How megadrought causes extensive mortality in a deep-rooted shrub species normally resistant to drought-induced dieback: The role of a biotic mortality agent.

Southern California experienced unprecedented megadrought between 2012 and 2018. During this time, Malosma laurina, a chaparral species normally resilient to single-year intense drought, developed extensive mortality exceeding 60% throughout low-elevation coastal populations of the Santa Monica Mountains. We assessed the physiological mechanisms by which the advent of megadrought predisposed M. laurina to extensive shoot dieback and whole-plant death. We found that hydraulic conductance of stem xylem (Ks, native ) was reduced seven to 11-fold in dieback adult and resprout branches, respectively. Staining of stem xylem vessels revealed that dieback plants experienced 68% solid-blockage, explaining the reduction in water transport. Following Koch's postulates, persistent isolation of a microorganism in stem xylem of dieback plants but not healthy controls indicated that the causative agent of xylem blockage was an opportunistic endophytic fungus, Botryosphaeria dothidea. We inoculated healthy M. laurina saplings with fungal isolates and compared hyphal elongation rates under well-watered, water-deficit, and carbon-deficit treatments. Relative to controls, we found that both water deficit and carbon-deficit increased hyphal extension rates and the incidence of shoot dieback.

De Novo SOX6 Variants Cause a Neurodevelopmental Syndrome Associated with ADHD, Craniosynostosis, and Osteochondromas.

SOX6 belongs to a family of 20 SRY-related HMG-box-containing (SOX) genes that encode transcription factors controlling cell fate and differentiation in many developmental and adult processes. For SOX6, these processes include, but are not limited to, neurogenesis and skeletogenesis. Variants in half of the SOX genes have been shown to cause severe developmental and adult syndromes, referred to as SOXopathies. We here provide evidence that SOX6 variants also cause a SOXopathy. Using clinical and genetic data, we identify 19 individuals harboring various types of SOX6 alterations and exhibiting developmental delay and/or intellectual disability; the individuals are from 17 unrelated families. Additional, inconstant features include attention-deficit/hyperactivity disorder (ADHD), autism, mild facial dysmorphism, craniosynostosis, and multiple osteochondromas. All variants are heterozygous. Fourteen are de novo, one is inherited from a mosaic father, and four offspring from two families have a paternally inherited variant. Intragenic microdeletions, balanced structural rearrangements, frameshifts, and nonsense variants are predicted to inactivate the SOX6 variant allele. Four missense variants occur in residues and protein regions highly conserved evolutionarily. These variants are not detected in the gnomAD control cohort, and the amino acid substitutions are predicted to be damaging. Two of these variants are located in the HMG domain and abolish SOX6 transcriptional activity in vitro. No clear genotype-phenotype correlations are found. Taken together, these findings concur that SOX6 haploinsufficiency leads to a neurodevelopmental SOXopathy that often includes ADHD and abnormal skeletal and other features.

A mutagenic analysis of NahE, a hydratase-aldolase in the naphthalene degradative pathway.

NahE is a hydratase-aldolase that converts o-substituted trans-benzylidenepyruvates (H, OH, or CO2-) to benzaldehyde, salicylaldehyde, or 2-carboxybenzaldehyde, respectively, and pyruvate. The enzyme is in a bacterial degradative pathway for naphthalene, which is a toxic and persistent environmental contaminant. Sequence, crystallographic, and mutagenic analysis identified the enzyme as a member of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. As such, it has a conserved lysine (Lys183) and tyrosine (Tyr155), for Schiff base formation, as well as a GXXGE motif for binding of the pyruvoyl carboxylate group. A crystal structure of the selenomethionine derivative of NahE shows these active site elements along with nearby residues that might be involved in the mechanism and/or specificity. Mutations of five active site amino acids (Thr65, Trp128, Tyr155, Asn157, and Asn281) were constructed and kinetic parameters measured in order to assess the effect(s) on catalysis. The results show that the two Trp128 mutants (Phe and Tyr) have the least effect on catalysis, whereas amino acids with bulky side chains at Thr65 (Val) and Asn281 (Leu) have the greatest effect. Changing Tyr155 to Phe and Asn157 to Ala also hinders catalysis, and the effects fall in between these extremes. These observations are put into a structural context using a crystal structure of the Schiff base of the reaction intermediate. Trapping experiments with substrate, Na(CN)BH3, and wild type enzyme and selected mutants mostly paralleled the kinetic analysis, and identified two salicylaldehyde-modified lysines: the active site lysine (Lys183) and one outside the active site (Lys279). The latter could be responsible for the observed inhibition of NahE by salicylaldehyde. Together, the results provide new insights into the NahE-catalyzed reaction.

Kinetic, Inhibition, and Structural Characterization of a Malonate Semialdehyde Decarboxylase-like Protein from Calothrix sp. PCC 6303: A Gateway to the non-Pro1 Tautomerase Superfamily Members.

The amino-terminal proline (Pro1) has long been thought to be a mechanistic imperative for tautomerase superfamily (TSF) enzymes, functioning as a general base or acid in all characterized reactions. However, a global examination of more than 11,000 nonredundant sequences of the TSF uncovered 346 sequences that lack Pro1. The majority (∼85%) are found in the malonate semialdehyde decarboxylase (MSAD) subgroup where most of the 294 sequences form a separate cluster. Four sequences within this cluster retain Pro1. Because these four sequences might provide clues to assist in the identification and characterization of activities of nearby sequences without Pro1, they were examined by kinetic, inhibition, and crystallographic studies. The most promising of the four (from Calothrix sp. PCC 6303 designated 437) exhibited decarboxylase and tautomerase activities and was covalently modified at Pro1 by 3-bromopropiolate. A crystal structure was obtained for the apo enzyme (2.35 Šresolution). The formation of a 3-oxopropanoate adduct with Pro1 provides clues to build a molecular model for the bound ligand. The modeled ligand extends into a region that allows interactions with three residues (Lys37, Arg56, Glu98), suggesting that these residues can play roles in the observed decarboxylation and tautomerization activities. Moreover, these same residues are conserved in 16 nearby, non-Pro1 sequences in a sequence similarity network. Thus far, these residues have not been implicated in the mechanisms of any other TSF members. The collected observations provide starting points for the characterization of the non-Pro1 sequences.

Symmetry of 4-Oxalocrotonate Tautomerase Trimers Influences Unfolding and Fragmentation in the Gas Phase.

The recent discovery of asymmetric arrangements of trimers in the tautomerase superfamily (TSF) adds structural diversity to this already mechanistically diverse superfamily. Classification of asymmetric trimers has previously been determined using X-ray crystallography. Here, native mass spectrometry (MS) and ultraviolet photodissociation (UVPD) are employed as an integrated strategy for more rapid and sensitive differentiation of symmetric and asymmetric trimers. Specifically, the unfolding of symmetric and asymmetric trimers initiated by collisional heating was probed using UVPD, which revealed unique gas-phase unfolding pathways. Variations in UVPD patterns from native-like, compact trimeric structures to unfolded, extended conformations indicate a rearrangement of higher-order structure in the asymmetric trimers that are believed to be stabilized by salt-bridge triads, which are absent from the symmetric trimers. Consequently, the symmetric trimers were found to be less stable in the gas phase, resulting in enhanced UVPD fragmentation overall and a notable difference in higher-order re-structuring based on the extent of hydrogen migration of protein fragments. The increased stability of the asymmetric trimers may justify their evolution and concomitant diversification of the TSF. Facilitating the classification of TSF members as symmetric or asymmetric trimers assists in delineating the evolutionary history of the TSF.

Kinetic and Structural Analysis of Two Linkers in the Tautomerase Superfamily: Analysis and Implications.

The tautomerase superfamily (TSF) is a collection of enzymes and proteins that share a simple ß-α-ß structural scaffold. Most members are constructed from a single-core ß-α-ß motif or two consecutively fused ß-α-ß motifs in which the N-terminal proline (Pro-1) plays a key and unusual role as a catalytic residue. The cumulative evidence suggests that a gene fusion event took place in the evolution of the TSF followed by duplication (of the newly fused gene) to result in the diversification of activity that is seen today. Analysis of the sequence similarity network (SSN) for the TSF identified several linking proteins ("linkers") whose similarity links subgroups of these contemporary proteins that might hold clues about structure-function relationship changes accompanying the emergence of new activities. A previously uncharacterized pair of linkers (designated N1 and N2) was identified in the SSN that connected the 4-oxalocrotonate tautomerase (4-OT) and cis-3-chloroacrylic acid dehalogenase (cis-CaaD) subgroups. N1, in the cis-CaaD subgroup, has the full complement of active site residues for cis-CaaD activity, whereas N2, in the 4-OT subgroup, lacks a key arginine (Arg-39) for canonical 4-OT activity. Kinetic characterization and nuclear magnetic resonance analysis show that N1 has activities observed for other characterized members of the cis-CaaD subgroup with varying degrees of efficiencies. N2 is a modest 4-OT but shows enhanced hydratase activity using allene and acetylene compounds, which might be due to the presence of Arg-8 along with Arg-11. Crystallographic analysis provides a structural context for these observations.

Defining the genotypic and phenotypic spectrum of X-linked MSL3-related disorder.

PURPOSE: We sought to delineate the genotypic and phenotypic spectrum of female and male individuals with X-linked, MSL3-related disorder (Basilicata-Akhtar syndrome). METHODS: Twenty-five individuals (15 males, 10 females) with causative variants in MSL3 were ascertained through exome or genome sequencing at ten different sequencing centers. RESULTS: We identified multiple variant types in MSL3 (ten nonsense, six frameshift, four splice site, three missense, one in-frame-deletion, one multi-exon deletion), most proven to be de novo, and clustering in the terminal eight exons suggesting that truncating variants in the first five exons might be compensated by an alternative MSL3 transcript. Three-dimensional modeling of missense and splice variants indicated that these have a deleterious effect. The main clinical findings comprised developmental delay and intellectual disability ranging from mild to severe. Autism spectrum disorder, muscle tone abnormalities, and macrocephaly were common as well as hearing impairment and gastrointestinal problems. Hypoplasia of the cerebellar vermis emerged as a consistent magnetic resonance image (MRI) finding. Females and males were equally affected. Using facial analysis technology, a recognizable facial gestalt was determined. CONCLUSION: Our aggregated data illustrate the genotypic and phenotypic spectrum of X-linked, MSL3-related disorder (Basilicata-Akhtar syndrome). Our cohort improves the understanding of disease related morbidity and allows us to propose detailed surveillance guidelines for affected individuals.

Structural Basis for the Asymmetry of a 4-Oxalocrotonate Tautomerase Trimer.

Tautomerase superfamily (TSF) members are constructed from a single ß-α-ß unit or two consecutively joined ß-α-ß units. This pattern prevails throughout the superfamily consisting of more than 11000 members where homo- or heterohexamers are localized in the 4-oxalocrotonate tautomerase (4-OT) subgroup and trimers are found in the other four subgroups. One exception is a subset of sequences that are double the length of the short 4-OTs in the 4-OT subgroup, where the coded proteins form trimers. Characterization of two members revealed an interesting dichotomy. One is a symmetric trimer, whereas the other is an asymmetric trimer. One monomer is flipped 180° relative to the other two monomers so that three unique protein-protein interfaces are created that are composed of different residues. A bioinformatics analysis of the fused 4-OT subset shows a further division into two clusters with a total of 133 sequences. The analysis showed that members of one cluster (86 sequences) have more salt bridges if the asymmetric trimer forms, whereas the members of the other cluster (47 sequences) have more salt bridges if the symmetric trimer forms. This hypothesis was examined by the kinetic and structural characterization of two proteins within each cluster. As predicted, all four proteins function as 4-OTs, where two assemble into asymmetric trimers (designated R7 and F6) and two form symmetric trimers (designated W0 and Q0). These findings can be extended to the other sequences in the two clusters in the fused 4-OT subset, thereby annotating their oligomer properties and activities.

Strategies to Reduce the Euthanasia of Impounded Dogs and Cats Used by Councils in Victoria, Australia.

Using euthanasia to manage dog and cat overpopulation causes health issues and emotional stress in employees involved, increases staff turnover, and has financial, moral and ethical ramifications for communities. Welfare agencies and local government agencies (councils) share responsibility for managing companion animal populations. This study investigated Australian councils in the state of Victoria, to identify strategies used to reduce euthanasia. Statistics regarding animal populations, registration, intake, reclaim, rehome and euthanasia were obtained from the Domestic Animal Management Plan of each council. Of the 79 Victorian councils, 74% achieved ≤10% euthanasia of impounded dogs, which is widely quoted as zero euthanasia of adoptable and treatable animals. The mean euthanasia rates for cats by the councils was 48%, with only one council achieving a euthanasia rate of ≤10% for cats. Mean reclaim rates for dogs were higher (73%) than for cats (13%), as was the mean proportion of unclaimed dogs rehomed (71%), compared to cats (45%). Telephone questionnaires were conducted with animal management officers from 35 councils (44%). Those with low euthanasia rates had high reclaim rates and/or rehome rates. Reclaim, rehome and euthanasia rates for dogs and cats were not significantly different between councils that operated their own pound facilities and those that utilized the services of welfare organizations to operate pounds on behalf of the council. More council managers believed they would never achieve ≤10% euthanasia for cats (49%) than for dogs (11%). A variety of strategies were used by councils to achieve high reclaim and rehoming rates.

Problems Associated with the Microchip Data of Stray Dogs and Cats Entering RSPCA Queensland Shelters.

A lack of published information documenting problems with the microchip data for the reclaiming of stray animals entering Australian shelters limits improvement of the current microchipping system. A retrospective study analysing admission data for stray, adult dogs (n = 7258) and cats (n = 6950) entering the Royal Society for the Prevention of Cruelty to Animals (RSPCA) Queensland between January 2012 and December 2013 was undertaken to determine the character and frequency of microchip data problems and their impact on outcome for the animal. Only 28% of dogs and 9% of cats were microchipped, and a substantial proportion (37%) had problems with their data, including being registered to a previous owner or organisation (47%), all phone numbers incorrect/disconnected (29%), and the microchip not registered (14%). A higher proportion of owners could be contacted when the microchip had no problems, compared to those with problems (dogs, 93% vs. 70%; cats, 75% vs. 41%). The proportion of animals reclaimed declined significantly between microchipped animals with no data problems, microchipped animals with data problems and non-microchipped animals-87%, 69%, and 37%, respectively, for dogs and 61%, 33%, and 5%, respectively, for cats. Strategies are needed to increase the accuracy of microchip data to facilitate the reclaiming of stray dogs and cats.