Time-to-progression after front-line fludarabine, cyclophosphamide, and rituximab chemoimmunotherapy for chronic lymphocytic leukaemia: a retrospective, multicohort study.

BACKGROUND: Fludarabine, cyclophosphamide, and rituximab (FCR) has become a gold-standard chemoimmunotherapy regimen for patients with chronic lymphocytic leukaemia. However, the question remains of how to treat treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia. We therefore aimed to develop and validate a gene expression signature to identify which of these patients are likely to achieve durable remissions with FCR chemoimmunotherapy. METHODS: We did a retrospective cohort study in two cohorts of treatment-naive patients (aged ≥18 years) with chronic lymphocytic leukaemia. The discovery and training cohort consisted of peripheral blood samples collected from patients treated at the University of Texas MD Anderson Cancer Center (Houston, TX, USA), who fulfilled the diagnostic criteria of the International Workshop on Chronic Lymphocytic Leukemia, had received at least three cycles of FCR chemoimmunotherapy, and had been treated between Oct 10, 2000, and Oct 26, 2006 (ie, the MDACC cohort). We did transcriptional profiling on samples obtained from the MDACC cohort to identify genes associated with time to progression. We did univariate Cox proportional hazards analyses and used significant genes to cluster IGHV-unmutated samples into two groups (intermediate prognosis and unfavourable prognosis). After using cross-validation to assess robustness, we applied the Lasso method to standardise the gene expression values to find a minimum gene signature. We validated this signature in an external cohort of treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia enrolled on the CLL8 trial of the German Chronic Lymphocytic Leukaemia Study Group who were treated between July 21, 2003, and April 4, 2006 (ie, the CLL8 cohort). FINDINGS: The MDACC cohort consisted of 101 patients and the CLL8 cohort consisted of 109 patients. Using the MDACC cohort, we identified and developed a 17-gene expression signature that distinguished IGHV-unmutated patients who were likely to achieve a long-term remission following front-line FCR chemoimmunotherapy from those who might benefit from alternative front-line regimens (hazard ratio 3·83, 95% CI 1·94-7·59; p<0·0001). We validated this gene signature in the CLL8 cohort; patients with an unfavourable prognosis versus those with an intermediate prognosis had a cause-specific hazard ratio of 1·90 (95% CI 1·18-3·06; p=0·008). Median time to progression was 39 months (IQR 22-69) for those with an unfavourable prognosis compared with 59 months (28-84) for those with an intermediate prognosis. INTERPRETATION: We have developed a robust, reproducible 17-gene signature that identifies a subset of treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia who might substantially benefit from treatment with FCR chemoimmunotherapy. We recommend testing the value of this gene signature in a prospective study that compares FCR treatment with newer alternative therapies as part of a randomised clinical trial. FUNDING: Chronic Lymphocytic Leukaemia Global Research Foundation and the National Institutes of Health/National Cancer Institute.

Covalent Bonding and Atomic-Level Plasticity Increase Adhesion in Silicon-Diamond Nanocontacts.

Nanoindentation and sliding experiments using single-crystal silicon atomic force microscope probes in contact with diamond substrates in vacuum were carried out in situ with a transmission electron microscope (TEM). After sliding, the experimentally measured works of adhesion were significantly larger than values estimated for pure van der Waals (vdW) interactions. Furthermore, the works of adhesion increased with both the normal stress and speed during the sliding, indicating that applied stress played a central role in the reactivity of the interface. Complementary molecular dynamics (MD) simulations were used to lend insight into the atomic-level processes that occur during these experiments. Simulations using crystalline silicon tips with varying degrees of roughness and diamond substrates with different amounts of hydrogen termination demonstrated two relevant phenomena. First, covalent bonds formed across the interface, where the number of bonds formed was affected by the hydrogen termination of the substrate, the tip roughness, the applied stress, and the stochastic nature of bond formation. Second, for initially rough tips, the sliding motion and the associated application of shear stress produced an increase in irreversible atomic-scale plasticity that tended to smoothen the tips' surfaces, which resulted in a concomitant increase in adhesion. In contrast, for initially smooth tips, sliding roughened some of these tips. In the limit of low applied stress, the experimentally determined works of adhesion match the intrinsic (van der Waals) work of adhesion for an atomically smooth silicon-diamond interface obtained from MD simulations. The results provide mechanistic interpretations of sliding-induced changes and interfacial adhesion and may help inform applications involving adhesive interfaces that are subject to applied shear forces and displacements.

Temperature-responsive silk-elastinlike protein polymer enhancement of intravesical drug delivery of a therapeutic glycosaminoglycan for treatment of interstitial cystitis/painful bladder syndrome.

Interstitial cystitis (IC), also known as painful bladder syndrome, is a debilitating chronic condition with many patients failing to respond to current treatment options. Rapid clearance, mucosal coating, and tight epithelium create strong natural barriers that reduce the effectiveness of many pharmacological interventions in the bladder. Intravesical drug delivery (IDD) is the administration of therapeutic compounds or devices to the urinary bladder via a urethral catheter. Previous work in improving IDD for IC has focused on the sustained delivery of analgesics within the bladder and other small molecule drugs which do not address underlying inflammation and bladder damage. Therapeutic glycosaminoglycans (GAG) function by restoring the mucosal barrier within the bladder, promoting healing responses, and preventing irritating solutes from reaching the bladder wall. There is an unmet medical need for a therapy that provides both acute relief of symptoms while alleviating underlying physiological sources of inflammation and promoting healing within the urothelium. Semi-synthetic glycosaminoglycan ethers (SAGE) are an emerging class of therapeutic GAG with intrinsic anti-inflammatory and analgesic properties. To reduce SAGE clearance and enhance its accumulation in the bladder, we developed a silk-elastinlike protein polymer (SELP) based system to enhance SAGE IDD. We evaluated in vitro release kinetics, rheological properties, impact on bladder function, pain response, and bladder inflammation and compared their effectiveness to other temperature-responsive polymers including Poloxamer 407 and poly(lactic-co-glycolic acid)-poly(ethylene glycol). SAGE delivered via SELP-enhanced intravesical delivery substantially improved SAGE accumulation in the urothelium, provided a sustained analgesic effect 24 h after administration, and reduced inflammation.

Sociodemographic disparities in corticolimbic structures.

This study sought to examine the interactive relations of socioeconomic status and race to corticolimbic regions that may play a key role in translating stress to the poor health outcomes overrepresented among those of lower socioeconomic status and African American race. Participants were 200 community-dwelling, self-identified African American and White adults from the Healthy Aging in Neighborhoods of Diversity across the Life Span SCAN study. Brain volumes were derived using T1-weighted MP-RAGE images. Socioeconomic status by race interactions were observed for right medial prefrontal cortex (B = .26, p = .014), left medial prefrontal cortex (B = .26, p = .017), left orbital prefrontal cortex (B = .22, p = .037), and left anterior cingulate cortex (B = .27, p = .018), wherein higher socioeconomic status Whites had greater volumes than all other groups. Additionally, higher versus lower socioeconomic status persons had greater right and left hippocampal (B = -.15, p = .030; B = -.19, p = .004, respectively) and amygdalar (B = -.17, p = .015; B = -.21; p = .002, respectively) volumes. Whites had greater right and left hippocampal (B = -.17, p = .012; B = -.20, p = .003, respectively), right orbital prefrontal cortex (B = -.34, p < 0.001), and right anterior cingulate cortex (B = -.18, p = 0.011) volumes than African Americans. Among many factors, the higher levels of lifetime chronic stress associated with lower socioeconomic status and African American race may adversely affect corticolimbic circuitry. These relations may help explain race- and socioeconomic status-related disparities in adverse health outcomes.

Influence of Silica Nanoparticle Density and Flow Conditions on Sedimentation, Cell Uptake, and Cytotoxicity.

Careful evaluation of the toxicological response of engineered nanomaterials (ENMs) as a function of physicochemical properties can aid in the design of safe platforms for biomedical applications including drug delivery. Typically, in vitro ENM cytotoxicity assessments are performed under conventional static cell culture conditions. However, such conditions do not take into account the sedimentation rate of ENMs. Herein, we synthesized four types of similar size silica nanoparticles (SNPs) with modified surface roughness, charge, and density and characterized their cytotoxicity under static and dynamic conditions. Influence of particle density on sedimentation and diffusion velocities were studied by comparing solid dense silica nanoparticles of approximately 350 nm in diameter with hollow rattle shape particles of similar size. Surface roughness and charge had negligible impact on sedimentation and diffusion velocities. Lower cellular uptake and toxicity was observed by rattle particles and under dynamic conditions. Dosimetry of ENMs are primarily reported by particle concentration, assuming homogeneous distribution of nanoparticles in cell culture media. However, under static conditions, nanoparticles tend to sediment at a higher rate due to gravitational forces and hence increase effective doses of nanoparticles exposed to cells. By introducing shear flow to SNP suspensions, we reduced sedimentation and nonhomogeneous particle distribution. These results have implications for design of in vitro cytotoxicity assessment of ENMs and suggest that among other factors, sedimentation of nanoparticles in toxicity assessment should be carefully considered.

Soil microbial communities and elk foraging intensity: implications for soil biogeochemical cycling in the sagebrush steppe.

Foraging intensity of large herbivores may exert an indirect top-down ecological force on soil microbial communities via changes in plant litter inputs. We investigated the responses of the soil microbial community to elk (Cervus elaphus) winter range occupancy across a long-term foraging exclusion experiment in the sagebrush steppe of the North American Rocky Mountains, combining phylogenetic analysis of fungi and bacteria with shotgun metagenomics and extracellular enzyme assays. Winter foraging intensity was associated with reduced bacterial richness and increasingly distinct bacterial communities. Although fungal communities did not respond linearly to foraging intensity, a greater ß-diversity response to winter foraging exclusion was observed. Furthermore, winter foraging exclusion increased soil cellulolytic and hemicellulolytic enzyme potential and higher foraging intensity reduced chitinolytic gene abundance. Thus, future changes in winter range occupancy may shape biogeochemical processes via shifts in microbial communities and subsequent changes to their physiological capacities to cycle soil C and N.

The evolution of inflorescence diversity in the nightshades and heterochrony during meristem maturation.

One of the most remarkable manifestations of plant evolution is the diversity for floral branching systems. These "inflorescences" arise from stem cell populations in shoot meristems that mature gradually to reproductive states in response to environmental and endogenous signals. The morphology of the shoot meristem maturation process is conserved across distantly related plants, raising the question of how diverse inflorescence architectures arise from seemingly common maturation programs. In tomato and related nightshades (Solanaceae), inflorescences range from solitary flowers to highly branched structures bearing hundreds of flowers. Since reproductive barriers between even closely related Solanaceae have precluded a genetic dissection, we captured and compared meristem maturation transcriptomes from five domesticated and wild species reflecting the evolutionary continuum of inflorescence complexity. We find these divergent species share hundreds of dynamically expressed genes, enriched for transcription factors. Meristem stages are defined by distinct molecular states and point to modified maturation schedules underlying architectural variation. These modified schedules are marked by a peak of transcriptome expression divergence during the reproductive transition, driven by heterochronic shifts of dynamic genes, including transcriptional regulators with known roles in flowering. Thus, evolutionary diversity in Solanaceae inflorescence complexity is determined by subtle modifications of transcriptional programs during a critical transitional window of meristem maturation, which we propose underlies similar cases of plant architectural variation. More broadly, our findings parallel the recently described transcriptome "inverse hourglass" model for animal embryogenesis, suggesting both plant and animal morphological variation is guided by a mid-development period of transcriptome divergence.

In Vivo Magnetic Stimulation of Rat Sciatic Nerve With Centimeter- and Millimeter-Scale Solenoid Coils.

Previous reports of magnetic stimulation of the peripheral nervous system (PNS) used various coil geometries, all with outer diameters larger than 35 mm, and stimulation energies in the 50 J range to evoke neural excitation. Recent reports of central nervous system (CNS) activation used sub-mm-scale solenoid coils with mJ energy levels. The goal of this study was to translate the lower energy levels from the CNS to the PNS via using smaller coils placed in closer proximity to the neural tissue. Such a performance improvement would advance the state of the art of magnetic stimulation and provide a path towards new neuroprosthetic devices. Primarily, we investigated the range of coil outer diameters from 25 mm down to 5 mm to better understand the dependence of coil diameter on energy required for PNS activation. Nine cm- and mm-scale copper solenoid coils, with various resistances, inductances, inner and outer diameters, and heights were compared by quantizing neuromuscular responses to magnetic stimulation via capacitive discharge excitation of rat sciatic nerves in vivo. Additionally, the effects of stimulus duration and coil position were investigated. As opposed to prior work, this study compares a subset of stimulation parameters in an intact nerve preparation, and shows that magnetic stimulation with coils that abut the nerve is a reliable, effective method of neuromuscular stimulation. Although we observed different energies required for neuromuscular activation depending on the coil and excitation parameters used, for the experimental configuration, devices, and stimulus waveform shapes presented in this manuscript, no systematic dependence of PNS activation on coil diameter was found, even for the mm-scale coils investigated herein. However, there was a clear relationship between discharge circuit capacitance and energy required to evoke a neuromuscular response. Coils approximately 12 mm in outer diameter and larger consistently evoked responses, whereas coils 5 mm in outer diameter did not. Furthermore, we observed meaningful neuromuscular excitation when stimulating with energies as low as 20 J. Although this is an improvement over prior work, it is still orders of magnitude greater than the energy required for conventional electrical stimulation, suggesting that these devices are presently not suitable for use in an application requiring continued pulsed stimulation. Nevertheless, these devices are suitable for basic research and as clinical tools that infrequently stimulate, such as in diagnostic applications.

Exploitation of the Ornithine Effect Enhances Characterization of Stapled and Cyclic Peptides.

A method to facilitate the characterization of stapled or cyclic peptides is reported via an arginine-selective derivatization strategy coupled with MS/MS analysis. Arginine residues are converted to ornithine residues through a deguanidination reaction that installs a highly selectively cleavable site in peptides. Upon activation by CID or UVPD, the ornithine residue cyclizes to promote cleavage of the adjacent amide bond. This Arg-specific process offers a unique strategy for site-selective ring opening of stapled and cyclic peptides. Upon activation of each derivatized peptide, site-specific backbone cleavage at the ornithine residue results in two complementary products: the lactam ring-containing portion of the peptide and the amine-containing portion. The deguanidination process not only provides a specific marker site that initiates fragmentation of the peptide but also offers a means to unlock the staple and differentiate isobaric stapled peptides.

A µm-Scale Computational Model of Magnetic Neural Stimulation in Multifascicular Peripheral Nerves.

There has been recurring interest in using magnetic neural stimulation for implantable localized stimulation. However, the large stimulation voltages and energies necessary to evoke neuronal activity have tempered this interest. To investigate the potential of magnetic stimulation as a viable methodology and to provide the ability to investigate novel coil designs that can result in lower stimulation threshold voltages and energies, there is a need for a model that accurately predicts the magnetic field-tissue interaction that results in neuronal stimulation. In this study, we provide a computational framework to accurately estimate the stimulation threshold and have validated the model with in vivo magnetic stimulation experiments. To make such predictions, we developed a micrometer-resolution anatomically driven computational model of rat sciatic nerve and quantified the effect of tissue heterogeneity (i.e., fascicular organization, axon distribution, and density) and axonal membrane capacitance on the resulting threshold. Using the multiresolution impedance method, we computed the spatial-temporal distribution of the induced electric field in the nerve and applied this field to a Frankenhaeuser-Huxley axon model in NEURON to simulate the nonlinear mechanisms of the membrane channels. The computational model developed predicts the stimulation thresholds for four magnetic coil designs with different geometrical parameters within the 95% confidence interval (experiments count = 4) of measured in vivo stimulation thresholds for the rat sciatic nerve.

Neurological soft signs predict abnormal cerebellar-thalamic tract development and negative symptoms in adolescents at high risk for psychosis: a longitudinal perspective.

INTRODUCTION: There is an emerging consensus that neurological soft signs (NSS) may not be "soft" at all but rather may reflect neuropathy, particularly in the cerebellum and thalamus. However, our understanding of connective tract abnormalities is limited, and to date, there have been no investigations examining NSS and longitudinal white matter development during the prodrome. Mapping the correlates of NSS in ultrahigh-risk (UHR) youth offers potential for highlighting a viable biomarker as well as for advancing understanding of pathogenic processes during the adolescent risk period. METHODS: A total of 68 (33 UHR and 35 healthy control) adolescents were assessed with an NSS inventory, structured interviews, and diffusion tensor imaging. Fractional anisotropy (FA) of theoretically relevant cerebellar-thalamic tracts was calculated (left/right superior cerebellar peduncles [SCPs]). Twelve months later, a subset of 30 (15 UHR and 15 control) participants returned for follow-up diffusion tension imaging/clinical assessments. RESULTS: UHR youth exhibited elevated NSS across domains. While there were no group differences in the integrity of the SCPs at baseline, controls showed a normative increase while the UHR group showed a decrease in FA over 12 months. NSS predicted a longitudinal decrease in cerebellar-thalamic FA and elevations in negative but not positive symptoms 12 months later. DISCUSSION: Findings of abnormal white matter development provide direct empirical evidence to support prominent neurodevelopmental theories. The predictive relationships between NSS and longitudinal cerebellar-thalamic tract integrity and negative symptom course provide insight into the role of cognitive dysmetria in the high-risk period and inform on a unique biomarker tied to core features underlying psychosis.

Magnetic stimulation of mammalian peripheral nerves in vivo: an alternative to functional electrical stimulation.

Functional electrical stimulation is the current gold standard for stimulating neuronal interfaces for functional neuromuscular and cortical applications, but it is not without its drawbacks. One such fault is the need to have direct electrical contact with the nerve tissue, and any side effects this causes. Functional magnetic stimulation, which works though electromagnetic induction, does not require electrical contact and may be a viable alternative to functional electrical stimulation. We are investigating the capabilities of magnetic stimulation with centimeter scale (< 2.5 cm) coils in feline and rodent sciatic nerves in vivo. We have shown that magnetic stimulation can consistently produce the same levels of neuromuscular activation as electrical stimulation. Additionally, the position of the coil relative to the nerve influences neuromuscular activation, suggesting the possibility of selective muscle activation.

Sleep dysfunction and thalamic abnormalities in adolescents at ultra high-risk for psychosis.

BACKGROUND: Sleep dysfunction is a pervasive, distressing characteristic of psychosis, yet little is known regarding sleep quality prior to illness onset. At present, it is unclear whether sleep dysfunction precedes the emergence of psychotic symptoms, signifying a core feature of the disorder, or if it represents a consequence of prolonged contact with aspects of schizophrenia and its treatment (e.g., medication use or neurotoxicity) or co-morbid symptoms (e.g., depressive and manic symptomatology). The current study examined sleep dysfunction in adolescents at ultra high-risk (UHR) for psychosis, relationships between sleep disturbances and psychosis symptoms, volume of an integral sleep-structure (thalamus), and associations between thalamic abnormalities and sleep impairment in UHR youth. METHOD: Thirty-three UHR youth and 33 healthy controls (HC) participated in a self-assessment of sleep functioning (Pittsburgh Sleep Quality Index; PSQI), self and parent-report clinical interviews, and structural magnetic resonance imaging (MRI). RESULTS: UHR adolescents displayed increased latency to sleep onset and greater sleep disturbances/disrupted continuity compared to HC youth, over and above concurrent mood symptoms. Among UHR youth, increased sleep dysfunction was associated with greater negative symptom severity but not positive symptoms. Compared to HC adolescents, UHR participants displayed decreased bilateral thalamus volume, which was associated with increased sleep dysfunction. CONCLUSIONS: Sleep dysfunction occurs during the pre-psychotic period, and may play a role in the etiology and pathophysiology of psychosis. In addition, the relationship of disrupted sleep to psychosis symptoms in UHR youth indicates that prevention and intervention strategies may be improved by targeting sleep stabilization in the pre-psychotic period.

Mining a cathepsin inhibitor library for new antiparasitic drug leads.

The targeting of parasite cysteine proteases with small molecules is emerging as a possible approach to treat tropical parasitic diseases such as sleeping sickness, Chagas' disease, and malaria. The homology of parasite cysteine proteases to the human cathepsins suggests that inhibitors originally developed for the latter may be a source of promising lead compounds for the former. We describe here the screening of a unique ∼ 2,100-member cathepsin inhibitor library against five parasite cysteine proteases thought to be relevant in tropical parasitic diseases. Compounds active against parasite enzymes were subsequently screened against cultured Plasmodium falciparum, Trypanosoma brucei brucei and/or Trypanosoma cruzi parasites and evaluated for cytotoxicity to mammalian cells. The end products of this effort include the identification of sub-micromolar cell-active leads as well as the elucidation of structure-activity trends that can guide further optimization efforts.

Evaluation of cryptolepine and huperzine derivatives as lead compounds towards new agents for the treatment of human African trypanosomiasis.

The alkaloid cryptolepine (1) and eight synthetic analogues (2-8) were assessed for in vitro activities against Trypanosoma brucei. Four of the analogues were found to be highly potent with IC50 values of less than 3 nM and three of these were assessed against T. brucei brucei infection in rats. The most effective compound was 2, 7-dibromocryptolepine (7); a single oral dose of 20 mg/kg suppressed parasitaemia and increased the mean survival time to 13.6 days compared with 8.4 days for untreated controls. In addition, four huperzine derivatives (9-12) were shown to have in vitro antitrypanosomal activities with IC50 values ranging from 303-377 nM.

The making of a compound inflorescence in tomato and related nightshades.

Variation in the branching of plant inflorescences determines flower number and, consequently, reproductive success and crop yield. Nightshade (Solanaceae) species are models for a widespread, yet poorly understood, program of eudicot growth, where short side branches are initiated upon floral termination. This "sympodial" program produces the few-flowered tomato inflorescence, but the classical mutants compound inflorescence (s) and anantha (an) are highly branched, and s bears hundreds of flowers. Here we show that S and AN, which encode a homeobox transcription factor and an F-box protein, respectively, control inflorescence architecture by promoting successive stages in the progression of an inflorescence meristem to floral specification. S and AN are sequentially expressed during this gradual phase transition, and the loss of either gene delays flower formation, resulting in additional branching. Independently arisen alleles of s account for inflorescence variation among domesticated tomatoes, and an stimulates branching in pepper plants that normally have solitary flowers. Our results suggest that variation of Solanaceae inflorescences is modulated through temporal changes in the acquisition of floral fate, providing a flexible evolutionary mechanism to elaborate sympodial inflorescence shoots.

Discovery of trypanocidal compounds by whole cell HTS of Trypanosoma brucei.

Chemotherapy against human African trypanosomiasis relies on four drugs that cause frequent and occasionally severe side-effects. Because human African trypanosomiasis is a disease of poor people in Africa, the traditional market-driven pathways to drug development are not available. One potentially rapid and cost-effective approach to identifying and developing new trypanocidal drugs would be high throughput-screening of existing drugs already approved for other uses, as well as clinical candidates in late development. We have developed an ATP-bioluminescence assay that could be used to rapidly and efficiently screen compound libraries against trypanosomes in a high throughput-screening format to validate this notion. We screened a collection of 2160 FDA-approved drugs, bioactive compounds and natural products to identify hits that were cytotoxic to cultured Trypanosoma brucei at a concentration of 1 mum or less. This meant that any hit identified would be effective at a concentration readily achievable by standard drug dosing in humans. From the screen, 35 hits from seven different drug categories were identified. These included the two approved trypanocidal drugs, suramin and pentamidine, several other drugs suspected but never validated as trypanocidal, and 17 novel trypanocidal drugs.

An easily dissociated 26 S proteasome catalyzes an essential ubiquitin-mediated protein degradation pathway in Trypanosoma brucei.

The 26 S proteasome, a complex between the 20 S proteasome and 19 S regulatory units, catalyzes ATP-dependent degradation of unfolded and ubiquitinated proteins in eukaryotes. We have identified previously 20 S and activated 20 S proteasomes in Trypanosoma brucei, but not 26 S proteasome. However, the presence of 26 S proteasome in T. brucei was suggested by the hydrolysis of casein by cell lysate, a process that requires ATP but is inhibited by lactacystin, and the lactacystin-sensitive turnover of ubiquitinated proteins in the intact cells. T. brucei cDNAs encoding the six proteasome ATPase homologues (Rpt) were cloned and expressed. Five of the six T. brucei Rpt cDNAs, except for Rpt2, were capable of functionally complementing the corresponding rpt deletion mutants of Saccharomyces cerevisiae. Immunoblots showed the presence in T. brucei lysate of the Rpt proteins, which co-fractionated with the yeast 19 S proteasome complex by gel filtration and localized in the 19 S fraction of a glycerol gradient. All the Rpt and putative 19 S non-ATPase (Rpn) proteins were co-immunoprecipitated from T. brucei lysate by individual anti-Rpt antibodies. Treatment of T. brucei cells with a chemical cross-linker resulted in co-immunoprecipitation of 20 S proteasome with all the Rpt and Rpn proteins that sedimented in a glycerol gradient to the position of 26 S proteasome. These data demonstrate the presence of 26 S proteasome in T. brucei cells, which apparently dissociate into 19 S and 20 S complexes upon cell lysis. RNA interference to block selectively the expression of proteasome 20 S core and Rpt subunits resulted in significant accumulation of ubiquitinated proteins accompanied by cessation of cell growth. Expression of yeast RPT2 gene in T. brucei Rpt2-deficient cells could not rescue the lethal phenotype, thus confirming the incompatibility between the two Rpt2s. The T. brucei 11 S regulator (PA26)-deficient RNA interference cells grew normally, suggesting the dispensability of activated 20 S proteasome in T. brucei.