In-cell Catalysis by Tethered Organo-Osmium Complexes Generates Selectivity for Breast Cancer Cells.

Anticancer agents that exhibit catalytic mechanisms of action offer a unique multi-targeting strategy to overcome drug resistance. Nonetheless, many in-cell catalysts in development are hindered by deactivation by endogenous nucleophiles. We have synthesised a highly potent, stable Os-based 16-electron half-sandwich ('piano stool') catalyst by introducing a permanent covalent tether between the arene and chelated diamine ligand. This catalyst exhibits antiproliferative activity comparable to the clinical drug cisplatin towards triple-negative breast cancer cells and can overcome tamoxifen resistance. Speciation experiments revealed Os to be almost exclusively albumin-bound in the extracellular medium, while cellular accumulation studies identified an energy-dependent, protein-mediated Os accumulation pathway, consistent with albumin-mediated uptake. Importantly, the tethered Os complex was active for in-cell transfer hydrogenation catalysis, initiated by co-administration of a non-toxic dose of sodium formate as a source of hydride, indicating that the Os catalyst is delivered to the cytosol of cancer cells intact. The mechanism of action involves the generation of reactive oxygen species (ROS), thus exploiting the inherent redox vulnerability of cancer cells, accompanied by selectivity for cancerous cells over non-tumorigenic cells.

Advances in the treatment of cytomegalovirus.

BACKGROUND: Human cytomegalovirus (HCMV) is a threat to immunologically weak patients. HCMV cannot yet be eliminated with a vaccine, despite recent advances. SOURCES OF DATA: Sources of data are recently published research papers and reviews about HCMV treatments. AREAS OF AGREEMENT: Current antivirals target the UL54 DNA polymerase and are limited by nephrotoxicity and viral resistance. Promisingly, letermovir targets the HCMV terminase complex and has been recently approved by the FDA and EMA. AREAS OF CONTROVERSY: Should we screen newborns for HCMV, and use antivirals to treat sensorineural hearing loss after congenital HCMV infection? GROWING POINTS: Growing points are developing drugs against latently infected cells. In addition to small molecule inhibitors, a chemokine-based fusion toxin protein, F49A-FTP, has shown promise in killing both lytically and latently infected cells. AREAS TIMELY FOR DEVELOPING RESEARCH: We need to understand what immune responses are required to control HCMV, and how best to raise these immune responses with a vaccine.

Severe extinction and rapid recovery of mammals across the Cretaceous-Palaeogene boundary, and the effects of rarity on patterns of extinction and recovery.

The end-Cretaceous mass extinction ranks among the most severe extinctions of all time; however, patterns of extinction and recovery remain incompletely understood. In particular, it is unclear how severe the extinction was, how rapid the recovery was and how sampling biases might affect our understanding of these processes. To better understand terrestrial extinction and recovery and how sampling influences these patterns, we collected data on the occurrence and abundance of fossil mammals to examine mammalian diversity across the K-Pg boundary in North America. Our data show that the extinction was more severe and the recovery more rapid than previously thought. Extinction rates are markedly higher than previously estimated: of 59 species, four survived (93% species extinction, 86% of genera). Survival is correlated with geographic range size and abundance, with widespread, common species tending to survive. This creates a sampling artefact in which rare species are both more vulnerable to extinction and less likely to be recovered, such that the fossil record is inherently biased towards the survivors. The recovery was remarkably rapid. Within 300 000 years, local diversity recovered and regional diversity rose to twice Cretaceous levels, driven by increased endemicity; morphological disparity increased above levels observed in the Cretaceous. The speed of the recovery tends to be obscured by sampling effects; faunas show increased endemicity, such that a rapid, regional increase in diversity and disparity is not seen in geographically restricted studies. Sampling biases that operate against rare taxa appear to obscure the severity of extinction and the pace of recovery across the K-Pg boundary, and similar biases may operate during other extinction events.

Diverse specificities, phenotypes, and antiviral activities of cytomegalovirus-specific CD8+ T cells.

UNLABELLED: CD8(+) T cells specific for pp65, IE1, and IE2 are present at high frequencies in human cytomegalovirus (HCMV)-seropositive individuals, and these have been shown to have phenotypes associated with terminal differentiation, as well as both cytokine and proliferative dysfunctions, especially in the elderly. However, more recently, T cell responses to many other HCMV proteins have been described, but little is known about their phenotypes and functions. Consequently, in this study, we chose to determine the diversity of HCMV-specific CD8(+) T cell responses to the products of 11 HCMV open reading frames (ORFs) in a cohort of donors aged 20 to 80 years old as well as the ability of the T cells to secrete gamma interferon (IFN-γ). Finally, we also tested their functional antiviral capacity using a novel viral dissemination assay. We identified substantial CD8(+) T cell responses by IFN-γ enzyme-linked immunospot (ELISPOT) assays to all 11 of these HCMV proteins, and across the cohort, individuals displayed a range of responses, from tightly focused to highly diverse, which were stable over time. CD8(+) T cell responses to the HCMV ORFs were highly differentiated and predominantly CD45RA(+), CD57(+), and CD28(-), across the cohort. These highly differentiated cells had the ability to inhibit viral spread even following direct ex vivo isolation. Taken together, our data argue that HCMV-specific CD8(+) T cells have effective antiviral activity irrespective of the viral protein recognized across the whole cohort and despite viral immune evasion. IMPORTANCE: Human cytomegalovirus (HCMV) is normally carried without clinical symptoms and is widely prevalent in the population; however, it often causes severe clinical disease in individuals with compromised immune responses. HCMV is never cleared after primary infection but persists in the host for life. In HCMV carriers, the immune response to HCMV includes large numbers of virus-specific immune cells, and the virus has evolved many mechanisms to evade the immune response. While this immune response seems to protect healthy people from subsequent disease, the virus is never eliminated. It has been suggested that this continuous surveillance by the immune system may have deleterious effects in later life. The study presented in this paper examined immune responses from a cohort of donors and shows that these immune cells are effective at controlling the virus and can overcome the virus' lytic cycle immune evasion mechanisms.

The effect of hand-arm vibration syndrome on quality of life.

BACKGROUND: It is important to determine how hand-arm vibration syndrome (HAVS), a common occupational condition, affects quality of life (QOL). AIMS: To measure the physical (SF12-P) and mental (SF12-M) components of QOL in workers with HAVS, using the SF12 questionnaire, and to determine the effect of the vascular, sensorineural and musculoskeletal components of HAVS on QOL. METHODS: Subjects were recruited consecutively from workers with HAVS attending an occupational medicine clinic. They were assessed to determine the Stockholm vascular and sensorineural scale stages as well as an upper extremity pain score, measured by the Borg scale, as an indication of musculoskeletal problems associated with the use of vibrating tools. The SF12-P and SF12-M were both compared with Canadian population normal values after adjusting for age and sex. Multiple linear regression was used to determine the effect of the various HAVS components on SF12-P and SF12-M as well as the effects of age and carpal tunnel syndrome. RESULTS: One hundred and forty-one subjects were recruited and 139 (99%) agreed to participate, including 134 men and 5 women. The SF12-P and SF12-M scores were significantly below the Canadian population mean values (P < 0.001), indicating lower QOL. In the multiple regression analysis, the predictor with the largest partial R (2) value for both the SF12-P and SF12-M was the upper extremity pain score. CONCLUSIONS: Both the physical and the mental QOL in workers with HAVS were below Canadian population normal values and subjects' upper extremity pain score had the greatest effect on their QOL.

The disparity of priapulid, archaeopriapulid and palaeoscolecid worms in the light of new data.

Priapulids and their extinct relatives, the archaeopriapulids and palaeoscolecids, are vermiform, carnivorous ecdysozoans with an armoured, extensible proboscis. These worms were an important component of marine communities during the Palaeozoic, but were especially abundant and diverse in the Cambrian. Today, they comprise just seven genera in four families. Priapulids were among the first groups used to test hypotheses concerning the morphological disparity of Cambrian fossils relative to the extant fauna. A previous study sampled at the generic level, concluding that Cambrian genera embodied marginally less morphological diversity than their extant counterparts. Here, we sample predominantly at the species level and include numerous fossils and some extant forms described in the last fifteen years. Empirical morphospaces for priapulids, archaeopriapulids and palaeoscolecids are relatively insensitive to changes in the taxon or character sample: their overall form has altered little, despite the markedly improved sampling. Cambrian and post-Cambrian genera occupy adjacent rather than broadly overlapping regions of these spaces, and Cambrian species still show lower morphological disparity than their post-Cambrian counterparts. Crucially, the significance of this difference has increased with improved taxon sampling over research time. In contrast with empirical morphospaces, the phylogeny of priapulids, archaeopriapulids and palaeoscolecids derived from morphological characters is extremely sensitive to details of taxon sampling and the manner in which characters are weighted. However, the extant Priapulidae and Halicryptidae invariably resolve as sister families, with this entire clade subsequently being sister group to the Maccabeidae. In our most inclusive trees, the extant Tubiluchidae are separated from these other living taxa by a number of small, intervening fossil clades.

DASH work module in workers with hand-arm vibration syndrome.

BACKGROUND: The Disabilities of the Arm, Shoulder and Hand work module (DASH-W) questionnaire has not previously been described in relation to hand-arm vibration syndrome (HAVS). AIMS: To measure work-related disability in workers with HAVS using the DASH-W questionnaire and to determine how the various components of HAVS affect the DASH-W score. METHODS: Workers with HAVS from a variety of industries were assessed over a 2-year period at the occupational health clinic, St Michael's Hospital, Toronto. Subjects completed the DASH-W questionnaire and were assessed by an occupational physician to determine their Stockholm sensorineural and vascular stages and upper extremity pain score measured by the Borg scale, as an indication of musculoskeletal problems associated with HAVS. The average DASH-W score was compared with the average value for the US population. Multiple linear regression was used to determine the contribution of the various components of HAVS to the DASH-W score. RESULTS: There were 139 (134 men and 5 women) participants. The subjects with HAVS had a mean DASH-W score of 54.7 (95% CI: 50.3-59.1), which was considerably higher than the average for the US population (P < 0.001). Statistically significant HAVS variables in the multiple linear regression included the Stockholm sensorineural stage (P < 0.05) and the upper extremity pain score (P < 0.001) with the pain score having the highest partial R (2) value. CONCLUSIONS: Workers with HAVS reported significant upper extremity work-related disability as measured by the DASH-W questionnaire, and the upper extremity pain score made the largest contribution to the DASH-W scores in these subjects.

Morphological disparity in the cambrian.

An analysis of the range of morphology among arthropods demonstrates that disparity among living arthropods is similar to that in Cambrian arthropods. The range of morphological design resulting from the Cambrian "explosion" has been overestimated, reflecting a tendency to separate as "problematic" taxa that cannot be accommodated in the classification on the basis of the living biota. Problematic taxa are largely an artifact of an inadequate taxonomy. Special evolutionary processes may not be necessary to explain the early radiation of the metazoans.

Fossil ghost ranges are most common in some of the oldest and some of the youngest strata.

Biologists routinely compare inferences about the order of evolutionary branching (phylogeny) with the order in which groups appear in the fossil record (stratigraphy). Where they conflict, ghost ranges are inferred: intervals of geological time where a fossil lineage should exist, but for which there is no direct evidence. The presence of very numerous and/or extensive ghost ranges is often believed to imply spurious phylogenies or a misleadingly patchy fossil record, or both. It has usually been assumed that the frequency of ghost ranges should increase with the age of rocks. Previous studies measuring ghost ranges for whole trees in just a small number of temporal bins have found no significant increase with antiquity. This study uses a much higher resolution approach to investigate the gappiness implied by 1,000 animal and plant cladograms over 77 series and stages of the Phanerozoic. It demonstrates that ghost ranges are indeed relatively common in some of the oldest strata. Surprisingly, however, ghost ranges are also relatively common in some of the youngest, fossil-rich rocks. This pattern results from the interplay between several complex factors and is not a simple function of the completeness of the fossil record. The Early Palaeozoic record is likely to be less organismically and stratigraphically complete, and its fossils -- many of which are invertebrates-may be more difficult to analyse cladistically. The Late Cenozoic is subject to the pull of the Recent, but this accounts only partially for the increased gappiness in the younger strata.

The association between body size, prostate volume and prostate-specific antigen.

Increasing prostate volume contributes to urinary tract symptoms and may obscure prostate cancer detection. We investigated the association between obesity and prostate volume, prostate-specific antigen (PSA) and PSA density among 753 men referred for prostate biopsy. Among men with a negative biopsy, prostate volume significantly increased approximately 25% from the lowest to highest body mass index (BMI), waist or hip circumference or height categories. PSA was 0.7 ng/ml lower with a high waist-to-hip ratio. These associations were less consistent among subjects diagnosed with high-grade prostatic intraepithelial neoplasia or cancer. Our data suggest that obesity and height are independently associated with prostate volume..

LPS promotes a monocyte phenotype permissive for human cytomegalovirus immediate-early gene expression upon infection but not reactivation from latency.

Human cytomegalovirus (HCMV) infection of myeloid cells is closely linked with the differentiation status of the cell. Haematopoietic progenitors and CD14+ monocytes are usually non-permissive for lytic gene expression which can lead to the establishment of latent infections. In contrast, differentiation to macrophage or dendritic cell (DC) phenotypes promotes viral reactivation or renders them permissive for lytic infection. The observation that high doses of Lipopolysaccharide (LPS) drove rapid monocyte differentiation in mice led us to investigate the response of human monocytes to HCMV following LPS stimulation in vitro. Here we report that LPS triggers a monocyte phenotype permissiveness for lytic infection directly correlating with LPS concentration. In contrast, addition of LPS directly to latently infected monocytes was not sufficient to trigger viral reactivation which is likely linked with the failure of the monocytes to differentiate to a DC phenotype. Interestingly, we observe that this effect on lytic infection of monocytes is transient, appears to be dependent on COX-2 activation and does not result in a full productive infection. Thus LPS stimulated monocytes are partially permissive lytic gene expression but did not have long term impact on monocyte identity regarding their differentiation and susceptibility for the full lytic cycle of HCMV.

Transient activation of human cytomegalovirus lytic gene expression during latency allows cytotoxic T cell killing of latently infected cells.

Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle. We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation. In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells. This approach of transiently inducing viral lytic gene expression by HDAC inhibition, in otherwise latently infected cells, offers a window of opportunity to target and purge the latent myeloid cell reservoir by making these normally immunologically undetectable cells visible to pre-existing host immune responses to viral lytic antigens.

Do u smoke after txt? Results of a randomised trial of smoking cessation using mobile phone text messaging.

OBJECTIVES: To determine the effectiveness of a mobile phone text messaging smoking cessation programme. DESIGN: Randomised controlled trial SETTING: New Zealand PARTICIPANTS: 1705 smokers from throughout New Zealand who wanted to quit, were aged over 15 years, and owned a mobile phone were randomised to an intervention group that received regular, personalised text messages providing smoking cessation advice, support, and distraction, or to a control group. All participants received a free month of text messaging; starting for the intervention group on their quit day to assist with quitting, and starting for the control group at six months to encourage follow up. Follow up data were available for 1624 (95%) at six weeks and 1265 (74%) at six months. MAIN OUTCOME MEASURES: The main trial outcome was current non-smoking (that is, not smoking in the past week) six weeks after randomisation. Secondary outcomes included current non-smoking at 12 and 26 weeks. RESULTS: More participants had quit at six weeks in the intervention compared to the control group: 239 (28%) v 109 (13%), relative risk 2.20 (95% confidence interval 1.79 to 2.70), p < 0.0001. This treatment effect was consistent across subgroups defined by age, sex, income level, or geographic location (p homogeneity > 0.2). The relative risk estimates were similar in sensitivity analyses adjusting for missing data and salivary cotinine verification tests. Reported quit rates remained high at six months, but there was some uncertainty about between group differences because of incomplete follow up. CONCLUSIONS: This programme offers potential for a new way to help young smokers to quit, being affordable, personalised, age appropriate, and not location dependent. Future research should test these findings in different settings, and provide further assessment of long term quit rates.

Hypercalcemia associated with chronic lymphocytic leukemia.

A patient with chronic lymphocytic leukemia (CLL) is described in whom hypercalcemia occurred in association with elevation of the peripheral lymphocyte count and expansion of total tumor mass. Hypercalcemia was ameliorated with the institution of chemotherapy for the leukemic process and subsequent fall in WBC count and decrease in total tumor burden; hypercalcemia recurred with relapse of the leukemic process. The serum immunoreactive parathyroid hormone (iPTH) concentration, when measured, was inappropriately elevated for the degree of hypercalcemia. The hypercalcemia would appear to be a direct consequence of the leukemia, and possibly involved secretion of a parathyroid hormone-like polypeptide by the CLL cells. Although a possible role for either an osteoclast-activating substance or prostaglandins was not excluded, they would not account for the elevated serum iPTH levels observed.

Physiological properties of the luteinizing hormone pulse signal: impact of intensive and extended venous sampling paradigms on its characterization in healthy men and women.

The pulsatile nature of the gonadotropin signal is a critical determinant of physiological activation of the gonadal axis. Nonetheless, major uncertainties exist regarding the exact patterns of LH secretion that constitute normal physiological profiles in man. To assess possible bases for the discrepancies in the literature, we sampled blood at 5-min intervals for 24 h in eight normal men and eight normal women in the early follicular phase of the menstrual cycle. The constituent 5-, 10-, 15-, and 20-min immunoactive LH series and the consituent 6-, 12-, or 24-h sampling durations provided ranges of sampling intensities and durations for analysis of significant LH pulses. A technique for minimizing the influence of false positive immunoassay errors on peak detection was used to aid in estimating apparent true positive LH pulse frequency. Nonlinear curve fitting of the relationship between sampling intensity and apparent true positive LH pulse frequency revealed a stable pulse frequency estimate at intensive rates of venous sampling, with values of 19.5 +/- 1.9 (+/- SEM) pulses/24 h (periodicity, 73.8 +/- 6.5 min/pulse) in men and 20.6 +/- 3.6 pulses/24 h (periodicity, 70.0 +/- 10 min/pulse) in women. Further analyses indicated that sampling every 3.1 and 2.0 min for 24 h would be required to capture 90% of the LH pulses in men and women, respectively. Moreover, even with a 5-min sampling rate, the statistical counting errors of the LH pulse frequency estimates varied markedly with sampling duration; for example, in men sampled in 6-, 12-, and 24-hr sessions, the values were, respectively, 49%, 35%, and 24% of the observed pulse frequencies. Similar variations were found in women. Counting errors were 30-50% higher for conventional 20-min sampling rates than for 5-min sampling intervals. Measured interpulse intervals varied widely from 10-330 min (median, 60 min; n = 131 LH pulses) in men and from 10-340 min (median, 65 min; n = 125 LH pulses) in women. In addition, absolute LH pulse amplitudes varied from 1-28 mIU/ml (median, 4.1 mIU/ml) in men and from 1-24 mIU/ml (median, 3.6 mIU/ml) in women. These estimates were associated with a median number of points identified within each pulse of 6.0 in men and 4.0 in women. In summary, the present exhaustive sampling studies demonstrate for the first time stable estimates of physiological LH pulsations in normal men and women, and document broad ranges of normal LH pulse amplitudes and interpulse intervals.(ABSTRACT TRUNCATED AT 400 WORDS)

Rickets in adult cystic fibrosis with myopathy, pancreatic insufficiency and proximal renal tubular dysfunction.

Rickets is reported in a 19 year old white man with cystic fibrosis in whom pancreatic and hepatic involvement was advanced. There was evidence of secondary hyperparathyroidism with proximal renal tubular acidosis, aminoaciduria, phosphaturia and hypophosphatemia. Treatment with oral pancreatic and parenteral vitamin D supplements led to full recovery of the rachitic syndrome and the proximal renal tubular dysfunction.

Partial atrophy in prostate needle cores: another diagnostic pitfall for the surgical pathologist.

We have seen in consultation a variant of atrophy, which is frequently confused with well-differentiated adenocarcinoma of the prostate. We have designated this entity as partial atrophy to distinguish it from its more common counterpart of fully developed atrophy. Partial atrophy is defined as benign prostate glands with relatively scant cytoplasm, yet the glands are not fully atrophic in that they do not appear basophilic at low magnification. Fifty-one cases of partial atrophy were identified (4 from Johns Hopkins Hospital, 47 from consultation). Within the partial atrophy focus, irregular (crinkled) nuclei were frequent in 23.5% and occasionally present in 33.3% of cases. Visible nucleoli were frequent in 25.4% of cases. Basal cells were not identifiable in 27.4% of cases or were hard to identify in 35.3% of cases. No intraluminal crystalloids or blue-tinged mucinous secretion was identified in partial atrophy. Adenocarcinoma or glands suspicious for cancer were present in other cores in 15.6% of cases. More fully developed atrophy was present in simultaneously obtained needle cores in 35.3% of cases. In the cases in which regular atrophy was the only coexisting condition, it was present within one 10x field from the partial atrophy in 22.2%, farther than one 10x field from the partial atrophy in 11.1%, and present in the same gland as the partial atrophy in 66.7%. Partial atrophy may be confused with low-grade adenocarcinoma because of the focus of crowded glands, irregular nuclei, and visible nucleoli. Clues for recognizing partial atrophy include relatively scant cytoplasm, distinct crinkled nuclei, pale cytoplasm similar to adjacent, more recognizably benign glands, and association with more fully developed benign atrophy.

How often are diagnostic features missed with less extensive histologic sampling of prostate needle biopsy specimens?

The authors determined whether clinically relevant diagnostic information would be lost by examination of <3 levels per tissue core in prostate needle biopsy specimens. They evaluated 439 consecutive sextant biopsy specimens for the following three histopathologic features: presence of adenocarcinoma involving one core, Gleason pattern 4 in cases of grade 3 + 4 = 7 adenocarcinoma, and perineural invasion (PNI) by carcinoma. For all cases, 3 levels from each involved core were reviewed for the presence or absence of these three features. In 50 cases with adenocarcinoma involving only 1 core, diagnostic carcinoma was present on all 3 levels in 43 cores (86%). Carcinoma was present on only 2 levels in 3 cores (6%), present only on 1 level in 3 cores (6%), and present only on additional cut-downs, not on the original 3 levels in 1 core (2%). Among 32 cases, 51 cores were identified that contained Gleason grade 3 + 4 = 7 adenocarcinoma. In 41 cores (80%), pattern 4 was identified in all 3 levels. In 5 cores (10%), pattern 4 was identified on only 2 levels, and in another 5 cores (10%), pattern 4 was present on only 1 level. Among 36 cases, 69 tissue cores were identified that contained perineural invasion (PNI). In 54 cores (78%), PNI was present on all 3 levels. In 7 cores (10%), PNI was present on only 2 of 3 levels, while in 7 other cores (10%), PNI was present on only 1 of 3 levels. In 1 core (1.5%), PNI was noted only on additional cutdowns, not on the original 3 levels. We estimated that reducing the number of levels to 1 per core could result in the misdiagnosis of PNI, grading, or carcinoma in approximately 8-11% of cores with these features and could have changed the case diagnosis in 9 of 439 cases. If only 2 levels were reviewed, we predict misdiagnosis in 5% to 6% of cores with these features and a change in the case diagnosis in 5 of 439 cases. Misdiagnosis of clinically relevant features on prostate biopsy specimens can be minimized with histologic review of 3 levels per tissue core.

Aging and cholinergic responses in bovine trachealis muscle.

1. The relative potencies of muscarinic agonists on bovine tracheal smooth muscle were unchanged as a consequence of aging and were carbachol greater than oxotremorine greater than muscarine greater than pilocarpine greater than McNeil A-343. 2. During aging, the potencies of carbachol, oxotremorine, McNeil A-343 and pilocarpine, but not muscarine, were reduced. 3. Maximal induced tensions to all the agents studied were reduced as a consequence of age. 4. Irreversible antagonism with benzilylcholine mustard showed that agonist efficacy was significantly reduced during aging. 5. Estimated receptor occupancy at the EC50 was significantly greater in tracheal tissues from the mature versus immature cows for every agonist studied. 6. The dissociation constants for full agonists (carbachol, oxotremorine and methacholine) were decreased with maturation while the converse was observed with partial agonists (McNeil A-343, pilocarpine). 7. We conclude that there are significant changes in the properties and coupling of muscarinic receptors during aging. These changes may contribute to the reduced airway reactivity seen in vivo.