To sum up, ASC is a competitive candidate to treat wastewater with high fluoride and phosphate content, such as phosphogypsum leachate.

TFEB-Mediated Lysosomal Restoration Alleviates High Glucose-Induced Cataracts Via Attenuating Oxidative Stress.

Purpose: Diabetic cataract (DC) is a visual disorder arising from diabetes mellitus (DM). Autophagy, a prosurvival intracellular process through lysosomal fusion and degradation, has been implicated in multiple diabetic complications. Herein, we performed in vivo and in vitro assays to explore the specific roles of the autophagy-lysosome pathway in DC. Methods: Streptozotocin-induced DM and incubation in high glucose (HG) led to rat lens opacification. Protein Simple Wes, Western blot, and immunoassay were utilized to investigate autophagic changes in lens epithelial cells (LECs) and lens fiber cells (LFCs). RNA-sequencing (RNA-seq) was performed to explore genetic changes in the lenses of diabetic rats. Moreover, autophagy-lysosomal functions were examined using lysotracker, Western blot, and immunofluorescence analyses in HG-cultured primary rabbit LECs. Results: First, DM and HG culture led to fibrotic LECs, swelling LFCs, and eventually cataracts. Further analysis showed aberrant autophagic degradation in LECs and LFCs during cataract formation. RNA-seq data revealed that the differentially expressed genes (DEGs) were enriched in the lysosome pathway. In primary LECs, HG treatment resulted in decreased transcription factor EB (TFEB) and cathepsin B (CTSB) activity, and increased lysosomal size and pH values. Moreover, TFEB-mediated dysfunctional lysosomes resulted from excessive oxidative stress in LECs under HG conditions. Furthermore, TFEB activation by curcumin analog C1 alleviated HG-induced cataracts through enhancing lysosome biogenesis and activating protective autophagy, thereby attenuating HG-mediated oxidative damage. Conclusions: In summary, we first identified that ROS-TFEB-dependent lysosomal dysfunction contributed to autophagy blockage in HG-induced cataracts. Additionally, TFEB-mediated lysosomal restoration might be a promising therapeutic method for preventing and treating DC through mitigating oxidative stress.

Autophagy inhibition attenuates TGF-ß2-induced epithelial-mesenchymal transition in lens epithelial cells.

AIMS: Autophagy has been reported to play an essential role in fibrotic disorders. Known as fibrotic cataract, posterior capsular opacification (PCO) result from pathological epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). This study aims to identify the role and potential mechanism of autophagy in TGF-ß2-induced EMT in LECs. MAIN METHODS: Primary rabbit LECs were treated with TGF-ß2 to induce EMT as a model of fibrotic cataract in vitro. 3-methyladenine, chloroquine, bafilomycin A1, and gene silencing of autophagy-related protein 7 (ATG7) were treated in LECs for autophagy inhibition, while rapamycin was utilized for autophagy activation. The expression levels of EMT/autophagy-associated markers were analyzed by qRT-PCR, western blotting, immunofluorescence and transmission electron microscopy. We additionally examined cell migration ability with transwell migration assay and wound healing assay. KEY FINDINGS: TGF-ß2 promoted autophagy flux during EMT progression of LECs in a time-dependent manner. Autophagy activation by rapamycin enhanced TGF-ß2-triggered fibrogenic responses and cell migration in LECs, whereas pharmacological inhibition of autophagy alleviated TGF-ß2-induced increases of EMT markers and cell migration of LECs. In addition, the phosphorylation of Smad2/3 induced by TGF-ß2 was suppressed through autophagy inhibition, while it was promoted upon autophagy activation, indicating that TGF-ß2/Smad signaling was involved in the modulation of autophagy on EMT in LECs. Furthermore, ATG7-silenced LECs exerted anti-fibrosis effect induced by TGF-ß2 through downregulation of autophagy. SIGNIFICANCE: Intervention/inhibition of autophagy could attenuate TGF-ß2-induced EMT in LECs, which provides autophagy-related insights on preventing and treating the fibrotic cataract or other fibrotic diseases.

Renin inhibitors for the treatment of hypertension: design and optimization of a novel series of tertiary alcohol-bearing piperidines.

The design and optimization of a novel series of renin inhibitor is described herein. Strategically, by committing the necessary resources to the development of synthetic sequences and scaffolds that were most amenable for late stage structural diversification, even as the focus of the SAR campaign moved from one end of the molecule to another, highly potent renin inhibitors could be rapidly identified and profiled.

Renin inhibitors for the treatment of hypertension: design and optimization of a novel series of pyridone-substituted piperidines.

An SAR campaign aimed at decreasing the overall lipophilicity of renin inhibitors such as 1 is described herein. It was found that replacement of the northern appendage in 1 with an N-methyl pyridone and subsequent re-optimization of the benzyl amide handle afforded compounds with in vitro and in vivo profiles suitable for further profiling. An unexpected CV toxicity in dogs observed with compound 20 led to the employment of a time and resource sparing rodent model for in vivo screening of key compounds. This culminated in the identification of compound 31 as an optimized renin inhibitor.

Identification of adulterants in a Chinese herbal medicine by LC-HRMS and LC-MS-SPE/NMR and comparative in vivo study with standards in a hypertensive rat model.

Based on anecdotal evidence of anti-hypertensive effect of Gold Nine Soft Capsules, an in vivo study of this complex Chinese "herbal-based" medicine was initiated. Dosage of the content of Gold Nine capsules in spontaneous hypertensive rats showed a remarkably good effect. This led to further investigation of the components of the preparation and eventual identification of three known anti-hypertensive drugs; amlodipine, indapamide and valsartan, which were not declared on the label. Compounds were rapidly identified using LC-HRMS and LC-MS-SPE/NMR, quantified by HPLC, and the in vivo activity of a combination of commercially purchased standards was shown to be equivalent to that of the capsule content. Adulteration of herbal remedies and dietary supplements with synthetic drugs is an increasing problem that may lead to serious adverse effects. LC-MS-SPE/NMR as a method for the rapid identification of such adulterants is highlighted in this case study.

The C3a anaphylatoxin receptor is a key mediator of insulin resistance and functions by modulating adipose tissue macrophage infiltration and activation.

OBJECTIVE: Significant new data suggest that metabolic disorders such as diabetes, obesity, and atherosclerosis all posses an important inflammatory component. Infiltrating macrophages contribute to both tissue-specific and systemic inflammation, which promotes insulin resistance. The complement cascade is involved in the inflammatory cascade initiated by the innate and adaptive immune response. A mouse genomic F2 cross biology was performed and identified several causal genes linked to type 2 diabetes, including the complement pathway. RESEARCH DESIGN AND METHODS: We therefore sought to investigate the effect of a C3a receptor (C3aR) deletion on insulin resistance, obesity, and macrophage function utilizing both the normal-diet (ND) and a diet-induced obesity mouse model. RESULTS: We demonstrate that high C3aR expression is found in white adipose tissue and increases upon high-fat diet (HFD) feeding. Both adipocytes and macrophages within the white adipose tissue express significant amounts of C3aR. C3aR(-/-) mice on HFD are transiently resistant to diet-induced obesity during an 8-week period. Metabolic profiling suggests that they are also protected from HFD-induced insulin resistance and liver steatosis. C3aR(-/-) mice had improved insulin sensitivity on both ND and HFD as seen by an insulin tolerance test and an oral glucose tolerance test. Adipose tissue analysis revealed a striking decrease in macrophage infiltration with a concomitant reduction in both tissue and plasma proinflammatory cytokine production. Furthermore, C3aR(-/-) macrophages polarized to the M1 phenotype showed a considerable decrease in proinflammatory mediators. CONCLUSIONS: Overall, our results suggest that the C3aR in macrophages, and potentially adipocytes, plays an important role in adipose tissue homeostasis and insulin resistance.

A caspase active site probe reveals high fractional inhibition needed to block DNA fragmentation.

Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [(125)I]M808, which acts as a caspase active site probe. [(125)I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [(125)I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies.

Late onset Tay-Sachs disease in mice with targeted disruption of the Hexa gene: behavioral changes and pathology of the central nervous system.

Tay-Sachs disease is an autosomal recessive neurodegenerative disease resulting from a block in the hydrolysis of GM2 ganglioside, an intermediate in ganglioside catabolism. The mouse model of Tay-Sachs disease (Hexa -/-) has been described as behaviorally indistinguishable from wild type until at least 1 year of age due to a sialidase-mediated bypass of the metabolic defect that reduces the rate of GM2 ganglioside accumulation. In this study, we have followed our mouse model to over 2 years of age and have documented a significant disease phenotype that is reminiscent of the late onset, chronic form of human Tay-Sachs disease. Onset occurs at 11-12 months of age and progresses slowly, in parallel with increasing storage of GM2 ganglioside. The disease is characterized by hind limb spasticity, weight loss, tremors, abnormal posture with lordosis, possible visual impairment, and, late in the disease, muscle weakness, clasping of the limbs, and myoclonic twitches of the head. Immunodetection of GM2 ganglioside showed that storage varies widely in different regions, but is most intense in pyriform cortex, hippocampus (CA3 field, subiculum), amygdala, hypothalamus (paraventricular supraoptic, ventromedial and arcuate nuclei, and mammilary body), and the somatosensory cortex (layer V) in 1- to 2-year-old mutant mice. We suggest that the Tay-Sachs mouse model is a phenotypically valid model of disease and may provide for a reliable indicator of the impact of therapeutic strategies, in particular geared to the late onset, chronic form of human Tay-Sachs disease.

Differential efficacy of caspase inhibitors on apoptosis markers during sepsis in rats and implication for fractional inhibition requirements for therapeutics.

A rodent model of sepsis was used to establish the relationship between caspase inhibition and inhibition of apoptotic cell death in vivo. In this model, thymocyte cell death was blocked by Bcl-2 transgene, indicating that apoptosis was predominantly dependent on the mitochondrial pathway that culminates in caspase-3 activation. Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly different efficacy for different cell death markers. Inhibition of DNA fragmentation required substantially higher levels of caspase-3 attenuation than that required for blockade of other apoptotic events such as spectrin proteolysis and phosphatidylserine externalization. These data indicate a direct relationship between caspase inhibition and some apoptotic manifestations but that small quantities of uninhibited caspase-3 suffice to initiate genomic DNA breakdown, presumably through the escape of catalytic quantities of caspase-activated DNase. These findings suggest that putative caspase-independent apoptosis may be overestimated in some systems since blockade of spectrin proteolysis and other cell death markers does not accurately reflect the high degrees of caspase-3 inhibition needed to prevent DNA fragmentation. Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.