A de novo 2.3 kb structural variant in MITF explains a novel splashed white phenotype in a Thoroughbred family.

Splashed white in horses is characterized by extensive white patterning on the legs, face and abdomen and may be accompanied by deafness. To date, seven variants in microphthalmia-associated transcription factor (MITF) and two variants in Paired Box 3 (PAX3) have been identified to explain this phenotype. A splashed white Thoroughbred stallion, whose sire and dam were not patterned, was hypothesized to have a de novo variant leading to his white coat pattern. A whole-genome sequencing candidate gene approach identified two single nucleotide variants (SNVs) in SOX10, four SNVs in MITF and a 2.3 kb deletion in MITF with the alternative allele present in this stallion but absent in the other 18 horses analyzed. All six SNVs were annotated as modifiers and were not further considered. The deletion in MITF (NC_009159.3:g.21555811_21558139delinsAAAT) encompasses exon 9 encoding a part of the helix-loop-helix domain required for DNA binding. Sanger sequencing and parentage testing confirmed that this deletion was a de novo mutation of maternal origin. Consistent with the published nomenclature, we denote this likely causal variant as SW8. Genotyping three of this stallion's offspring identified SW8 only in the nearly all-white foal that was confirmed deaf by brainstem auditory evoked response testing. This foal was also a compound heterozygote for dominant white variants (W20/W22), but to date, W variants alone have not been connected to deafness. SW8 marks the fourth de novo MITF variant in horses reported to cause white patterning. The link between deafness and all MITF variants with and without other variants impacting melanocyte development and function needs to be further explored.

Mental health over nine months during the SARS-CoV2 pandemic: Representative cross-sectional survey in twelve waves between April and December 2020 in Austria.

BACKGROUND: There is accumulating evidence about detrimental impacts of the pandemic on population mental health, but knowledge on risk of groups specifically affected by the pandemic and variations across time is still limited. METHODS: We surveyed approximately n=1,000 Austrian residents in 12 waves between April and December 2020 (n=12,029). Outcomes were suicidal ideation (Beck Suicidal Ideation Scale), depressive symptoms (Patient Health Questionnaire-9), anxiety (Hospital Anxiety Depression Scale), and domestic violence. We also assessed the perceived burden from the pandemic. Demographic and Covid-19 specific occupational and morbidity-related variables were used to explain outcomes in multivariable regression analyses, controlling for well-established risk factors of mental ill-health, and variations over time were analyzed. RESULTS: Young age, working in healthcare or from home, and own Covid-19 illness were consistent risk factors controlling for a wide range of known mental health risk factors. Time patterns in the perceived burden from Covid-19-related measures were consistent with the time sequence of restrictions and relaxations of governmental measures. Depressive and anxiety symptoms were relatively stable over time, with some increase of depression during the second phase of lockdowns. Domestic violence increased immediately after both hard lockdowns. Suicidal ideation decreased slightly over time, with a low during the second hard lockdown. Mental health indicators for women and young people showed some deterioration over time, whereas those reporting own Covid-19 illness improved. LIMITATIONS: Data from before the pandemic were not available. CONCLUSIONS: Among mental health outcomes, increases in domestic violence and, to some smaller extent, depressive symptoms, appeared most closely related to the timing of hard lockdowns. Healthcare staff, individuals working from home, those with Covid-19, as well as young people and women are non-traditional risk groups who warrant heightened attention in prevention during and in the aftermath of the pandemic.

Effects of media stories featuring coping with suicidal crises on psychiatric patients: Randomized controlled trial.

BACKGROUND: Accumulating evidence suggests beneficial effects of media stories featuring individuals mastering their suicidal crises, but effects have not been assessed for psychiatric patients. METHODS: We randomized n = 172 adult psychiatric patients (n = 172, 97.1% inpatients) to read an educative article featuring a person mastering a suicidal crisis (n = 92) or an unrelated article (n = 80) in a single-blind randomized controlled trial. Questionnaire data were collected before (T1) and after exposure (T2) as well as 1 week later (study end-point, T3). The primary outcome was suicidal ideation as assessed with the Reasons for Living Inventory; secondary outcomes were help-seeking intentions, mood, hopelessness, and stigmatization. Differences between patients with affective versus other diagnoses were explored based on interaction tests. RESULTS: We found that patients with affective disorders (n = 99) experienced a small-sized reduction of suicidal ideation at 1-week follow up (mean difference to control group [MD] at T3 = -0.17 [95% CI -0.33, -0.03], d = -0.15), whereas patients with nonaffective diagnoses (n = 73) experienced a small-sized increase (T2: MD = 0.24 [95% CI 0.06, 0.42], d = 0.19). Intervention group participants further experienced a nonsustained increase of help-seeking intentions (T2: MD = 0.53 [95% CI 0.11, 0.95], d = 0.19) and a nonsustained deterioration of mood (T2: MD = -0.14 [95% CI -0.27, -0.02], d = -0.17). CONCLUSIONS: This study suggests that patients with affective disorders appear to benefit from media materials featuring mastery of suicidal crises. More research is needed to better understand which patient groups are at possible risk of unintended effects.

News media representations of women who kill their newly born children.

This paper presents a first quantitative analysis of language in media reports of neonaticide and a comparative examination of language use within the reports. One thousand twenty-seven Austrian print media reports from 2004 to 2014 were retrieved; after exclusion, 331 were analysed using the Linguistic Inquiry and Word Count (LIWC) software. After a preliminary analysis, a comparative analysis was carried out between reports on the Graz case and all other cases. The preliminary analysis revealed that a majority of media reports were related to one repeat neonaticide case (Graz) despite not being clinically different from other cases identified for the same period. The comparative linguistic analysis shows some statistically significant differences relating to the domains of emotional words (less words of anxiety, sadness) and family and in the category of insight and certainty (more words). The unexpected media attention on the Graz case and the ensuing verdict, which was in contradiction with the Austrian infanticide act, might have been influenced by the way language was used by journalists and the media. The authors suggest guidelines on sensitive media reporting are required.

Influence of rate of inclusion of microalgae on the sensory characteristics and fatty acid composition of cheese and performance of dairy cows.

Modification of milk and cheese fat to contain long-chain n-3 fatty acids (FA) by feeding microalgae (ALG) to dairy cows has the potential to improve human health, but the subsequent effect on the sensory attributes of dairy products is unclear. The objective was to determine the effect of feeding dairy cows different amounts of ALG that was rich in docosahexaenoic acid (DHA) on milk and cheese FA profile, cheese sensory attributes, and cow performance. Twenty Holstein dairy cows were randomly allocated to 1 of 4 dietary treatments in a 4 × 4 row and column design, with 4 periods of 28 d, with cheddar cheese production and animal performance measurements undertaken during the final 7 d of each period. Cows were fed a basal diet that was supplemented with ALG (Schizochytrium limancinum) at 4 rates: 0 (control, C), 50 (LA), 100 (MA), or 150 g (HA) of ALG per cow per day. We found that both milk and cheese fat content of DHA increased linearly with ALG feed rate and was 0.29 g/100 g FA higher in milk and cheese from cows fed HA compared with C. Supplementation with ALG linearly reduced the content of saturated FA and the ratio of n-6:n-3 FA in milk and cheese. Supplementation with ALG altered 20 out of the 32 sensory attributes, with a linear increase in cheese air holes, nutty flavor, and dry mouth aftertaste with ALG inclusion. Creaminess of cheese decreased with ALG inclusion rate and was positively correlated with saturated FA content. We also observed a quadratic effect on fruity odor, which was highest in cheese from cows fed HA and lowest in LA, and firmness and crumbliness texture, being highest in MA and lowest in HA. Supplementation with ALG had no effect on the dry matter intake, milk yield, or live weight change of the cows, with mean values of 23.1, 38.5, and 0.34 kg/d respectively, but milk fat content decreased linearly, and energy-corrected milk yield tended to decrease linearly with rate of ALG inclusion (mean values of 39.6, 38.4, 37.1, and 35.9 g/kg, and 41.3, 41.3, 40.5, and 39.4 kg/d for C, LA, MA, and HA, respectively). We conclude that feeding ALG to high-yielding dairy cows improved milk and cheese content of DHA and altered cheese taste but not cow performance, although milk fat content reduced as inclusion rate increased.

Increased Neuronal Differentiation of Neural Progenitor Cells Derived from Phosphovimentin-Deficient Mice.

Vimentin is an intermediate filament (also known as nanofilament) protein expressed in several cell types of the central nervous system, including astrocytes and neural stem/progenitor cells. Mutation of the vimentin serine sites that are phosphorylated during mitosis (VIM SA/SA ) leads to cytokinetic failures in fibroblasts and lens epithelial cells, resulting in chromosomal instability and increased expression of cell senescence markers. In this study, we investigated morphology, proliferative capacity, and motility of VIM SA/SA astrocytes, and their effect on the differentiation of neural stem/progenitor cells. VIM SA/SA astrocytes expressed less vimentin and more GFAP but showed a well-developed intermediate filament network, exhibited normal cell morphology, proliferation, and motility in an in vitro wound closing assay. Interestingly, we found a two- to fourfold increased neuronal differentiation of VIM SA/SA neurosphere cells, both in a standard 2D and in Bioactive3D cell culture systems, and determined that this effect was neurosphere cell autonomous and not dependent on cocultured astrocytes. Using BrdU in vivo labeling to assess neural stem/progenitor cell proliferation and differentiation in the hippocampus of adult mice, one of the two major adult neurogenic regions, we found a modest increase (by 8%) in the fraction of newly born and surviving neurons. Thus, mutation of the serine sites phosphorylated in vimentin during mitosis alters intermediate filament protein expression but has no effect on astrocyte morphology or proliferation, and leads to increased neuronal differentiation of neural progenitor cells.

Neural Progenitor Cells in Cerebral Cortex of Epilepsy Patients do not Originate from Astrocytes Expressing GLAST.

Adult neurogenesis in human brain is known to occur in the hippocampus, the subventricular zone, and the striatum. Neural progenitor cells (NPCs) were reported in the cortex of epilepsy patients; however, their identity is not known. Since astrocytes were proposed as the source of neural progenitors in both healthy and diseased brain, we tested the hypothesis that NPCs in the epileptic cortex originate from reactive, alternatively, de-differentiated astrocytes that express glutamate aspartate transporter (GLAST). We assessed the capacity to form neurospheres and the differentiation potential of cells dissociated from fresh cortical tissue from patients who underwent surgical treatment for pharmacologically intractable epilepsy. Neurospheres were generated from 57% of cases (8/14). Upon differentiation, the neurosphere cells gave rise to neurons, oligodendrocytes, and astrocytes. Sorting of dissociated cells showed that only cells negative for GLAST formed neurospheres. In conclusion, we show that cells with neural stem cell properties are present in brain cortex of epilepsy patients, and that these cells are not GLAST-positive astrocytes.

Predictors of psychological improvement on non-professional suicide message boards: content analysis.

BACKGROUND: Suicide message boards have been at the core of debates about negative influences of the Internet on suicidality. Nothing is currently known about communication styles that may help users to psychologically improve in these settings. METHOD: In all, 1182 archival threads with 20 499 individual postings from seven non-professional suicide message boards supporting an 'against-suicide', 'neutral' or 'pro-suicide' attitude were randomly selected and subject to content analysis. Initial needs of primary posters (i.e. individual who open a thread), their psychological improvement by the end of the thread, their responses received and indicators of suicidality were coded. Differences between 'pro-suicide', 'neutral' and 'against suicide' boards, and correlations between primary posters and respondents in terms of suicidality were assessed. Logistic regression was used to test associations with psychological improvement. RESULTS: 'Pro-suicide' boards (n = 4) differed from 'neutral' (n = 1) and 'against-suicide' (n = 2) boards in terms of communicated contents. Indicators of suicidality correlated moderately to strongly between primary posters and respondents on 'pro-suicide' message boards, but less on other boards. Several communicative strategies were associated with psychological improvement in primary posters, including the provision of constructive advice [adjusted odds ratio (aOR) 4.10, 95% confidence interval (CI) 2.40-7.03], active listening (aOR 1.60, 95% CI 1.12-2.27), sympathy towards the poster (aOR 2.22, 95% CI 1.68-2.95) and provision of alternatives to suicide (aOR 2.30, 95% CI 1.67-3.18). CONCLUSIONS: Respondents resemble primary posters with regard to suicidality in 'pro-suicide' boards, which may hinder psychological improvement. Still, opportunities to intervene in these settings using simple communication techniques exist and need to be taken and evaluated.

Bendamustine, etoposide and dexamethasone to mobilize peripheral blood hematopoietic stem cells for autologous transplantation in patients with multiple myeloma.

Chemotherapeutic agents without cross-resistance to prior therapies may enhance PBSC collection and improve patient outcomes by exacting a more potent direct antitumor effect before autologous stem cell transplant. Bendamustine has broad clinical activity in transplantable lymphoid malignancies, but concern remains over the potential adverse impact of this combined alkylator-nucleoside analog on stem cell mobilization. We performed a prospective, nonrandomized phase II study including 34 patients with multiple myeloma (MM) (n=34; International Staging System (ISS) stages I (35%), II (29%) and III (24%); not scored (13%)) to evaluate bendamustine's efficacy and safety as a stem cell mobilizing agent. Patients received bendamustine (120 mg/m2 IV days 1, 2), etoposide (200 mg/m2 IV days 1-3) and dexamethasone (40 mg PO days 1- 4) (bendamustine, etoposide and dexamethasone (BED)) followed by filgrastim (10 µg/kg/day SC; through collection). All patients (100%) successfully yielded stem cells (median of 21.60 × 106/kg of body weight; range 9.24-55.5 × 106/kg), and 88% required a single apheresis. Six nonhematologic serious adverse events were observed in 6 patients including: neutropenic fever (1, grade 3), bone pain (1, grade 3) and renal insufficiency (1, grade 1). In conclusion, BED safely and effectively mobilizes hematopoietic stem cells.

Maintenance rituximab after autologous stem cell transplantation in patients with mantle cell lymphoma.

BACKGROUND: High-dose therapy and autologous stem cell transplantation (ASCT) improves outcomes for patients with mantle cell lymphoma (MCL), but relapse ultimately occurs in most patients. Recently presented interim results from a phase III prospective trial suggest maintenance rituximab (MR) after ASCT for MCL improves progression-free survival (PFS). The maturation of these data and any benefit of MR on overall survival (OS) remain to be defined. PATIENTS AND METHODS: In this retrospective study, we examined a cohort of consecutive patients with MCL that underwent ASCT for MCL at our center and evaluated their outcomes according to whether they received MR after ASCT (n = 50) or did not (n = 107). MR was treated as a time-dependent covariate to account for variation in timing of its initiation. RESULTS: MR was associated with an improved PFS [hazard ratio (HR) 0.44; confidence interval (CI) (0.24-0.80), P = 0.007] and overall survival (OS; HR 0.46; CI 0.23-0.93, P = 0.03) following a multivariate adjustment for confounding factors with a median follow-up of ∼5 years. Grade 4 neutropenia was increased (34% versus 18%, P = 0.04) in the MR group, but no effect on the rate of mortality unrelated to relapse was observed. CONCLUSIONS: These data support that MR after ASCT for MCL confers a benefit in PFS and additionally suggest it may improve OS. General application of this strategy will require confirmation of benefit in prospective randomized trials.

Classification of subpopulations of cells within human primary brain tumors by single cell gene expression profiling.

Brain tumors are heterogeneous with respect to genetic and histological properties of cells within the tumor tissue. To study subpopulations of cells, we developed a protocol for obtaining viable single cells from freshly isolated human brain tissue for single cell gene expression profiling. We evaluated this technique for characterization of cell populations within brain tumor and tumor penumbra. Fresh tumor tissue was obtained from one astrocytoma grade IV and one oligodendroglioma grade III tumor as well as the tumor penumbra of the latter tumor. The tissue was dissociated into individual cells and the expression of 36 genes was assessed by reverse transcription quantitative PCR followed by data analysis. We show that tumor cells from both the astrocytoma grade IV and oligodendroglioma grade III tumor constituted cell subpopulations defined by their gene expression profiles. Some cells from the oligodendroglioma grade III tumor proper shared molecular characteristics with the cells from the penumbra of the same tumor suggesting that a subpopulation of cells within the oligodendroglioma grade III tumor consisted of normal brain cells. We conclude that subpopulations of tumor cells can be identified by using single cell gene expression profiling.

HB-EGF affects astrocyte morphology, proliferation, differentiation, and the expression of intermediate filament proteins.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a vascular-derived trophic factor, belongs to the epidermal growth factor (EGF) family of neuroprotective, hypoxia-inducible proteins released by astrocytes in CNS injuries. It was suggested that HB-EGF can replace fetal calf serum (FCS) in astrocyte cultures. We previously demonstrated that in contrast to standard 2D cell culture systems, Bioactive3D culture system, when used with FCS, minimizes the baseline activation of astrocytes and preserves their complex morphology. Here, we show that HB-EGF induced EGF receptor (EGFR) activation by Y1068 phosphorylation, Mapk/Erk pathway activation, and led to an increase in cell proliferation, more prominent in Bioactive3D than in 2D cultures. HB-EGF changed morphology of 2D and Bioactive3D cultured astrocytes toward a radial glia-like phenotype and induced the expression of intermediate filament and progenitor cell marker protein nestin. Glial fibrillary acidic protein (GFAP) and vimentin protein expression was unaffected. RT-qPCR analysis demonstrated that HB-EGF affected the expression of Notch signaling pathway genes, implying a role for the Notch signaling in HB-EGF-mediated astrocyte response. HB-EGF can be used as a FCS replacement for astrocyte expansion and in vitro experimentation both in 2D and Bioactive3D culture systems; however, caution should be exercised since it appears to induce partial de-differentiation of astrocytes.

A novel method for three-dimensional culture of central nervous system neurons.

Neuronal signal transduction and communication in vivo is based on highly complex and dynamic networks among neurons expanding in a three-dimensional (3D) manner. Studies of cell-cell communication, synaptogenesis, and neural network plasticity constitute major research areas for understanding the involvement of neurons in neurodegenerative diseases, such as Huntington's, Alzheimer's, and Parkinson's disease, and in regenerative neural plasticity responses in situations, such as neurotrauma or stroke. Various cell culture systems constitute important experimental platforms to study neuronal functions in health and disease. A major downside of the existing cell culture systems is that the alienating planar cell environment leads to aberrant cell-cell contacts and network formation and increased reactivity of cell culture-contaminating glial cells. To mimic a suitable 3D environment for the growth and investigation of neuronal networks in vitro has posed an insurmountable challenge. Here, we report the development of a novel electrospun, polyurethane nanofiber-based 3D cell culture system for the in vitro support of neuronal networks, in which neurons can grow freely in all directions and form network structures more complex than any culture system has so far been able to support. In this 3D system, neurons extend processes from their cell bodies as a function of the nanofiber diameter. The nanofiber scaffold also minimizes the reactive state of contaminating glial cells.

Bioactive 3D cell culture system minimizes cellular stress and maintains the in vivo-like morphological complexity of astroglial cells.

We tested the hypothesis that astrocytes grown in a suitable three-dimensional (3D) cell culture system exhibit morphological and biochemical features of in vivo astrocytes that are otherwise lost upon transfer from the in vivo to a two-dimensional (2D) culture environment. First, we report development of a novel bioactively coated nanofiber-based 3D culture system (Bioactive3D) that supports cultures of primary mouse astrocytes. Second, we show that Bioactive3D culture system maintains the in vivo-like morphological complexity of cultured cells, allows movement of astrocyte filopodia in a way that resembles the in vivo situation, and also minimizes the cellular stress, an inherent feature of standard 2D cell culture systems. Third, we demonstrate that the expression of gap junctions is reduced in astrocytes cultured in a 3D system that supports well-organized cell-cell communication, in contrast to the enforced planar tiling of cells in a standard 2D system. Finally, we show that astrocytes cultured in the Bioactive3D system do not show the undesired baseline activation but are fully responsive to activation-inducing stimuli. Thus, astrocytes cultured in the Bioactive3D appear to more closely resemble astrocytes in vivo and represent a superior in vitro system for assessing (patho)physiological and pharmacological responses of these cells and potentially also in co-cultures of astrocytes and other cell types.

t-PA-specific modulation of a human blood-brain barrier model involves plasmin-mediated activation of the Rho kinase pathway in astrocytes.

Tissue-type plasminogen activator (t-PA) can modulate permeability of the neurovascular unit and exacerbate injury in ischemic stroke. We examined the effects of t-PA using in vitro models of the blood-brain barrier. t-PA caused a concentration-dependent increase in permeability. This effect was dependent on plasmin formation and potentiated in the presence of plasminogen. An inactive t-PA variant inhibited the t-PA-mediated increase in permeability, whereas blockade of low-density lipoprotein receptors or exposed lysine residues resulted in similar inhibition, implying a role for both a t-PA receptor, most likely a low-density lipoprotein receptor, and a plasminogen receptor. This effect was selective to t-PA and its close derivative tenecteplase. The truncated t-PA variant reteplase had a minor effect on permeability, whereas urokinase and desmoteplase were ineffective. t-PA also induced marked shape changes in both brain endothelial cells and astrocytes. Changes in astrocyte morphology coincided with increased F-actin staining intensity, larger focal adhesion size, and elevated levels of phosphorylated myosin. Inhibition of Rho kinase blocked these changes and reduced t-PA/plasminogen-mediated increase in permeability. Hence plasmin, generated on the cell surface selectively by t-PA, modulates the astrocytic cytoskeleton, leading to an increase in blood-brain barrier permeability. Blockade of the Rho/Rho kinase pathway may have beneficial consequences during thrombolytic therapy.

Species differences in reactivity of mouse and rat astrocytes in vitro.

Reactive astrogliosis constitutes a major obstacle to neuronal regeneration and is characterized by rearrangement and upregulation of expression of cytoskeletal proteins, increased proliferation and hypertrophy. Many approaches have been attempted to mimic astrogliosis by inducing reactive astrocytes in vitro. Such research is usually performed using astrocytes derived from Mus musculus or Rattus norvegicus, and results compared between species on the assumption that these cells behave equivalently. Therefore, we compared reactivity between mouse and rat astrocytes in scratch wound assays to gain further insight into how comparable these cell culture models are. Proliferation and migration, as well as expression of the cytoskeletal proteins glial fibrillary acidic protein (GFAP) and vimentin, were compared by immunocytochemistry and immunoblot. Further, we investigated migration of proliferating cells by 5-ethynyl-2'-deoxyuridine staining. Substantial differences in GFAP expression and proliferation between astrocytes of the two species were found: rat astrocytes showed different cytoskeletal morphology, expressed significantly more GFAP and vimentin of different molecular size and were more proliferative than comparable mouse astrocytes. Our results suggest that rat and mouse astrocytes may respond differently to various reactivity-triggering stimuli, which needs to be considered when general conclusions are drawn regarding effects of factors regulating astrocyte reactivity.

Eph receptor tyrosine kinases regulate astrocyte cytoskeletal rearrangement and focal adhesion formation.

EphA4 null mice have impaired astrocytic gliosis following spinal cord injury. This may be because of altered cytoskeletal regulation and is examined herein using cultured astrocytes from wildtype and EphA4 null mice. Under basal conditions EphA4 null astrocytes appeared relatively normal but following stimuli resulting in cytoskeletal rearrangement, EphA4 null cells responded more slowly. When F-actin stress fibers were collapsed using the Rho kinase inhibitor HA1077, fewer EphA4 null cells showed stress fiber collapse in response to HA1077 and recovered stress fibers more slowly following HA1077 removal. EphA4 null astrocytes were less adherent and had smaller focal adhesions, while activation of Eph receptors with ephrin-A5-Fc increased the numbers of focal adhesions in both wildtype and knockout astrocytes following serum starvation. Using scratch wound assays, EphA4 null astrocytes invading the scratch showed impaired glial fibrillary acidic protein expression, particularly in proliferative cells. Astrocytes did not express Ephexin, a major Eph-interacting Rho guanine exchange factor, but they expressed Vav proteins, with lower levels of phospho-Vav in EphA4 null compared to wildtype astrocytes. This may contribute to the slower cytoskeletal responses generally observed in the EphA4 null astrocytes. Eph receptor signaling therefore regulates astrocyte reactivity through modulation of cytoskeletal responses.

CRS1 is a novel group II intron splicing factor that was derived from a domain of ancient origin.

Protein-dependent group II intron splicing provides a forum for exploring the roles of proteins in facilitating RNA-catalyzed reactions. The maize nuclear gene crs1 is required for the splicing of the group II intron in the chloroplast atpF gene. Here we report the molecular cloning of the crs1 gene and an initial biochemical characterization of its gene product. Several observations support the notion that CRS1 is a bona fide group II intron splicing factor. First, CRS1 is found in a ribonucleoprotein complex in the chloroplast, and cofractionation data provide evidence that this complex includes atpF intron RNA. Second, CRS1 is highly basic and includes a repeated domain with features suggestive of a novel RNA-binding domain. This domain is related to a conserved free-standing open reading frame of unknown function found in both the eubacteria and archaea. crs1 is the founding member of a gene family in plants that was derived by duplication and divergence of this primitive gene. In addition to its previously established role in atpF intron splicing, new genetic data implicate crs1 in chloroplast translation. The chloroplast splicing and translation functions of crs1 may be mediated by the distinct protein products of two crs1 mRNA forms that result from alternative splicing of the crs1 pre-mRNA.