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Our recent Communication suggested that ammonia in aqueous solution may preferentially destabilize large cages in methane clathrate hydrates.

In this Account, we highlight recent work in the developing field of mineralogy of Saturn's moon Titan, focusing on binary co-crystals of small organic molecules. Titan has a massive inventory of organic molecules on its surface that are formed via photochemistry in the atmosphere and likely processing on the surface as well. Physical processes both in the atmosphere and on the surface can lead to molecules interacting at cryogenic temperatures. Recent laboratory work has demonstrated that co-crystals between two or more molecules can form under these conditions. In the organic-rich environment of Titan, such co-crystals are naturally occurring minerals and a critical area of research to understand the physical, chemical, and possibly even biological and prebiotic processes occurring in this alien world.With a future NASA mission, Dragonfly, slated to land on Titan in the next decade, much work is needed to understand organic mineralogy in order to properly interpret the data from this and past Titan missions, such as Cassini-Huygens. By cataloging Titan minerals and their properties, we can begin to connect these behaviors to large-scale surface features observed on Titan (labyrinth terrain, lake evaporites, karst, dunes, etc.), and possible processes leading to their formation (erosion, deposition, etc.). To date, seven co-crystals (aside from clathrates and hydrates) have been experimentally reported to form under Titan-relevant conditions, with an eighth predicted by theoretical modeling. This Account will summarize the formation and properties of these cryominerals and discuss the implications for surface processes on Titan. Enhanced thermal expansion and decreased crystal size, for example, may lead to fracturing and/or more rapid erosion of co-crystal-based deposits; density changes upon co-crystal formation may also play a role in organic diagenesis and metamorphism on Titan. Some cryominerals with stability only under certain conditions may preserve the evidence of Titan's history, such as cryovolcanic activity, ethane fluvial/pluvial exposure, and outgassing of CO2 from the interior of the moon.In this Account, we will also highlight areas of future work, such as the characterization of pure molecular solids and the search for ternary (and more complex) co-crystals. We note that on Titan, organic chemistry dominates, which gives a unique opportunity for chemists to play an even more significant role in planetary science discoveries and likewise in discoveries motivated by planetary science to inform fundamental organic and physical chemistry research.

Titan, the largest satellite of Saturn, possesses a complex photochemical cycle producing a broad inventory of organic molecules in its thick atmosphere and on its surface. Two of the most common m...

Titan, Saturn’s largest satellite, is the only icy moon with a dense atmosphere. This atmosphere is composed mainly of N2. Methane, the second most abundant constituent, would be depleted in only 3...

A simple system is described for the introduction of gases into standard X-ray diffraction capillaries mounted in situ in the X-ray beam of laboratory X-ray diffraction instruments. This system retains many of the advantages of the standard Norby cell, but does not require custom machining and has less stringent space restrictions. The system has been used to study the crystallization and interaction of volatile organics at cryogenic temperatures, but gas–solid interactions could also be studied at elevated temperatures using this approach.

BackgroundCoformulated sodium phenylbutyrate/taurursodiol (PB/TURSO) was shown to prolong survival and slow functional decline in amyotrophic lateral sclerosis (ALS).ObjectiveDetermine whether PB/TURSO prolonged tracheostomy/ventilation-free survival and/or reduced first hospitalisation in participants with ALS in the CENTAUR trial.MethodsAdults with El Escorial Definite ALS ≤18 months from symptom onset were randomised to PB/ TURSO or placebo for 6 months. Those completing randomised treatment could enrol in an open-label extension (OLE) phase and receive PB/TURSO for ≤30 months. Times to the following individual or combined key events were compared in the originally randomised treatment groups over a period spanning trial start through July 2020 (longest postrandomisation follow-up, 35 months): death, tracheostomy, permanent assisted ventilation (PAV) and first hospitalisation.ResultsRisk of any key event was 47% lower in those originally randomised to PB/TURSO (n=87) versus placebo (n=48, 71% of whom received delayed-start PB/TURSO in the OLE phase) (HR=0.53; 95% CI 0.35 to 0.81; p=0.003). Risks of death or tracheostomy/PAV (HR=0.51; 95% CI 0.32 to 0.84; p=0.007) and first hospitalisation (HR=0.56; 95% CI 0.34 to 0.95; p=0.03) were also decreased in those originally randomised to PB/TURSO.ConclusionsEarly PB/TURSO prolonged tracheostomy/PAV-free survival and delayed first hospitalisation in ALS.Trial registration numberNCT03127514;NCT03488524.

Titan hosts a complex chemical engine producing a rich inventory of organic molecules in its thick atmosphere and on its surface. Some of these organics may be deposited in the liquid hydrocarbon lakes in the polar regions and form evaporite features when the lakes dry out as part of Titan’s methane/ethane cycle that is analogous to Earth’s hydrologic cycle. Modeling suggests that acetylene and butane would be the main components of such evaporite deposits. We have previously demonstrated that some organic molecules (such as benzene and ethane) readily form co-crystals in Titan-relevant conditions. We report here Raman spectroscopic evidence for a new co-crystal between acetylene and butane, which could be the most common organic co-crystal discovered so far of direct relevance to Titan’s surface. Intermolecular interactions such as those in the acetylene-butane co-crystal could modify the kinetics and equilibria of various processes (dissolution, reprecipitation, etc.) and therefore may play a key role i...

The global ocean believed to exist beneath Europa's thick ice shell is often cited as one of the most likely places in the solar system to find evidence of extraterrestrial habitable environments or even extant life. However, given the technical challenges and the costs associated with outer solar system exploration, direct analysis of the ocean appears unlikely in the foreseeable future. Therefore, constraints on the chemical composition of the ocean will need to be inferred, for example, from analysis of the surface ice, either via remote sensing or landed surface missions. In this paper, we combine our recent body of work studying the chemistry of frozen putative Europa ocean brines with new experiments to develop predictions of the sequence by which the hydrated minerals form when a four-ionic component ocean (Na+, Cl−, Mg2+, and SO42−) freezes as a function of relative ionic concentrations and pH. This in turn provides a means to begin linking observed surface chemistry and the chemical environment of the subsurface ocean as well as insight into endogenic versus exogenic origin of detected species.

Prominent bright spots containing natrite (Na2CO3) and smaller amounts of NH4Cl or NH4HCO3 observed in Ceres' Occator crater have been proposed to originate from an internal reservoir, where subsurface brines freeze upon reaching the surface. Our group has previously examined the freezing of putative subsurface brines containing ammonium, sodium, carbonate, and chloride ions at a cooling rate of 30 K/min (Vu et al., 2017), and found support for this hypothesis. The present study investigates this further by examining the formation of minerals on Ceres’ surface upon kinetic vs thermodynamic freezing. Using Infrared and Raman spectroscopies, flash frozen brines were found to form predominantly ammonium chloride and ammonium bicarbonate, even in sodium-dominated solutions. In contrast, hydrohalite (NaCl·2H2O) only formed when sodium and/or chloride were present in excess in the brine solutions. Additionally, the effect of vacuum exposure on these frozen brines was further analyzed to simulate the surface conditions of Ceres. It was observed that the vacuum exposure did not affect speciation, although it did clarify spectral features and confirm the presence of the aforementioned species. Our results provide evidence that (i) ammonium-bearing materials are kinetically-favored products under fast-freezing conditions with these brine mixtures, and (ii) the bright spots on Ceres's surface likely originate from subsurface liquids, mobilized either by a cryovolcanic process or by the Occator-forming impact, and not by direct excavation.

Data returned from Martian missions have revealed a wide diversity of surface mineralogies, including in geological structures interpreted to be sedimentary or altered by liquid water. These terrains are of great interest because of their potential to document the environment at a time when life may have appeared. Intriguingly, Martian sedimentary rocks show distinctly low thermal inertia values (i.e. 300 - 700 Jm−2K−1s−1/2, indicative of a combination of low thermal conductivity, specific heat capacity, and density). These low values are difficult to reconcile with their competent bedrock morphologies, whereas hundreds of bedrock occurrences, interpreted as volcanic in origin, have been mapped globally and display thermal inertia values > 1200 Jm−2K−1s−1/2. Bedrock thermal inertia values are generally assumed to be driven by their bulk thermal conductivity, which in turn is controlled by their micro- and macro-physical properties (i.e., degree and style of cementation in the case of detritic rocks, horizontal fractures and layering, etc.), and not by their density (well-known from terrestrial analog measurements, and with modest variability) or specific heat capacity (generally uncharacterized for non-basaltic materials below room temperature). In this paper, we demonstrate that specific heat capacity cannot be a potential cause for the differential thermophysical behavior between magmatic and sedimentary rocks through a series of experimental Cp(T) measurements at 100–350 K using differential scanning calorimetry. The results on 20 Martian-relevant minerals investigated in this work indicate that these materials exhibit very similar specific heats, ranging from 0.3–0.7 Jg−1K−1 at 100 K to 0.6–1.7 Jg−1K−1 at 350 K. When used in a Martian thermal model, this range of Cp values translate to very small surface temperature differences, indicating that uncertainty in composition (and its effect on the specific heat) is not a noticeable source of thermal inertia variability for indurated units on Mars. We therefore conclude that the low thermal inertia value of sedimentary rocks compared to magmatic/volcanic rocks is likely due to their low apparent bulk conductivity, which bears information on their internal physical structure. Future work combining the analysis of thermal observations acquired at various local times and seasons will help further characterize this heterogeneity.

Technologies are being developed to enable a robotic spacecraft to directly access Europa’s ocean by penetrating its kilometers-thick ice crust. Until such time comes, however, our ability to characterize it will be limited to observations of its expression on the surface. Further, the ability to place meaningful constraints on the composition of the source brines will depend on our understanding of the chemistry associated with their freezing and emplacement. Among the body of research focused on improving this understanding, recent laboratory studies have shown that Mg-bearing salts, previously proposed as constituents of Europa’s surface, have a propensity to form vitreous hydrates when flash frozen from their parent brines. The possibility that such vitreous species could form on Europa is important as their presence will be a confounding factor in their detection and identification of the underlying salt hydrates. Further, since glass formation can protect biological molecules and even microorganisms from damage caused by crystallization, they may prove to be important targets of astrobiological interest. This work explores the conditions under which vitreous salt hydrates form in order to better assess their potential for forming on Europa. Specifically, solutions of Mg- and Na-bearing salts were frozen over a range of freezing rates and interrogated with Raman spectroscopy to determine whether vitreous or crystalline species (including hydration state) formed. These results show that vitreous salt hydrates can form at freezing rates as low as ∼10 K minute−1, thus expanding their potential for occurrence on Europa beyond extreme flash-freezing scenarios such as plume deposition.

The incorporation of ammonia inside methane clathrate hydrate is of great interest to the hydrate chemistry community. We investigated the phase behavior of methane clathrate formed from aqueous ammonia solution. Ammonia's presence decreases methane occupancy in the large cages, without definitive Raman spectroscopic evidence for its incorporation inside the structure.

Titan, the largest moon of Saturn, likely supports a rich organic mineralogy that we are only beginning to understand. Photochemistry in the upper atmosphere generates a complex array of organic molecules from the simple precursors N2 and CH4. These organics continue to react and combine, forming aerosol layers and ultimately depositing on the surface. Organics are transported via pluvial (rain) and fluvial (rivers/flooding) processes into the methane-based hydrocarbon lakes, where evaporation of volatile liquids can create evaporite deposits of remnant dissolved molecules. Within such deposits, chemical and physical processes may be occurring even at low temperatures. We have demonstrated in previous work that benzene and ethane rapidly form a unique and stable co-crystal at Titan surface temperatures (90–95 K), akin to a salt on Earth, where the weak van der Waals interactions in the benzene-ethane co-crystal are analogous to the ionic bonds in a salt. Here, we report the formation of a second co-crysta...

Titan’s icy shell is expected to contain predominantly methane clathrate hydrates, water ice Ih, and possibly ammonia hydrates, beneath a cover of diverse organics formed via atmospheric photochemistry. The dissociation of clathrate hydrates has long been inferred as a potential replenishment mechanism for atmospheric methane; however, pure methane clathrates would be stable all the way to the surface. The melting of ammonia hydrates and subsequent interaction with methane clathrates could favor the dissociation of clathrates at much lower temperatures, due to the strong antifreeze effect of ammonia. To better understand the phase behavior of clathrate hydrates in the presence of ammonia, we have developed phase diagrams for the ternary system water–ammonia–tetrahydrofuran at 1 bar and in the temperature range 77–280 K via differential scanning calorimetry and Raman spectroscopy. We have been able to determine how ammonia promotes the start of a partial dissociation of THF–clathrates at temperatures far c...

According to clues left by the Cassini mission, Titan, one of the two Solar System bodies with a hydrologic cycle, may harbor liquid hydrocarbon-based analogs of our terrestrial aquifers, referred to as "alkanofers". On the Earth, petroleum and natural gas reservoirs show a vertical gradient in chemical composition, established over geological timescales. In this work, we aim to investigate the conditions under which Titan's processes could lead to similar situations. We built numerical models including barodiffusion and thermodiffusion (Soret's effect) in N_2+CH_4+C_2H_6 liquid mixtures, which are relevant for Titan's possible alkanofers. Our main assumption is the existence of reservoirs of liquids trapped in a porous matrix with low permeability. Due to the small size of the molecule, nitrogen seems to be more sensitive to gravity than ethane, even if the latter has a slightly larger mass. This behavior, noticed for an isothermal crust, is reinforced by the presence of a geothermal gradient. Vertical composition gradients, formed over timescales of between a fraction of a mega-year to several tens of mega-years, are not influenced by molecular diffusion coefficients. We find that ethane does not accumulate at the bottom of the alkanofers under diffusion, leaving the question of why ethane is not observed on Titan's surface unresolved. If the alkanofer liquid was in contact with water-ice, we checked that N_2 did not, in general, impede the clathration of C_2H_6, except in some layers. Interestingly, we found that noble gases could easily accumulate at the bottom of an alkanofer., Molecular dynamics simulations data available at: https://zenodo.org/record/4975047 (DOI: 10.5281/zenodo.4975047), PC-SAFT Fortran 2008 implementation can be downloaded from: https://github.com/dcordiercnrs/pcsaft-titan and also https://zenodo.org/record/5085305 (DOI: 10.5281/zenodo.5085305), paper in press in A&A

Observations of bright deposits in Ceres’ Occator crater have revealed large quantities of natrite (Na 2 CO 3 ) and smaller amounts of NH 4 Cl or NH 4 HCO 3 . These materials have been suggested to originate from the crystallization of brines that reached the surface from below. Here we report a systematic study of the chemistry of frozen brines containing sodium, ammonium, chloride, and carbonate ions via micro-Raman spectroscopy. Natrite and hydrohalite (NaCl·2H 2 O) are found to form preferentially, even in ammonium-dominated solutions. Additionally, NH 4 Cl could only form when NH 4 + or Cl - (or both) are present in excess in the brine solutions. The detection of NH 4 Cl on Ceres’ surface may thus imply an early subsurface reservoir rich in ammonium and/or chloride, placing an important constraint on modeling of the liquid composition.

Importance Many patients with generalized myasthenia gravis (gMG) have substantial clinical disability, persistent disease burden, and adverse effects attributable to chronic immunosuppression. Therefore, there is a significant need for targeted, well-tolerated therapies with the potential to improve disease control and enhance quality of life. Objective To evaluate the clinical effects of zilucoplan, a subcutaneously (SC) self-administered macrocyclic peptide inhibitor of complement component 5, in a broad population of patients with moderate to severe gMG. Design, Setting, and Participants This randomized, double-blind, placebo-controlled phase 2 clinical trial at 25 study sites across North America recruited participants between December 2017 and August 2018. Fifty-seven patients were screened, of whom 12 did not meet inclusion criteria and 1 was lost to follow-up after randomization but before receiving study drug, resulting in a total of 44 acetylcholine receptor autoantibody (AChR-Ab)–positive patients with gMG with baseline Quantitative Myasthenia Gravis (QMG) scores of at least 12, regardless of treatment history. Interventions Patients were randomized 1:1:1 to a daily SC self-injection of placebo, 0.1-mg/kg zilucoplan, or 0.3-mg/kg zilucoplan for 12 weeks. Main Outcomes and Measures The primary and key secondary end points were the change from baseline to week 12 in QMG and MG Activities of Daily Living scores, respectively. Significance testing was prespecified at a 1-sided α of .10. Safety and tolerability were also assessed. Results The study of 44 patients was well balanced across the 3 treatment arms with respect to key demographic and disease-specific variables. The mean age of patients across all 3 treatment groups ranged from 45.5 to 54.6 years and most patients were white (average proportions across 3 treatment groups: 78.6%-86.7%). Clinically meaningful and statistically significant improvements in primary and key secondary efficacy end points were observed. Zilucoplan at a dose of 0.3 mg/kg SC daily resulted in a mean reduction from baseline of 6.0 points in the QMG score (placebo-corrected change, –2.8;P = .05) and 3.4 points in the MG Activities of Daily Living score (placebo-corrected change, –2.3;P = .04). Clinically meaningful and statistically significant improvements were also observed in other secondary end points, the MG Composite and MG Quality-of-Life scores. Outcomes for the 0.1-mg/kg SC daily dose were also statistically significant but slower in onset and less pronounced than with the 0.3-mg/kg dose. Rescue therapy (intravenous immunoglobulin or plasma exchange) was required in 3 of 15, 1 of 15, and 0 of 14 participants in the placebo, 0.1-mg/kg zilucoplan, and 0.3-mg/kg zilucoplan arms, respectively. Zilucoplan was observed to have a favorable safety and tolerability profile. Conclusions and Relevance Zilucoplan yielded rapid, meaningful, and sustained improvements over 12 weeks in a broad population of patients with moderate to severe AChR-Ab–positive gMG. Near-complete complement inhibition appeared superior to submaximal inhibition. The observed safety and tolerability profile of zilucoplan was favorable. Trial Registration ClinicalTrials.gov Identifier:NCT03315130.

The vibrational signatures for the υ(2) C≡C and υ(1) symmetric C─H stretches of acetylene in cubic structure I clathrate, synthesized under ambient pressure, are reported for the first time. The most diagnostic features are at 1966 for υ(2) and 3353 cm(−1) for υ(1), respectively, and are assigned to acetylene trapped in the large 5(12)6(2) cages. In addition, the υ(2) mode for acetylene occupying the small 5(12) cages is observed at 1972.5 cm(−1), a red shift of 1.5 cm(−1) from its gas phase frequency. Unit cell parameters and thermal expansion coefficients are determined via powder X-ray diffraction between 195 and 225 K and are found to be in good correlation with previous single crystal data at 143 K. The calculated density for acetylene clathrate is also reported, with values ranging from 0.985 g/cm(3) at 195 K to 0.976 g/cm(3) at 225 K. These results are relevant for spectral detection of acetylene-containing compounds on planetary bodies, as well as providing additional insights on the thermal behavior and physical properties of acetylene clathrate.

Chloride salts have been proposed to exist on the surface of Europa based on some geochemical predictions of the ocean composition as well as some interpretations of data from the Galileo spacecraft. To help elucidate our understanding of Europa’s surface composition, we have conducted a study of frozen chloride salt brines prepared under simulated Europa surface conditions (vacuum and temperature) using both near-infrared (NIR) and Raman spectroscopies. The latter was used to determine the hydration states of various chloride salts as a function of the temperature, while NIR spectroscopy of identically prepared samples was used to provide reference reflectance spectra of the identified hydrated salts. We further investigated the stability of the formed hydrated salts under vacuum and ultraviolet (UV) irradiation. Our results indicate that, at temperatures ranging from 80 to 233 K, (i) NaCl·2H2O is formed from the freezing of NaCl brines, (ii) freezing of KCl solutions does not form KCl hydrates, (iii) fr...

The composition of Europa's subsurface ocean is of great interest for understanding the internal geochemistry and potential habitability of this icy body. However, current constraints on the ocean composition need to rely largely on its expression on the surface. In this work, we combine chemical divide modeling with cryogenic Raman and X-ray diffraction experiments to examine freezing of a simple putative brine system containing Na+, Mg2+, Cl−, and SO42− across a range of ionic concentrations and freezing rates to assess the feasibility of inferring the ocean's chemical composition via in-situ techniques. The results suggest that multiple hydrated salts not predicted by chemical models are frequently encountered in the final solid phase, making accurate quantification of the subsurface liquid composition via surface observables rather challenging. In addition, flash freezing of diluted brines often produces water ice together with vitreous Mg-bearing salt hydrates, which may significantly hinder their detection. These findings can help inform both analytical protocols for a Raman spectrometer onboard a Europa surface lander as well as potential locations for exploration, in order to best provide meaningful constraints on emplacement mechanisms and the composition of frozen salt minerals on the surface.

A recent article reported experimental observations of methane and CO2 clathrate formation at conditions similar to the interstellar medium (ISM), namely 10-30 K and 10-10 mbar. The authors conducted time-dependent reflection-absorption infrared spectroscopy (RAIRS) of vapor-deposited H2O:CH4 and H2O:CO2 mixtures and interpreted new blue and red -shifted peaks from those of trapped CH4 and CO2 in amorphous ice, respectively, as indicative of clathrate formation. In this Letter to the Editor, we point out potential pitfalls and caution against the implications drawn for the ISM., Comment: Letter to the Editor, Proceedings of the National Academy of Sciences

Benzene is found on Titan and is a likely constituent of the putative evaporite deposits formed around the hydrocarbon lakes. We have recently demonstrated the formation of a benzene-ethane co-crystal under Titan-like surface conditions. Here we investigate the kinetics of formation of this new structure as a function of temperature. We show that the formation process would reach completion under Titan surface conditions in ~18 h and that benzene precipitates from liquid ethane as the co-crystal. This suggests that benzene-rich evaporite basins around ethane/methane lakes and seas may not contain pure crystalline benzene, but instead benzene-ethane co-crystals. This co-crystalline form of benzene with ethane represents a new class of materials for Titan's surface, analogous to hydrated minerals on Earth. This new structure may also influence evaporite characteristics such as particle size, dissolution rate, and infrared spectral properties.

An expanding number of autosomal diseases has been associated with mitochondrial DNA (mtDNA) depletion and multiple deletions. These disorders have been classified as defects of intergenomic communication because mutations of the nuclear DNA are thought to disrupt the normal cross-talk that regulates the integrity and quantity of mtDNA. In 1989, autosomal dominant progressive external ophthalmoplegia with multiple deletions of mitochondrial DNA was the first of these disorders to be identified. Two years later, mtDNA depletion syndrome was initially reported in infants with severe hepatopathy or myopathy. The causes of these diseases are still unclear, but genetic linkage studies have identified three chromosomal loci for AD-PEO. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disorder associated with both mtDNA depletion and multiple deletions, is now known to be due to loss-of-function mutations in the gene encoding thymidine phosphorylase. Increased plasma thymidine levels in MNGIE patients suggest that imbalanced nucleoside and nucleotide pools in mitochondria may lead to impaired replication of mtDNA. Future research will certainly lead to the identification of additional genetic causes of intergenomic communication defects and will likely provide insight into the normal "dialogue" between the two genomes.

The composition of Europa's subsurface ocean is a critical determinant of its habitability. However, our current understanding of the ocean composition is limited to its expression on the surface. This work investigates experimentally the composition of mixed sodium–magnesium–sulfate–chloride solutions when frozen to 100 K, simulating conditions that likely occur as ocean fluids are emplaced onto Europa's surface. Micro-Raman spectroscopy is used to characterize phase composition of the frozen brines at 100 K. Our results show that solutions containing Na+, Cl−, Mg2+, and preferentially crystallize into Na2SO4 and MgCl2 hydrated minerals upon freezing, even at elevated [Mg2+]/[Na+] ratios. The detection of epsomite (MgSO4•7H2O) on Europa's surface, if confirmed, may thus imply a relatively sodium-poor ocean composition or a radiolytic process that converts MgCl2 to MgSO4 as suggested by Brown & Hand. The formation of NaCl on the surface, while dependent upon a number of factors such as freezing rate, may indicate an ocean significantly more concentrated in sodium than in magnesium.

The phylogenetic distributions of sigma- and phencyclidine receptors in neural tissues of 13 species and the pharmacological characteristics of these receptors in whole sea anemone and neural tissues of the guinea pig, chicken, and frog were studied. Specific binding of [3H]haloperidol and [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine, ligands that bind with high affinity to sigma- and phencyclidine receptors, respectively, was detected in all organisms examined. The order of potencies of various ligands to inhibit 1 nM [3H]haloperidol binding in brains of frogs and guinea pigs or 1 nM [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine in chicken or guinea pig brain homogenates was very similar. However, the characteristics and stereospecificity of binding of the two radioligands in sea anemone were different than in higher organisms. The results suggest that sigma- and phencyclidine binding sites are evolutionarily old, as the characteristics of the two sites are well preserved over a range of vertebrate phyla.

Hepatic oncocytes with abundant granular, eosinophilic cytoplasm due to mitochondrial hyperplasia are seen in various chronic liver diseases, particularly chronic hepatitis and cirrhosis. Increased mitochondria in oncocytes are thought to be a compensatory mechanism for deficiencies in the hepatocellular respiratory chain, although the pathogenesis of these deficiencies has been uncertain. We selected seven cases of cirrhosis (six with oncocytes, one without) for the following analysis: histoenzymatic and immunohistochemical staining of several mitochondrial DNA (mtDNA)- and nuclear DNA (nDNA)-encoded respiratory chain enzymes; immunostaining using antibodies against double-strand-DNA (anti-DNA) and against Ki-67 (a cell proliferation marker); and Southern blot analysis for mtDNA and nDNA. Eighty percent of oncocytes showed histoenzymatic and immunohistochemical deficiencies of cytochrome c oxidase and the mtDNA-encoded subunit I of complex IV, with preserved expression of nDNA-encoded succinate dehydrogenase and the iron-sulfur subunit of complex III (FeS). Cytoplasmic (but not nuclear) anti-DNA staining was partially or completely absent in approximately 50% of oncocytes. Three cases with oncocytes studied by Southern blot showed mtDNA reductions of 66%, 71%, and 85%. In conclusion, hepatic oncocytes demonstrate significant deficiencies of mtDNA and mtDNA-encoded respiratory chain enzymes. We propose that mtDNA depletion plays an important role in hepatocellular oncocytic transformation.

Since the first reports of disorders associated with mitochondrial DNA (mtDNA) defects more than a decade ago, the small mtDNA circle has been a Pandora's box of pathogenic mutations associated with human diseases. The "morbidity map" of mtDNA has gone from one point mutation and a few deletions in 1988 to more than 110 point mutations as of September, 2001. Nuclear DNA defects affecting mitochondrial function and mtDNA replication and integrity have also been identified in the past few years and more are expected. As a result, human "mitochondrial" diseases have evolved beyond the novelty diagnoses of a decade ago into an important area of medicine, and thus, the diagnostic principles of these disorders ought to be familiar to the clinician. In this article, the authors, we summarize the principles of mitochondrial genetics and discuss the common phenotypes, general diagnostic approach, and possible therapeutic venues for these fascinating disorders.

Mitochondrial DNA depletion syndrome is a clinically heterogeneous group of disorders characterized by a reduction in mitochondrial DNA copy number. The recent discovery of mutations in the deoxyguanosine kinase (dGK) gene in patients with the hepatocerebral form of mitochondrial DNA depletion syndrome prompted us to screen 21 patients to determine the frequency of dGK mutations, further characterize the clinical spectrum, and correlate genotypes with phenotypes. We detected mutations in three patients (14%). One patient had a homozygous GATT duplication (nucleotides 763-766), and another had a homozygous GT deletion (nucleotides 609-610); both mutations lead to truncated proteins. The third patient was a compound heterozygote for two missense mutations (R142K and E227K) that affect critical residues of the protein. These mutations were associated with variable phenotypes, and their low frequencies suggests that dGK is not the only gene responsible for mitochondrial DNA depletion in liver. The patient with the missense mutations had isolated liver failure and responded well to liver transplantation, which may be a therapeutic option in selected cases.

The postnatal developmental patterns of sigma (sigma) and phencyclidine (PCP) binding sites were compared in the rat brain. The results show diametrically different ontogenic patterns for the sites. While both the affinity and the density of sigma sites remain constant throughout the developmental period tested (postnatal day 1 to 1 year), the density of PCP binding sites increases from the time of birth, reaching the adult level by postnatal day 14. The differences in developmental patterns provide evidence for distinctive properties of cerebral sigma and PCP binding sites.

In the present study, the temporal appearance and distribution of substance P within individual subnuclei has been examined during the development of the rat interpeduncular nucleus (IPN). The prenatal organization as well as migration pattern of individual IPN subnuclei are also described. The IPN was distinguishable on embryonic day (E) 19, near the ventral mesencephalon. At this age, the IPN was organized into individual subnuclei like the adult, except for a bilateral distribution of presumptive rostral neurons. Rostral neurons were merged into a single, midline subnucleus by the day of birth, thereby completing an adult pattern of subnuclear organization. SP immunoreactivity, restricted to the lateral subnuclei, was first detected at E20. The intensity of SP-positive fibers in the lateral subnucleus increased with age, and appeared to become selectively distributed along both the medial and lateral borders of this subnucleus. Additional SP-positive fibers became evident postnatally in a thin band overlying both central and intermediate subnuclei, and within the dorsal medial, central and apical subnuclei. SP-positive cell bodies were present in the rostral subnucleus on postnatal day 28, thereby completing the development of an adult pattern of SP immunoreactivity within the IPN. IPN subnuclei Substance P development Prenatal and postnatal periods Immunocytochemistry