A low-tech, low-cost method to capture point-source ammonia emissions and their potential use as a nitrogen fertiliser.

Rising global energy prices have led to increased costs of nitrogen (N) fertilisers for farmers, but N pollution (losses) from agricultural activities can account for over 50% of the nitrogen applied. This study assesses the feasibility of a low-cost and low-tech method of NH3 emission capture from an agricultural point source (chicken manure) using a water column bubbling technique, and its application as a fertiliser to several plant types. Solutions of i) nitric acid (HNO3), ii) calcium nitrate (Ca(NO3)2), iii) a mixture of Ca(NO3)2 and HNO3 and iv) deionised H2O were used to scrub NH3 from air pumped from a storage container containing chicken manure. We conclude that NH3 can be captured from manure using low-tech methods, and that solutions of common fertiliser compounds such as ammonium nitrate and calcium ammonium nitrate can be replicated by binding captured NH3 to solutions of nitrate. Our results suggest that dissolved calcium nitrate is just as effective at scrubbing NH3 from the atmosphere as nitric acid at low concentrations, but could do so at a near neutral pH. For use on common silage grass for livestock feed, all of the captured ammonium solutions significantly increased yields, including the ammonium only solution. However, the aquatic plants (Taxiphyllum Barbieri and Salvinia auriculata) did not respond favourably to a high ratio of NH4+ in solution, and in the case of Salvinia auriculata, the plant was significantly damaged by the ammonium only solution. In conclusion, we highlight that the capture and utilisation of NH3 emissions from point sources is possible using very basic apparatus and that if used correctly, this captured nitrogen can be stored and applied to crops in a variety of forms which could reduce reliance and cost of mineral fertiliser use.

Measurements of methane and nitrous oxide in human breath and the development of UK scale emissions.

Exhaled human breath can contain small, elevated concentrations of methane (CH4) and nitrous oxide (N2O), both of which contribute to global warming. These emissions from humans are not well understood and are rarely quantified in global greenhouse gas inventories. This study investigated emissions of CH4 and N2O in human breath from 104 volunteers in the UK population, to better understand what drives these emissions and to quantify national-scale estimates. A total of 328 breath samples were collected, and age, sex, dietary preference, and smoking habits were recorded for every participant. The percentage of methane producers (MPs) identified in this study was 31%. The percentage of MPs was higher in older age groups with 25% of people under the age of 30 classified as MPs compared to 40% in the 30+ age group. Females (38%) were more likely to be MPs than males (25%), though overall concentrations emitted from both MP groups were similar. All participants were found to emit N2O in breath, though none of the factors investigated explained the differences in emissions. Dietary preference was not found to affect CH4 or N2O emissions from breath in this study. We estimate a total emission of 1.04 (0.86-1.40) Gg of CH4 and 0.069 (0.066-0.072) Gg of N2O in human breath annually in the UK, the equivalent of 53.9 (47.8-60.0) Gg of CO2. In terms of magnitude, these values are approximately 0.05% and 0.1% of the total emissions of CH4 and N2O reported in the UK national greenhouse gas inventories.

Temperature driven variations in VOC emissions from plastic products and their fate indoors: A chamber experiment and modelling study.

Plastic products are ubiquitous in our homes, but we know very little about emissions from these products and their subsequent impact on indoor air quality. This is the first study to systematically determine temperature-dependent emissions of volatile organic compounds from commonly used plastic consumer products found in the home. The plastic types included high-density polyethylene (HDPE), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS) and polyester rubber. Plastic samples were exposed to increasing temperatures (between 18 and 28 °C) in controlled environmental chambers, connected to a proton-transfer-reaction time-of-flight mass-spectrometer (PTR-ToF-MS), where real-time emissions were detected. Average emission rates were determined and used to initialise an indoor air chemistry model (INCHEM-Py) at the highest and lowest experimental temperatures, to explore the impact these product emissions have on the indoor air chemistry. The PS tubing plastic proved to be the highest emitting polymer per surface area. Almost all selected VOC emissions were found to have a linear relationship with temperature. Upon observing the impacts of primary VOC emissions from plastics in modelled simulations, the hydroxyl radical concentration decreased by an average of 1.6 and 10 % relative to the baseline (with no plastics included) at 18 °C and 28 °C respectively. On the other hand, formaldehyde concentrations increased by 29 and 31.6 % relative to the baseline conditions at 18 °C and 28 °C respectively. The presence of plastic products indoors, therefore, has the potential to impact the indoor air quality.

Opioid-Limiting Pain Control After Transurethral Resection of the Prostate: A Randomized Controlled Trial.

OBJECTIVE: To assess whether a multimodal opioid-limiting protocol and patient education intervention can reduce postoperative opioid use following transurethral resection of the prostate. METHODS: This prospective, non-blinded, single-institution, randomized controlled trial (NCT04102566) assigned 50 patients undergoing a transurethral resection of the prostate to either a standard of care control (SOC) or multimodal experimental group (MMG). The intervention included adding ibuprofen to the postoperative pain regimen, promoting appropriate opioid use while hospitalized, an educational intervention, and discharging without opioid prescription. Data regarding demographics, operative data, opioid use, pain scores, and patient satisfaction were compared. RESULTS: A total of 47 patients were included, n = 23 (MMG) and n = 24 (SOC). Demographic and operative findings were similar. Statistical analysis for noninferiority demonstrated non-inferior inpatient pain control (mean pain score 2.5 MMG vs 2.4 SOC, P = 0.0003). The multimodal group used significantly fewer morphine milligram equivalents after discharge (0 vs 4.1, P = 0.04). Inpatient use was reduced but did not reach statistical significance (6.0 vs 9.8, P = 0.2). Mean satisfaction scores with pain control were similar (9.6 MMG vs 9.2 SOC, P = 0.32). No opioid prescriptions were requested after discharge. Adverse events and medication side effects were infrequent and largely similar between groups. CONCLUSION: Implementation of an opioid-limiting postoperative pain protocol and patient education resulted in no outpatient opioid use while maintaining patient satisfaction with pain control. Eliminating opioids following a common urologic procedure will decrease risk of opioid-related adverse events and have a positive downstream impact.

Randomized controlled trial of a quadratus lumborum block with liposomal bupivacaine for postoperative analgesia in laparoscopic donor nephrectomy.

Perioperative pain management is an important consideration in early recovery and patient satisfaction following laparoscopic donor nephrectomy. Transmuscular quadratus lumborum block has been described to reduce pain and opioid usage following several abdominal surgeries. In this prospective single-blind randomized controlled trial, we compared 52 patients who adhered to our institutional donor nephrectomy Early Recovery After Surgery pathway, which includes a laparoscopic-guided transversus abdominus plane block, to 40 patients who additionally received a transmuscular quadratus lumborum block with liposomal bupivacaine. Compared to control patients, those who received the block spent longer in the operating room prior to the surgical start (65.4 vs. 51.6 min, P < .001). Both groups had similar total hospital length of stay (33.3 h vs. 34.4 h, P = .61). Pain scores from postoperative days 0-30, number of patients requiring opioids, postoperative nausea, and pain management satisfaction were similar between both groups. Patients who received the block consumed less opioid on postoperative day 1 compared to controls (P = .006). No complications were attributable to the block. The quadratus lumborum block provides a safe pain management adjunct for some patients, and may reduce opioid use in the early postoperative period when combined with our standard institutional protocol for kidney donors.

A survey of heavy metal contents of rural and urban roadside dusts: comparisons at low, medium and high traffic sites in Central Scotland.

Roadside dust can contain particulates enriched with potentially toxic elements (PTEs) as a result of the degradation of mechanical vehicular parts, tyre wear and combustion processes. To assess the potential accumulation of these metals in roadside areas, a snapshot study was carried out, investigating metal content at rural and urban locations in central Scotland. Samples of road dust were collected at six sites representing low, medium and high traffic intensity at rural and urban locations. The samples were separated based on particle size and analysed for heavy metal content using inductively coupled plasma optical emission spectroscopy (ICP-OES) after acid digestion. The metals analysed were aluminium (Al), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), lead (Pb) and zinc (Zn). The rural area measurements were carried out in West Lothian, approximately 13 to 17 miles west of the city of Edinburgh (UK). The urban area measurements were carried out in the southern part of the Edinburgh city district (UK). Concentrations of Cu, Cr and Zn were found to correlate with traffic intensity, although only Cu and Zn concentrations exceed recommended EC directive 86/278/EEC guidelines for urban runoff materials. The metal concentrations of small particles (0.45-20 µm) were exceedingly high in both Cu and Zn at areas of high traffic intensity, indicating potential areas of concern regarding health impacts for pedestrians and cyclists who are exposed to roadside dust on a regular basis.

Public transportation and transmission of viral respiratory disease: Evidence from influenza deaths in 121 cities in the United States.

One important concern around the spread of respiratory infectious diseases has been the contribution of public transportation, a space where people are in close contact with one another and with high-use surfaces. While disease clearly spreads along transportation routes, there is limited evidence about whether public transportation use itself is associated with the overall prevalence of contagious respiratory illnesses at the local level. We examine the extent of the association between public transportation and influenza mortality, a proxy for disease prevalence, using city-level data on influenza and pneumonia mortality and public transit use from 121 large cities in the United States (US) between 2006 and 2015. We find no evidence of a positive relationship between city-level transit ridership and influenza/pneumonia mortality rates, suggesting that population level rates of transit use are not a singularly important factor in the transmission of influenza.

Nitrous oxide emission factors of mineral fertilisers in the UK and Ireland: A Bayesian analysis of 20 years of experimental data.

In this study, we analysed datasets of N2O emission factors (EFs) from 21 separate studies carried out on arable and managed grasslands across the UK and Ireland over the past 20 years. A total of 641 separate events were collated from 40 experimental field sites. Individual EFs ranged over an order of magnitude (0-12% of applied N) for each fertiliser type, following a log-normal distribution in all cases. Our study shows that a Bayesian approach can provide a robust statistical method that is capable of performing uncertainty analysis on log-normal distributed data in a more defensible manner than conventional statistical methods allow. This method allowed for a national scale comparison of EFs between the most commonly applied mineral fertilisers based solely on previously published data (UK and Ireland in this case). The study shows that ammonium nitrate (AN) and Calcium ammonium nitrate (CAN) are the largest emitting fertiliser types by mass across the British Isles (temperate climate zone), with EFs of 1.1 (1.0-1.2) % and 1.0 (0.7-1.3) % for all recorded events, respectively; however, emissions from AN applications were significantly lower for applications to arable fields (0.6%) than to grasslands (1.3%). EFs associated with urea (CO(NH2)2) were significantly lower than AN for grasslands with an EF of 0.6 (0.5-0.7) %, but slightly higher for arable fields with an EF of 0.7 (0.4-1.4) %. The study highlights the potential effectiveness of microbial inhibitors at reducing emissions of N2O from mineral fertilisers, with Dicyandiamide (DCD) treated AN reducing emissions by approximately 28% and urea treated with either DCD or N-(n)-butyl) thiophosphorictriamide (NBTP) reducing emissions by approximately 40%. Although limited by a relatively small sample size (n = 11), urea treated with both DCD and NBPT appeared to have the lowest EF of all treatments at 0.13 (0.08-0.21) %, highlighting the potential to significantly reduce N2O emissions at regional scales if applied instead of conventional nitrogen fertilisers.

Temporal characteristics and vertical distribution of atmospheric ammonia and ammonium in winter in Beijing.

To understand the temporal characteristics and vertical distributions of ammonia (NH3) and ammonium (NH4) in urban Beijing, we conducted ground-based and tower-based measurements of gaseous NH3 and submicron aerosol composition. The average mixing ratio of NH3 was 16.5 ±â€¯7.4 ppb, ranging from 3.8 to 36.9 ppb. Gas-to-particle partitioning of NHx (=NH3 + NH4) played a significant role on NH3 concentration as the molar ratio of NH3 to NHx decreased as a function of NH4 concentration. The NH3 concentrations increased as a function of PM1 at lower levels (<125 µg m-3), but remained relatively constant at higher PM and NH4 levels, indicating an enhanced gas-to-particle conversion of NH3 during highly polluted conditions. The potential sources of NHx were found to include fossil fuel combustion and biomass burning. Regional transport could also play an important role on NH3 concentration during the formation stage of haze episodes due to particle-to-gas conversion. Four distinctive types of vertical profiles (87% of the time) of both NH3 and fine particle light extinction coefficient (bext) were observed and they were associated with well-mixed atmosphere, fast accumulation of local emissions, regional transport aloft, and the formation of low urban boundary layer, respectively. However, the vertical profiles of NH3 typically (96% of the time) showed a more homogeneous characteristic than those of bext below 260 m, except periods with both strong temperature inversion and large aerosol gradient, the formation of urban boundary layer shall cause a significant transition in the vertical distribution of NH3 below 260 m. During highly polluted situations (PM1 > 125 µg m-3), the strong effect of gas-to-particle partitioning of NHx sometimes (7% of the time) caused opposite trends in vertical profiles of NH3 and bext.

Mutations in MAST1 Cause Mega-Corpus-Callosum Syndrome with Cerebellar Hypoplasia and Cortical Malformations.

Corpus callosum malformations are associated with a broad range of neurodevelopmental diseases. We report that de novo mutations in MAST1 cause mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations (MCC-CH-CM) in the absence of megalencephaly. We show that MAST1 is a microtubule-associated protein that is predominantly expressed in post-mitotic neurons and is present in both dendritic and axonal compartments. We further show that Mast1 null animals are phenotypically normal, whereas the deletion of a single amino acid (L278del) recapitulates the distinct neurological phenotype observed in patients. In animals harboring Mast1 microdeletions, we find that the PI3K/AKT3/mTOR pathway is unperturbed, whereas Mast2 and Mast3 levels are diminished, indicative of a dominant-negative mode of action. Finally, we report that de novo MAST1 substitutions are present in patients with autism and microcephaly, raising the prospect that mutations in this gene give rise to a spectrum of neurodevelopmental diseases.

Donating Another Person's Kidney: Avoiding the Discard of Organs by Retransplantation.

BACKGROUND: Procurement and retransplantation of a previously transplanted kidney reclaim a functioning organ that would otherwise have been discarded. METHODS: Case series of 3 retransplantation cases within the course of 1 calendar year. RESULTS: These cases illustrate how to overcome the immunological, logistical, and technical barriers that have thus far limited the potential of this approach. Within this series, we report kidney reuse weeks and years after the original transplantation, as well as the previously undescribed "living donation of a deceased donor kidney". CONCLUSIONS: Retransplantation of previously transplanted kidneys can be performed successfully and should be considered in the face of the current organ shortage.

Mutations in TUBB4B Cause a Distinctive Sensorineural Disease.

Leber congenital amaurosis (LCA) is a neurodegenerative disease of photoreceptor cells that causes blindness within the first year of life. It occasionally occurs in syndromic metabolic diseases and plurisystemic ciliopathies. Using exome sequencing in a multiplex family and three simplex case subjects with an atypical association of LCA with early-onset hearing loss, we identified two heterozygous mutations affecting Arg391 in ß-tubulin 4B isotype-encoding (TUBB4B). Inspection of the atomic structure of the microtubule (MT) protofilament reveals that the ß-tubulin Arg391 residue contributes to a binding pocket that interacts with α-tubulin contained in the longitudinally adjacent αß-heterodimer, consistent with a role in maintaining MT stability. Functional analysis in cultured cells overexpressing FLAG-tagged wild-type or mutant TUBB4B as well as in primary skin-derived fibroblasts showed that the mutant TUBB4B is able to fold, form αß-heterodimers, and co-assemble into the endogenous MT lattice. However, the dynamics of growing MTs were consistently altered, showing that the mutations have a significant dampening impact on normal MT growth. Our findings provide a link between sensorineural disease and anomalies in MT behavior and describe a syndromic LCA unrelated to ciliary dysfunction.

Uner Tan syndrome caused by a homozygous TUBB2B mutation affecting microtubule stability.

The integrity and dynamic properties of the microtubule cytoskeleton are indispensable for the development of the mammalian brain. Consequently, mutations in the genes that encode the structural component (the α/ß-tubulin heterodimer) can give rise to severe, sporadic neurodevelopmental disorders. These are commonly referred to as the tubulinopathies. Here we report the addition of recessive quadrupedalism, also known as Uner Tan syndrome (UTS), to the growing list of diseases caused by tubulin variants. Analysis of a consanguineous UTS family identified a biallelic TUBB2B mutation, resulting in a p.R390Q amino acid substitution. In addition to the identifying quadrupedal locomotion, all three patients showed severe cerebellar hypoplasia. None, however, displayed the basal ganglia malformations typically associated with TUBB2B mutations. Functional analysis of the R390Q substitution revealed that it did not affect the ability of ß-tubulin to fold or become assembled into the α/ß-heterodimer, nor did it influence the incorporation of mutant-containing heterodimers into microtubule polymers. The 390Q mutation in S. cerevisiae TUB2 did not affect growth under basal conditions, but did result in increased sensitivity to microtubule-depolymerizing drugs, indicative of a mild impact of this mutation on microtubule function. The TUBB2B mutation described here represents an unusual recessive mode of inheritance for missense-mediated tubulinopathies and reinforces the sensitivity of the developing cerebellum to microtubule defects.

A patient with lissencephaly, developmental delay, and infantile spasms, due to de novo heterozygous mutation of KIF2A.

BACKGROUND: Microtubules are dynamic polymers of α/ß tubulin heterodimers that play a critical role in cerebral cortical development, by regulating neuronal migration, differentiation, and morphogenesis. Mutations in genes that encode either α- or ß-tubulin or a spectrum of proteins involved in the regulation of microtubule dynamics lead to clinically devastating malformations of cortical development, including lissencephaly. METHODS: This is a single case report or a patient with lissencephaly, developmental delay, nystagmus, persistent hyperplastic primary vitreous, and infantile spasms, and undertook a neurogenetic workup. We include studies of mutant function in Escherichia coli and HeLa cells. RESULTS: The patient was found to have a novel de novo mutation in kinesin family member 2A (KIF2A). This mutation results in a substitution of isoleucine at a highly conserved threonine residue within the ATP-binding domain. The KIF2A p.Thr320Ile mutant protein exhibited abnormal solubility, and KIF2A p.Thr320Ile overexpression in cultured cells led to the formation of aberrant microtubule networks. CONCLUSION: Findings support the pathogenic link between KIF2A mutation and lissencephaly, and expand the range of presentation to include infantile spasms and congenital anomalies.

Mutations in TUBB8 cause a multiplicity of phenotypes in human oocytes and early embryos.

BACKGROUND: TUBB8 is a primate-specific ß-tubulin isotype whose expression is confined to oocytes and the early embryo. We previously found that mutations in TUBB8 caused oocyte maturation arrest. The objective was to describe newly discovered mutations in TUBB8 and to characterise the accompanying spectrum of phenotypes and modes of inheritance. METHODS AND RESULTS: Patients with oocyte maturation arrest were sequenced with respect to TUBB8. We investigated the effects of identified mutations in vitro, in cultured cells and in mouse oocytes. Seven heterozygous missense and two homozygous mutations were identified. These mutations cause a range of folding defects in vitro, different degrees of microtubule disruption upon expression in cultured cells and interfere to varying extents in the proper assembly of the meiotic spindle in mouse oocytes. Several of the newly discovered TUBB8 mutations result in phenotypic variability. For example, oocytes harbouring any of three missense mutations (I210V, T238M and N348S) could extrude the first polar body. Moreover, they could be fertilised, although the ensuing embryos became developmentally arrested. Surprisingly, oocytes from patients harbouring homozygous TUBB8 mutations that in either case preclude the expression of a functional TUBB8 polypeptide nonetheless contained identifiable spindles. CONCLUSIONS: Our data substantially expand the range of dysfunctional oocyte phenotypes incurred by mutation in TUBB8, underscore the independent nature of human oocyte meiosis and differentiation, extend the class of genetic diseases known as the tubulinopathies and provide new criteria for the qualitative evaluation of meiosis II (MII) oocytes for in vitro fertilization (IVF).

Mutations in TUBB8 and Human Oocyte Meiotic Arrest.

Background Human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. Methods We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other ß-tubulin isotypes was assessed in human oocytes, early embryos, sperm cells, and several somatic tissues by means of a quantitative reverse-transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one ß-tubulin polypeptide (α/ß-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. Results We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed ß-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/ß-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. Conclusions TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility. (Funded by the National Basic Research Program of China and others.).

Infantile neurodegenerative disorder associated with mutations in TBCD, an essential gene in the tubulin heterodimer assembly pathway.

Mutation in a growing spectrum of genes is known to either cause or contribute to primary or secondary microcephaly. In primary microcephaly the genetic determinants frequently involve mutations that contribute to or modulate the microtubule cytoskeleton by causing perturbations of neuronal proliferation and migration. Here we describe four patients from two unrelated families each with an infantile neurodegenerative disorder characterized by loss of developmental milestones at 9­24 months of age followed by seizures, dystonia and acquired microcephaly. The patients harboured homozygous missense mutations (A475T and A586V) in TBCD, a gene encoding one of five tubulin-specific chaperones (termed TBCA-E) that function in concert as a nanomachine required for the de novo assembly of the α/ß tubulin heterodimer. The latter is the subunit from which microtubule polymers are assembled. We found a reduced intracellular abundance of TBCD in patient fibroblasts to about 10% (in the case of A475T) or 40% (in the case of A586V) compared to age-matched wild type controls. Functional analyses of the mutant proteins revealed a partially compromised ability to participate in the heterodimer assembly pathway. We show via in utero shRNA-mediated suppression that a balanced supply of tbcd is critical for cortical cell proliferation and radial migration in the developing mouse brain. We conclude that TBCD is a novel functional contributor to the mammalian cerebral cortex development, and that the pathological mechanism resulting from the mutations we describe is likely to involve compromised interactions with one or more TBCD-interacting effectors that influence the dynamics and behaviour of the neuronal cytoskeleton.

Mutations in Either TUBB or MAPRE2 Cause Circumferential Skin Creases Kunze Type.

Circumferential skin creases Kunze type (CSC-KT) is a specific congenital entity with an unknown genetic cause. The disease phenotype comprises characteristic circumferential skin creases accompanied by intellectual disability, a cleft palate, short stature, and dysmorphic features. Here, we report that mutations in either MAPRE2 or TUBB underlie the genetic origin of this syndrome. MAPRE2 encodes a member of the microtubule end-binding family of proteins that bind to the guanosine triphosphate cap at growing microtubule plus ends, and TUBB encodes a ß-tubulin isotype that is expressed abundantly in the developing brain. Functional analyses of the TUBB mutants show multiple defects in the chaperone-dependent tubulin heterodimer folding and assembly pathway that leads to a compromised yield of native heterodimers. The TUBB mutations also have an impact on microtubule dynamics. For MAPRE2, we show that the mutations result in enhanced MAPRE2 binding to microtubules, implying an increased dwell time at microtubule plus ends. Further, in vivo analysis of MAPRE2 mutations in a zebrafish model of craniofacial development shows that the variants most likely perturb the patterning of branchial arches, either through excessive activity (under a recessive paradigm) or through haploinsufficiency (dominant de novo paradigm). Taken together, our data add CSC-KT to the growing list of tubulinopathies and highlight how multiple inheritance paradigms can affect dosage-sensitive biological systems so as to result in the same clinical defect.

Tubulin-specific chaperones: components of a molecular machine that assembles the α/ß heterodimer.

The tubulin heterodimer consists of one α- and one ß-tubulin polypeptide. Neither protein can partition to the native state or assemble into polymerization competent heterodimers without the concerted action of a series of chaperone proteins including five tubulin-specific chaperones (TBCs) termed TBCA-TBCE. TBCA and TBCB bind to and stabilize newly synthesized quasi-native ß- and α-tubulin polypeptides, respectively, following their generation via multiple rounds of ATP-dependent interaction with the cytosolic chaperonin. There is free exchange of ß-tubulin between TBCA and TBCD, and of α-tubulin between TBCB and TBCE, resulting in the formation of TBCD/ß and TBCE/α, respectively. The latter two complexes interact, forming a supercomplex (TBCE/α/TBCD/ß). Discharge of the native α/ß heterodimer occurs via interaction of the supercomplex with TBCC, which results in the triggering of TBC-bound ß-tubulin (E-site) GTP hydrolysis. This reaction acts as a switch for disassembly of the supercomplex and the release of E-site GDP-bound heterodimer, which becomes polymerization competent following spontaneous exchange with GTP. The tubulin-specific chaperones thus function together as a tubulin assembly machine, marrying the α- and ß-tubulin subunits into a tightly associated heterodimer. The existence of this evolutionarily conserved pathway explains why it has never proved possible to isolate α- or ß-tubulin as stable independent entities in the absence of their cognate partners, and implies that each exists and is maintained in the heterodimer in a nonminimal energy state. Here, we describe methods for the purification of recombinant TBCs as biologically active proteins following their expression in a variety of host/vector systems.

Mutations in TUBG1, DYNC1H1, KIF5C and KIF2A cause malformations of cortical development and microcephaly.

The genetic causes of malformations of cortical development (MCD) remain largely unknown. Here we report the discovery of multiple pathogenic missense mutations in TUBG1, DYNC1H1 and KIF2A, as well as a single germline mosaic mutation in KIF5C, in subjects with MCD. We found a frequent recurrence of mutations in DYNC1H1, implying that this gene is a major locus for unexplained MCD. We further show that the mutations in KIF5C, KIF2A and DYNC1H1 affect ATP hydrolysis, productive protein folding and microtubule binding, respectively. In addition, we show that suppression of mouse Tubg1 expression in vivo interferes with proper neuronal migration, whereas expression of altered γ-tubulin proteins in Saccharomyces cerevisiae disrupts normal microtubule behavior. Our data reinforce the importance of centrosomal and microtubule-related proteins in cortical development and strongly suggest that microtubule-dependent mitotic and postmitotic processes are major contributors to the pathogenesis of MCD.