In utero antidepressant exposure not associated with ADHD in the offspring: A case control sibling design.

Recent studies have reported an association between antidepressant (AD) use during pregnancy and the risk to develop attention-deficit/hyperactivity disorder (ADHD) in the offspring. However, the association might be confounded by risk factors in the pregnant parent. To control for unmeasured factors between pregnancies carried by the same parent, we set up a case-control sibling study using the University of Groningen prescription database IADB.nl. Children receiving medication for ADHD (cases) before the age of 16 years were matched to siblings not receiving such medication (controls). Exposure was defined as at least two prescriptions for any AD during pregnancy, i.e., the period of 39 weeks before the birth date of the offspring. Secondary analyses were performed to assess the effects of the degree of exposure (the amount of Defined Daily Doses) and the type of AD exposed to. Univariate and multivariate logistic regression was used to estimate odds ratios (ORs) with corresponding 95% confidence intervals (CI). In total, 2,833 children (1,304 cases and 1,529 controls) were included in the analysis. Exposure rate to ADs among cases and controls was 2.2% and 2.4%, respectively. After adjusting for the birth date of the child (as a proxy for the date of pregnancy), age of the pregnant parent at birth, use of psychostimulants, opioids, and antiepileptic drugs by the pregnant parent in the 15 months before birth of the child, an adjusted OR of 1.11 (95% CI 0.67-1.83) was found for the risk of ADHD in the offspring when exposed in utero to ADs. This indicates no increased risk of ADHD in offspring following in utero exposure to ADs. The secondary analyses revealed no statistically significant associations either. The present study provides further evidence that an association between in utero AD exposure and ADHD in offspring might not exist. This perceived association may be caused (at least partially) by confounding by indication. The extent to which depression in the pregnant parent could cause mental disorders such as ADHD in offspring, and the mechanisms involved, should be investigated in further studies.

Subtyping psychological distress in the population: a semi-parametric network approach.

AIMS: The mechanisms underlying both depressive and anxiety disorders remain poorly understood. One of the reasons for this is the lack of a valid, evidence-based system to classify persons into specific subtypes based on their depressive and/or anxiety symptomatology. In order to do this without a priori assumptions, non-parametric statistical methods seem the optimal choice. Moreover, to define subtypes according to their symptom profiles and inter-relations between symptoms, network models may be very useful. This study aimed to evaluate the potential usefulness of this approach. METHODS: A large community sample from the Canadian general population (N = 254 443) was divided into data-driven clusters using non-parametric k-means clustering. Participants were clustered according to their (co)variation around the grand mean on each item of the Kessler Psychological Distress Scale (K10). Next, to evaluate cluster differences, semi-parametric network models were fitted in each cluster and node centrality indices and network density measures were compared. RESULTS: A five-cluster model was obtained from the cluster analyses. Network density varied across clusters, and was highest for the cluster of people with the lowest K10 severity ratings. In three cluster networks, depressive symptoms (e.g. feeling depressed, restless, hopeless) had the highest centrality. In the remaining two clusters, symptom networks were characterised by a higher prominence of somatic symptoms (e.g. restlessness, nervousness). CONCLUSION: Finding data-driven subtypes based on psychological distress using non-parametric methods can be a fruitful approach, yielding clusters of persons that differ in illness severity as well as in the structure and strengths of inter-symptom relationships.

Venetoclax, bendamustine, and rituximab in patients with relapsed or refractory NHL: a phase Ib dose-finding study.

Background: Venetoclax is a selective, potent inhibitor of the anti-apoptotic B-cell leukemia/lymphoma-2 protein approved for treatment of chronic lymphocytic leukemia. We conducted a dose-finding study of venetoclax in combination with bendamustine-rituximab (BR) in patients with relapsed/refractory non-Hodgkin's lymphoma (NHL). Patients and methods: BR was given for six cycles at standard doses. Intermittent and continuous oral venetoclax administration was explored at 50-1200 mg daily doses. Co-primary objectives included safety, pharmacokinetics (PKs), maximum-tolerated dose (MTD), and recommended phase II dose (RP2D); secondary objective was preliminary efficacy. Results: Sixty patients were enrolled: 32 with follicular lymphoma, 22 with diffuse large B-cell lymphoma, and 6 with marginal zone lymphoma. Nausea (70%), neutropenia (68%), diarrhea (55%), and thrombocytopenia (52%) were the most frequent adverse events (AEs). Most common grade 3/4 AEs were neutropenia (60%) and lymphopenia (38%). Serious AEs were reported in 24 patients; the most frequent were febrile neutropenia and disease progression (8% each). Five patients died from either disease progression (n = 4) or respiratory failure (n = 1). MTD was not reached; RP2D for venetoclax-BR combination was established as 800 mg daily continuously. Venetoclax PK exposure with and without BR was comparable. For all patients, overall response rate was 65%. Median duration of overall response, overall survival, and progression-free survival was 38.3 months [95% confidence interval (CI) 10.4-NR], not yet reached, and 10.7 months (95% CI 4.3-21.0), respectively. Conclusions: This study established the safety profile of venetoclax in combination with BR, and results demonstrated tolerability and preliminary efficacy of the combination. Additional follow-up is needed to better determine the future role of BR plus venetoclax in the treatment of relapsed/refractory B-cell NHL. Trial registered: Clinicaltrials.gov, NCT01594229.

International Working Group consensus response evaluation criteria in lymphoma (RECIL 2017).

In recent years, the number of approved and investigational agents that can be safely administered for the treatment of lymphoma patients for a prolonged period of time has substantially increased. Many of these novel agents are evaluated in early-phase clinical trials in patients with a wide range of malignancies, including solid tumors and lymphoma. Furthermore, with the advances in genome sequencing, new "basket" clinical trial designs have emerged that select patients based on the presence of specific genetic alterations across different types of solid tumors and lymphoma. The standard response criteria currently in use for lymphoma are the Lugano Criteria which are based on [18F]2-fluoro-2-deoxy-D-glucose positron emission tomography or bidimensional tumor measurements on computerized tomography scans. These differ from the RECIST criteria used in solid tumors, which use unidimensional measurements. The RECIL group hypothesized that single-dimension measurement could be used to assess response to therapy in lymphoma patients, producing results similar to the standard criteria. We tested this hypothesis by analyzing 47 828 imaging measurements from 2983 individual adult and pediatric lymphoma patients enrolled on 10 multicenter clinical trials and developed new lymphoma response criteria (RECIL 2017). We demonstrate that assessment of tumor burden in lymphoma clinical trials can use the sum of longest diameters of a maximum of three target lesions. Furthermore, we introduced a new provisional category of a minor response. We also clarified response assessment in patients receiving novel immune therapy and targeted agents that generate unique imaging situations.

Distinct gene signature revealed in white blood cells, CD4(+) and CD8(+) T cells in (NZBx NZW) F1 lupus mice after tolerization with anti-DNA Ig peptide.

Tolerizing mice polygenically predisposed to lupus-like disease (NZB/NZW F1 females) with a peptide mimicking anti-DNA IgG sequences containing MHC class I and class II T cell determinants (pConsensus, pCons) results in protection from full-blown disease attributable in part to the induction of CD4(+)CD25(+)Foxp3+ and CD8(+)Foxp3+ regulatory T cells. We compared 45 000 murine genes in total white blood cells (WBC), CD4(+) T cells, and CD8(+) T cells from splenocytes of (NZBxNZW) F1 lupus-prone mice tolerized with pCons vs untreated naïve mice and found two-fold or greater differential expression for 448 WBC, 174 CD4, and 60 CD8 genes. We identified differentially expressed genes that played roles in the immune response and apoptosis. Using real-time PCR, we validated differential expression of selected genes (IFI202B, Bcl2, Foxp3, Trp-53, CCR7 and IFNar1) in the CD8(+)T cell microarray and determined expression of selected highly upregulated genes in different immune cell subsets. We also determined Smads expression in different immune cell subsets, including CD4(+) T cells and CD8(+) T cells, to detect the effects of TGF-beta, known to be the major cytokine that accounts for the suppressive capacity of CD8(+) Treg in this system. Silencing of anti-apoptotic gene Bcl2 or interferon genes (IFI202b and IFNar1 in combination) in CD8(+) T cells from tolerized mice did not affect the expression of the other selected genes. However, silencing of Foxp3 reduced expression of Foxp3, Ifi202b and PD1-all of which are involved in the suppressive capacity of CD8(+) Treg in this model.

Psychological causes of non-compliance with electronically monitored occlusion therapy for amblyopia.

AIM: To analyse psychological causes for low compliance with occlusion therapy for amblyopia. METHOD: In a randomised trial, the effect of an educational programme on electronically measured compliance had been assessed. 149 families who participated in this trial completed a questionnaire based on the Protection Motivation Theory after 8 months of treatment. Families with compliance less than 20% of prescribed occlusion hours were interviewed to better understand their cause for non-compliance. RESULTS: Poor compliance was most strongly associated with a high degree of distress (p<0.001), followed by low perception of vulnerability (p = 0.014), increased stigma (p = 0.017) and logistical problems with treatment (p = 0.044). Of 44 families with electronically measured compliance less than 20%, 28 could be interviewed. The interviews confirmed that lack of knowledge, distress and logistical problems resulted in non-compliance. CONCLUSION: Poor parental knowledge, distress and difficulties implementing treatment seemed to be associated with non-compliance. For the same domains, the scores were more favourable for families who had received the educational programme than for those who had not.

Bortezomib in patients with relapsed or refractory mantle cell lymphoma: updated time-to-event analyses of the multicenter phase 2 PINNACLE study.

BACKGROUND: We previously reported results of the phase 2, multicenter PINNACLE study, which confirmed the substantial single-agent activity of bortezomib in patients with relapsed or refractory mantle cell lymphoma (MCL). MATERIALS AND METHODS: We report updated time-to-event data, in all patients and by response to treatment, after extended follow-up (median 26.4 months). RESULTS: Median time to progression (TTP) was 6.7 months. Median time to next therapy (TTNT) was 7.4 months. Median overall survival (OS) was 23.5 months. In responding patients, median TTP was 12.4 months, median duration of response (DOR) was 9.2 months, median TTNT was 14.3 months, and median OS was 35.4 months. Patients achieving complete response had heterogeneous disease characteristics; among these patients, median TTP and DOR were not reached, and median OS was 36.0 months. One-year survival rate was 69% overall and 91% in responding patients. Median OS from diagnosis was 61.1 months, after median follow-up of 63.7 months. Activity was seen in patients with refractory disease and patients relapsing following high-intensity treatment. Toxicity was generally manageable. CONCLUSIONS: Single-agent bortezomib is associated with lengthy responses and notable survival in patients with relapsed or refractory MCL, with considerable TTP and TTNT in responding patients, suggesting substantial clinical benefit.

Effects of dietary wheat bran on absorption and accumulation of PCBs in rats.

Polychlorinated biphenyls (PCBs) are toxic environmental contaminants, which tend to accumulate in the food chain. Since dietary intake is the most important exposure route, PCB body burden may be affected by taking proper dietary measures. In the present study, diets were supplemented with either wheat bran or its cellulose based placebo in order to study the effect of bran consumption on the absorption of dietary PCBs and the excretion of initially stored PCBs. During the period of PCB intake, faecal PCB excretion was elevated by consumption of wheat bran as compared to the placebo. Hence, apparent faecal PCB digestibilities as well as PCB retentions in the whole body were lower in the wheat bran consuming rats. After ending PCB consumption, dietary wheat bran had only a minor effect on faecal PCB output while accumulation in the body was not affected. When PCBs were consumed for a longer time, a small but significant reduction of apparent faecal PCB digestibility was found. However, PCB content in the body kept increasing while PCB retention as percent of intake remained almost constant. Furthermore, differences among individual PCB congeners in metabolic susceptibility and hydrophobic characteristics had an impact on their accumulation in the body.

Systolic and diastolic ventricular function in the normal and extra-embryonic venous clipped chicken embryo of stage 24: a pressure-volume loop assessment.

OBJECTIVES: Fluid mechanical forces affect cardiac development. In the chicken embryo, permanent obstruction of the right lateral vitelline vein by clipping reduces the mechanical load on the embryonic myocardium, which has been shown to induce a spectrum of outflow tract anomalies. Insight into the effects of this intervention on the mechanical function of the developing myocardium could contribute to a better understanding of the relationship between hemodynamics and cardiac morphogenesis. We aimed to explore the effects of clipping on intrinsic systolic and diastolic ventricular function at stage 24 in the chicken embryo METHODS: Cardiac pressure-volume relationships enable load-independent quantification of intrinsic ventricular systolic and diastolic properties. We determined ventricular function by pressure-volume loop analysis of in-ovo stage-24 chicken embryos (n = 15) 2 days after venous obstruction at 2.5 days of incubation (stage 17, venous clipped embryos). Control embryos (n = 15) were used for comparison. RESULTS: End-systolic volume was significantly higher in clipped embryos (0.36 +/- 0.02 microL vs. 0.29 +/- 0.02 microL, P = 0.002). End-systolic and end-diastolic pressure were also increased compared with control animals (2.93 +/- 0.07 mmHg vs. 2.70 +/- 0.08 mmHg, P = 0.036 and 1.15 +/- 0.06 mmHg vs. 0.82 +/- 0.05 mmHg, P < 0.001, respectively). No significant differences were demonstrated for other baseline hemodynamic parameters. Analysis of pressure-volume relationships showed a significantly lower end-systolic elastance in the clipped embryos (slope of end-systolic pressure-volume relationship: 2.91 +/- 0.24 mmHg/microL vs. 7.53 +/- 0.66 mmHg/microL, P < 0.005) indicating reduced contractility. Diastolic stiffness was significantly increased in the clipped embryos (slope of end-diastolic pressure-volume relationship: 1.54 +/- 0.21 vs. 0.60 +/- 0.08, P < 0.005), indicating reduced compliance. CONCLUSION: Venous obstruction apparently interferes with normal myocardial development, resulting in impaired intrinsic systolic and diastolic ventricular function. These changes in ventricular function may precede morphological derangements observed in later developmental stages.

Polychlorinated biphenyl distribution and faecal excretion in rats fed wheat bran.

Polychlorinated biphenyls (PCBs) are abundant and persistent environmental contaminants, which tend to accumulate through the food chain. Because of the toxic potential of these compounds, body burden should be kept as low as possible e.g. by taking dietary measures. In the present report, the effect of wheat bran consumption on absorption of dietary PCBs as well as on excretion of previously absorbed PCBs was investigated in rats. Moreover, the accumulation of 7 reference PCB congeners in liver and abdominal adipose tissue was studied. Faecal excretion of dietary PCBs was significantly higher in rats fed wheat bran compared to its placebo. As a result, apparent PCB digestibility was diminished, but not enough to significantly affect PCB accumulation in liver and abdominal adipose tissue. Furthermore, excretion of previously absorbed PCBs following switching of the rats to a control diet without added PCBs was enhanced by wheat bran fibre intake, although to a much lesser extent than excretion of PCBs originating directly from the diet. Consequently, stimulation of PCB clearance from liver and abdominal adipose tissue due to wheat bran consumption was not detectable. Although no preferential absorption of PCB congeners was observed, PCB patterns in tissues obviously differed from the dietary PCB pattern. This was mainly due to PCBs 52 and 101, which were metabolised in the body. Moreover, reduced levels of PCB 138 were found in liver, while PCB 28 and 138 were predominantly present in adipose tissue. The experiment also demonstrated that PCB redistribution from the liver to the adipose tissue occurs.

Digestibility, retention and incorporation of low-level dietary PCB contents in laying hens.

Polychlorinated biphenyls (PCBs) are persistent and hazardous environmental contaminants, which tend to bioaccumulate in the food chain. In the present report the long-term effect of low-level dietary PCB concentrations was studied on performance, egg quality, apparent PCB digestibility, apparent PCB retention and PCB accumulation in laying hens that were fed experimental diets for 41 weeks. The tested dietary concentrations of supplemented PCBs, based on the sum of seven reference congeners, were 0, 1.5 and 6 ng/g. PCB ingestion did not significantly affect performance or egg quality parameters. The PCB concentration in egg yolk reached a nearly constant level after approximately 40 and 70 days of consumption of the diets containing 1.5 and 6 ng PCBs/g, respectively. Apparent faecal PCB digestibility and apparent retention were not influenced by dietary levels of added fat varying between 1.5% and 4.5%, but were significantly higher in hens fed diets containing added PCBs. Moreover, apparent PCB digestibility and retention increased significantly with age. Among the seven individual PCB congeners, no systematically significant differences with regard to apparent faecal digestibility were observed throughout the experiment. Accumulation of PCBs in the fat fraction of egg yolk, abdominal adipose tissue and thigh and breast muscle greatly depended upon PCB intake, but never exceeded the maximally allowed concentration of 200 ng/g. As PCBs 52 and 101 were hardly found in egg yolks and hen tissues, it was concluded that both congeners were greatly metabolised. Comparison of relative contents of individual PCB congeners revealed that PCBs 118, 138 and 153 were preferentially incorporated in yolk and body tissues.

Accumulation and tissue distribution of selected polychlorinated biphenyl congeners in chickens.

During one to three consecutive periods of 2 weeks, broiler chickens (n = 108) received test dies to which different amount of PCBs (7 congeners) were added. The relationship between exposure time and accumulation of individual congeners in different chicken tissues, such as breast, thigh and abdominal fat tissue, was observed. In all tissues, the vast majority of the PCB accumulation occurred during the first 2 weeks of exposures. After that, PCB concentrations only increased in the abdominal fat tissue of the animals. The individual PCBs were distributed differently in the various tissues. While CBs 28, 118, 138, 153 and 180 accumulated in the chickens, CBs 52 and 101 were metabolized, but no methyl sulphone metabolites of these congeners could be detected. Our results provide information on the absorption, tissue distribution and biotransformation of the individual PCB congeners and confirm the structure-activity relationships for metabolism of PCBs in birds, which are different from those in fish or mammalian species.

Polychlorinated biphenyls in broiler diets: their digestibility and incorporation in body tissues.

The influence of dietary amounts of polychlorinated biphenyls (PCBs) was studied on performance, apparent PCB digestibility and PCB accumulation in broiler chickens that were maintained until 42 days of age. Dietary concentrations of supplemented PCBs, based on the sum of seven reference congeners, ranged from 0 to 12 ng/g, which was below the legal maximum of 200 ng PCBs/g fat in Belgian feeds. PCB ingestion did not significantly affect body weight and feed intake. Apparent PCB digestibility was not influenced by dietary levels of added fat varying between 4% and 8%, but was significantly higher in broilers fed diets containing added PCBs. Accumulation of PCBs in the fat fraction of abdominal adipose tissue and breast and thigh muscle greatly depended upon PCB intake. However, PCB contents in the various body fat fractions within the same animal differed, even within muscle tissues, indicating an unequal PCB distribution in body fats.

Hydrophobic core manipulations in ribonuclease T1.

Differential scanning calorimetry, urea denaturation, and X-ray crystallography were combined to study the structural and energetic consequences of refilling an engineered cavity in the hydrophobic core of RNase T1 with CH(3), SH, and OH groups. Three valines that cluster together in the major hydrophobic core of T1 were each replaced with Ala, Ser, Thr, and Cys. Compared to the wild-type protein, all these mutants reduce the thermodynamic stability of the enzyme considerably. The relative order of stability at all three positions is as follows: Val > Ala approximately equal to Thr > Ser. The effect of introducing a sulfhydryl group is more variable. Surprisingly, a Val --> Cys mutation in a hydrophobic environment can be as or even more destabilizing than a Val --> Ser mutation. Furthermore, our results reveal that the penalty for introducing an OH group into a hydrophobic cavity is roughly the same as the gain obtained from filling the cavity with a CH(3) group. The inverse equivalence of the behavior of hydroxyl and methyl groups seems to be crucial for the unique three-dimensional structure of the proteins. The importance of negative design elements in this context is highlighted.

Evidence for the utility of the Bm86 antigen from Boophilus microplus in vaccination against other tick species.

The Bm86 antigen, as originally identified in Boophilus microplus, is the basis of commercial tick vaccines against this tick species. The potential for using this antigen or homologues of the antigen in vaccination against other tick species has been assessed. We have conducted vaccine trials in cattle using the B. microplus-derived recombinant Bm86 vaccine (TickGARD) using pairs of vaccinated calves and control calves. These were infested with B. microplus and Boophilus decoloratus larvae simultaneously. For both species, the numbers of engorged female adult ticks, their weight and egg-laying capacity were all reduced, leading to a reduction in reproductive capacity of 74% for B. microplus and 70% for B. decoloratus. Hyalomma anatolicum anatolicum ticks were fed both as immatures as well as adults on vaccinated calves and non-vaccinated controls. There was an overall 50% reduction in the total weight of nymphs engorging on vaccinated calves, and a suggestion of a subsequent effect on feeding adults. For Hyalomma dromedarii there was a 95% reduction in the number of nymphs engorging and a further 55% reduction in weight of those ticks surviving. Rhipicephalus appendiculatus and Amblyomma variegatum ticks were fed simultaneously both as immatures and subsequently as adults. There was no evidence for a significant vaccination effect. Finally, the amino acid sequence of a Bm86 homologue found in H. a. anatolicum unequivocally demonstrated the conservation of this molecule in this species. Our strategy for the development of multivalent anti-tick vaccines is discussed in relation to these findings.

The contribution of metal ions to the conformational stability of ribonuclease T1: crystal versus solution.

In the crystalline state, ribonuclease T1 binds calcium ions at different lattice-dependent positions. In solution, its conformational stability is also remarkably increased in the presence of divalent metal ions. Combining urea unfolding studies and X-ray crystallography, we compared the presence of several metal ions at specific sites in the protein to their contribution to the overall stabilizing effect in solution. We constructed thermodynamic cycles involving particular metal ions and specific carboxylate functions. The resulting coupling energies indicate that some (but not all) metal ions found at lattice contacts in crystal structures may indeed significantly contribute to stability enhancement in the presence of metal ions in solution.

Altered apoptosis pathways in mantle cell lymphoma detected by oligonucleotide microarray.

An imbalance between cellular apoptosis and survival may be critical for the pathogenesis of lymphoma. Therefore, the gene expression pattern in lymph node preparations from patients with mantle cell lymphoma (MCL) was compared to the pattern in nonmalignant hyperplastic lymph nodes (HLs). Oligonucleotide microarray analysis was performed comparing 5 MCLs to 4 HLs using high-density microarrays. The expression data were analyzed using Genespring software. For confirmation, the expression of selected genes was analyzed by real-time polymerase chain reaction using the RNA extracted from 16 MCL and 12 HL samples. The focus was on 42 genes that were at least 3-fold down-regulated in MCL; in addition to the B-cell leukemia 2 (BCL2) system other apoptotic pathways were altered in MCL. The FAS-associated via death domain (FADD) gene that acts downstream of the FAS cascade as a key gene to induce apoptosis was more than 10-fold down-regulated in MCL. Furthermore, the death-associated protein 6 (DAXX) gene, the caspase 2 (CASP2) gene, and the RIPK1 domain containing adapter with death domain (RAIDD) gene, which are key genes in other proapoptotic pathways, were also decreased in the MCL samples. The suggestion is made that in addition to the known overexpression of cyclin D1, which drives entry into the cell cycle, disturbances of pathways associated with apoptosis contribute to the development of MCL. (Blood. 2001;98:787-794)

Parents' fear regarding fever and febrile seizures.

In order to improve the effectiveness of information, we studied parents' perceptions and knowledge about fever and febrile seizures. A questionnaire study was carried out among the parents whose children (n = 230) participated in a randomized controlled trial of ibuprofen to prevent recurrent febrile seizures. Of the 230 parents, 181 (79%) responded to the questionnaire. Of all parents, 45% were afraid or very afraid of fever, which was strongly associated with being afraid of recurrent febrile seizures. Parents of children with a non-West European background were more afraid. The consequences of parental fear included frequent temperature measurements (25% measured five times per day or more), sleeping in the same room (24%) and 13% remained awake at night. Witnessing a febrile seizure is a frightening experience for parents; a majority thought that febrile seizures were harmful, because they look dangerous. Forty-seven percent thought that their child was dying during the initial febrile seizure. On the other hand, reassuring information may be helpful: 21% mentioned it as their reason to consider febrile seizures not harmful. We conclude that parental fear of fever and febrile seizures is a major problem with several negative consequences for daily family life. Adequate provision of information may reduce parental fear. We suggest that information about fever and febrile seizures should be provided to all parents, preferably during their contact with the providers of preventive healthcare. The parents of children with a non-West European origin need extra attention.

Conserved water molecules in a large family of microbial ribonucleases.

We systematically analyzed the crystallographically determined water molecules of all known structures of RNase T1 and compared them to the ordered solvent in a large number of related microbial nucleases. To assess the crystallographers' impact on the interpretation of the solvent structure, we independently refined five validation structures from diffraction data derived from five isomorphous crystals of RNase T1. We also compared the positions of water molecules found in 11 published isomorphous RNase T1 inhibitor complexes. These data suggest that the positions of most of the waters located on the surface of a protein and that are well-determined in the experimental electron density maps are determined primarily by crystal packing forces. Water molecules with less well-defined electron density are in general unique to one or a small number of crystal structures. Only a small number of the well-defined waters are found to be independent of the crystal environment. These waters have a low accessible surface area and B-factor, and tend to be conserved in the crystal structures of a number of evolutionary related ribonucleases as well. A single water molecule is found conserved in all known microbial ribonucleases.