UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis.

Ubiquinone (also known as coenzyme Q) is a ubiquitous lipid-soluble redox cofactor that is an essential component of electron transfer chains. Eleven genes have been implicated in bacterial ubiquinone biosynthesis, including ubiX and ubiD, which are responsible for decarboxylation of the 3-octaprenyl-4-hydroxybenzoate precursor. Despite structural and biochemical characterization of UbiX as a flavin mononucleotide (FMN)-binding protein, no decarboxylase activity has been detected. Here we report that UbiX produces a novel flavin-derived cofactor required for the decarboxylase activity of UbiD. UbiX acts as a flavin prenyltransferase, linking a dimethylallyl moiety to the flavin N5 and C6 atoms. This adds a fourth non-aromatic ring to the flavin isoalloxazine group. In contrast to other prenyltransferases, UbiX is metal-independent and requires dimethylallyl-monophosphate as substrate. Kinetic crystallography reveals that the prenyltransferase mechanism of UbiX resembles that of the terpene synthases. The active site environment is dominated by π systems, which assist phosphate-C1' bond breakage following FMN reduction, leading to formation of the N5-C1' bond. UbiX then acts as a chaperone for adduct reorientation, via transient carbocation species, leading ultimately to formation of the dimethylallyl C3'-C6 bond. Our findings establish the mechanism for formation of a new flavin-derived cofactor, extending both flavin and terpenoid biochemical repertoires.

The role of conserved residues in Fdc decarboxylase in prenylated flavin mononucleotide oxidative maturation, cofactor isomerization, and catalysis.

The UbiD family of reversible decarboxylases act on aromatic, heteroaromatic, and unsaturated aliphatic acids and utilize a prenylated flavin mononucleotide (prFMN) as cofactor, bound adjacent to a conserved Glu-Arg-Glu/Asp ionic network in the enzyme's active site. It is proposed that UbiD activation requires oxidative maturation of the cofactor, for which two distinct isomers, prFMNketimine and prFMNiminium, have been observed. It also has been suggested that only the prFMNiminium form is relevant to catalysis, which requires transient cycloaddition between substrate and cofactor. Using Aspergillus niger Fdc1 as a model system, we reveal that isomerization of prFMNiminium to prFMNketimine is a light-dependent process that is largely independent of the Glu277-Arg173-Glu282 network and accompanied by irreversible loss of activity. On the other hand, efficient catalysis was highly dependent on an intact Glu-Arg-Glu network, as only Glu → Asp substitutions retain activity. Surprisingly, oxidative maturation to form the prFMNiminium species is severely affected only for the R173A variant. In summary, the unusual irreversible isomerization of prFMN is light-dependent and probably proceeds via high-energy intermediates but is independent of the Glu-Arg-Glu network. Our results from mutagenesis, crystallographic, spectroscopic, and kinetic experiments indicate a clear role for the Glu-Arg-Glu network in both catalysis and oxidative maturation.

Oxidative Maturation and Structural Characterization of Prenylated FMN Binding by UbiD, a Decarboxylase Involved in Bacterial Ubiquinone Biosynthesis.

The activity of the reversible decarboxylase enzyme Fdc1 is dependent on prenylated FMN (prFMN), a recently discovered cofactor. The oxidized prFMN supports a 1,3-dipolar cycloaddition mechanism that underpins reversible decarboxylation. Fdc1 is a distinct member of the UbiD family of enzymes, with the canonical UbiD catalyzing the (de)carboxylation of para-hydroxybenzoic acid-type substrates. Here we show that the Escherichia coli UbiD enzyme, which is implicated in ubiquinone biosynthesis, cannot be isolated in an active holoenzyme form despite the fact active holoFdc1 is readily obtained. Formation of holoUbiD requires reconstitution in vitro of the apoUbiD with reduced prFMN. Furthermore, although the Fdc1 apoenzyme can be readily reconstituted and activated, in vitro oxidation to the mature prFMN cofactor stalls at formation of a radical prFMN species in holoUbiD. Further oxidative maturation in vitro occurs only at alkaline pH, suggesting a proton-coupled electron transfer precedes formation of the fully oxidized prFMN. Crystal structures of holoUbiD reveal a relatively open active site potentially occluded from solvent through domain motion. The presence of a prFMN sulfite-adduct in one of the UbiD crystal structures confirms oxidative maturation does occur at ambient pH on a slow time scale. Activity could not be detected for a range of putative para-hydroxybenzoic acid substrates tested. However, the lack of an obvious hydrophobic binding pocket for the octaprenyl tail of the proposed ubiquinone precursor substrate does suggest UbiD might act on a non-prenylated precursor. Our data reveals an unexpected variation occurs in domain mobility, prFMN binding, and maturation by the UbiD enzyme family.

The UbiX-UbiD system: The biosynthesis and use of prenylated flavin (prFMN).

The UbiX-UbiD system consists of the flavin prenyltransferase UbiX that produces prenylated FMN that serves as the cofactor for the (de)carboxylase UbiD. Recent developments have provided structural insights into the mechanism of both enzymes, detailing unusual chemistry in each case. The proposed reversible 1,3-dipolar cycloaddition between the cofactor and substrate serves as a model to explain many of the key UbiD family features. However, considerable variation exists in the many branches of the UbiD family tree.

Regioselective para-Carboxylation of Catechols with a Prenylated Flavin Dependent Decarboxylase.

The utilization of CO2 as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme-catalyzed para-carboxylation of catechols, employing 3,4-dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirmed that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMNiminium species. This study reports on the in vitro reconstitution and activation of a prFMN-dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving an intermediate with a single covalent bond between a quinoid adduct and cofactor is proposed, which is distinct from the mechanism of prFMN-associated 1,3-dipolar cycloadditions in related enzymes.

The Role of Parental ADHD in Sustaining the Effects of a Family-School Intervention for ADHD.

This study investigated the extent to which parental Attention-Deficit/Hyperactivity Disorder (ADHD) symptoms impact child and parent outcomes following a multimodal family-school intervention, the Family School Success (FSS) program, when compared to an active-control condition (CARE). Participants were 139 children with ADHD (67% male; 91% non-Hispanic; 77% Caucasian; Grades 2-6) and their primary caretaker (91% female; ages 26-59) who participated in a randomized clinical trial evaluating the efficacy of FSS. Associations were examined between parent-reported ADHD symptoms at baseline and intervention outcomes reported by parents and teachers after treatment and at a 3-month follow-up, including child homework and classroom impairments, child ADHD and oppositional defiant disorder symptoms, parenting behaviors, and parent-teacher relationship quality. Across both treatment conditions, parental ADHD was not associated with parent or child outcomes at postassessment. However, differences emerged between the two treatment groups at follow-up for parents with ADHD, particularly when an empirically supported symptom cutoff was used to identify parents at risk for having ADHD. In FSS, but not in CARE, parental ADHD was associated with declines in treatment gains in the quality of the parent-teacher relationship and the child's homework performance. Parents at risk for ADHD had difficulty maintaining treatment effects for themselves and their child in the FSS intervention but not in CARE. The supportive and educational components central to the CARE intervention may be helpful in promoting the sustainability of psychosocial interventions for children with ADHD who have parents with elevated ADHD symptoms.

Parent attendance and homework adherence predict response to a family-school intervention for children with ADHD.

This study examined the relative contribution of two dimensions of parent engagement, attendance and homework adherence, to parent and child treatment response and explored whether early engagement was a stronger predictor of outcomes than later engagement. The sample consisted of parents of participants (n = 92; M age = 9.4 years, SD = 1.27; 67% male, 69% White) in a 12-session evidence-based family-school intervention for children with attention-deficit/hyperactivity disorder. Attendance was assessed using clinician records, and homework adherence was measured by rating permanent products. Outcomes included parent and teacher ratings of family involvement in education, parenting practices, and child functioning. Accounting for the contributions of baseline scores and attendance, homework adherence was a significant predictor of parental self-efficacy, the parent-teacher relationship, parenting through positive involvement, and the child's inattention to homework and homework productivity. Accounting for the contribution of baseline scores and homework adherence, attendance was a significant predictor of one outcome, the child's academic productivity. Early homework adherence appeared to be more predictive of outcomes than later adherence, whereas attendance did not predict outcomes during either half of treatment. These results indicate that, even in the context of evidence-based practice, it is the extent to which parents actively engage with treatment, rather than the number of sessions they attend, that is most important in predicting intervention response. Because attendance is limited as an index of engagement and a predictor of outcomes, increased efforts to develop interventions to promote parent adherence to behavioral interventions for children are warranted.

Albistyla, a new genus of Neotropical Limosininae (Diptera: Sphaeroceridae) .

The genus Albistyla is described for a group of similar, highly distinctive Neotropical species that key out at couplet 55 of the key to Neotropical Sphaeroceridae in Marshall and Buck (2010). Albistyla is diagnosed by the combination of a single pair of strongly cruciate interfrontal bristles, two dorsocentral bristles, and the costa ending at the tip of R4+5. It is defined as a monophyletic group primarily on the basis of the unusually modified male sternite 5 and the distinctive white surstylus with broad apical scales. The genus is infrequently collected and only the type species is known from good series. Many of the specimens examined were taken in association with tree falls or similar habitats rich in green leaf litter.

A redefinition of the genus Hemichaeta Steyskal, new status (Diptera; Micropezidae; Taeniapterinae).

We here reconsider the status of subgenus Hemichaeta Steyskal, concluding that there is no justification to keep it in the genus Poecilotylus Hennig. Hemichaeta is thus removed from Poecilotylus and elevated to generic status with Hemichaeta scutellata (Cresson) as the type and only included species. Hemichaeta scutellata is fully redescribed, including the previously undescribed male and female terminalia.

A revision of the New World genus Aptilotella Duda (Sphaeroceridae: Limosininae).

The genus Aptilotella Duda (Diptera: Sphaeroceridae: Limosininae) is redefined and revised. The type species, Aptilotella borgmeieri Duda is redescribed, and twenty-seven species are described as new: Aptilotella andersoni sp. n., A. angela sp. n., A. caerulea sp. n., A. corona sp. n., A. diffisa sp. n., A. ebenea sp. n., A. erinacea sp. n., A. gemmula sp. n., A. germana sp. n., A. gladia sp. n., A. gloriosa sp. n., A. gracilis sp. n., A. hamata sp. n., A. involucris sp. n., A. macta sp. n., A. macula sp. n., A. pennifera sp. n., A. pichin chensis sp. n., A. pyropanda sp. n., A. quadrata sp. n., A. quatuorchela sp. n., A. radians sp. n., A. simplex sp. n., A. solaria sp. n., A. sphyra sp. n., A. umbracatus sp. n., and A. viva sp. n. A phylogenetic analysis and key to species are presented, with illustrations and distributional notes.

Bregmosina, a new Neotropical genus of Limosininae (Diptera: Sphaeroceridae).

Bregmosina, a new genus of Limosininae (Diptera, Sphaeroceridae), is described on the basis of five new species from Central and South America. The genus is characterized by a broad head, swollen face, large oral cavity, a single pair of very large cruciate interfrontal bristles, short, broad fore tarsomeres and swollen hind tarsomeres one and two. The unusual male genitalia, including a longitudinally split sternite 5, strongly indicate that the genus is monophyletic. All species are probably associated with treefalls.

Generic classification of the Archiborborinae (Diptera: Sphaeroceridae), with a revision of Antrops Enderlein, Coloantrops gen. nov., Maculantrops gen. nov., Photoantrops gen. nov., and Poecilantrops gen. nov.

The Archiborborinae comprise a diverse clade of flies in the family Sphaeroceridae. We here revise the generic classification, redefining the genus Antrops Enderlein and naming 5 new genera: Boreantrops gen. nov., Coloantrops gen. nov., Maculantrops gen. nov., Photoantrops gen. nov., and Poecilantrops gen. nov. The genus Archiborborus, until recently a paraphyletic assemblage including most of the described species in the subfamily, is treated as a junior synonym of Antrops (syn. nov.) We revise the genera Antrops (53 species, including 40 sp. nov.: Antrops anovariegatus, Antrops aurantifemur, Antrops baeza, Antrops bellavista, Antrops biflavus, Antrops bucki, Antrops carpishensis, Antrops cochabamba, Antrops cochinoca, Antrops coniobaptos, Antrops coroico, Antrops cotopaxi, Antrops didactylos, Antrops diversipennis, Antrops eurus, Antrops fulgiceps, Antrops fuliginosus, Antrops guandera, Antrops guaramacalensis, Antrops inca, Antrops juninensis, Antrops mucarensis, Antrops niger, Antrops papallacta, Antrops pecki, Antrops podocarpus, Antrops quadrilobus, Antrops siberia, Antrops sierrazulensis, Antrops tachira, Antrops tequendama, Antrops tetrastichus, Antrops tumbrensis, Antrops unduavi, Antrops variegatus, Antrops versabilis, Antrops vittatus, Antrops yungas, and Antrops zongo and the following comb. nov.: Antrops annulatus (Richards), Antrops chaetosus (Richards), Antrops femoralis (Blanchard), Antrops hirtus (Bigot), Antrops maculipennis (Duda), Antrops maximus (Richards), Antrops microphthalmus (Richards), Antrops quadrinotus (Bigot), Antrops setosus (Duda), Antrops simplicimanus (Richards), Antrops nitidicollis (Becker), and Antrops orbitalis (Duda)), Coloantrops (1 species: Coloantrops daedalus, sp. nov.), Maculantrops (2 species, Maculantrops hirtipes (Macquart) comb. nov. and Maculantrops altiplanus, sp. nov.), Photoantrops (1 species: Pho-toantrops echinus sp. nov.), and Poecilantrops (10 species: Poecilantrops baorucensis, Poecilantrops boraceiensis, Poecilantrops crocidosternum, Poecilantrops dominicus, Poecilantrops flavifemur, Poecilantrops marensis, Poecilantrops plaumanni, Poecilantrops psilosternum, Poecilantrops stellans, Poecilantrops vittifrons, all sp. nov.). Six additional species of Antrops and two of Poecilantrops known from females only are diagnosed but not described. The genus Boreantrops is described for two species (Boreantrops mexicanus (Steyskal) comb. nov., and Boreantrops calceatus (Duda) comb. nov.). Boreantrops includes a number of new species but is not fully revised here. New synonyms at the species level are: Antrops femoralis (Blanchard 1852) = Archiborborus argentinensis Papp 1977, and Archiborborus submaculatus Duda 1921, both syn. nov.; Antrops hirtus (Bigot 1888) = Archiborborus edwardsi Richards 1931, syn. nov.; Antrops quadrinotus (Bigot 1888) = Archiborborus albicans Richards 1931, Archiborborus chilensis Richards 1931, Archiborbo-rus koenigi Duda 1932, all syn. nov.; Maculantrops hirtipes (Macquart 1844) = Copromyza alternata Rondani 1868, syn. nov.

Development of a Family-School Intervention for Young Children With Attention Deficit Hyperactivity Disorder.

Although numerous studies have evaluated the effectiveness of multi-modal psychosocial interventions for children with attention deficit hyperactivity disorder, these programs are limited in that there has not beeti an explicit focus on the connection between fatnily and school. This study was designed to develop and pilot test a family-school ititervention, Family-School Success-Early Elementary (FSS-EE), for kindergarten and first-grade studetits with attention deficit hyperactivity disorder. Key components of FSS-EE were family-school behavioral consultatioti, daily report cards, and strategies to improve parent-child relationships atid family involvement in educatioti. FSS-EE was developed using a multistep iterative process. The piloted version consisted of 12 weekly sessions including 6 group meetings, 4 individualized family sessions, and 2 school-based consultations. Families participating in the study were given the choice of placing their childreti on medication; 25% of children were on medication at the time of random assignmetit. Childreti (n = 61) were randomly assigned to FSS-EE or a comparison group controlling for nonspecific treatment effects. Outcomes were assessed at post interventioti and 2-month follow-up. Study findings indicated that FSS-EE was feasible to implement and acceptable to paretits atid teachers. In addition, the findings provided preliminary evidence that FSS-EE is effective in improving parenting practices, child behavior at school, and the student-teacher relationship.

Amapeza and Nigripeza, new genera of Neotropical micropezid flies (Diptera, Micropezidae, Taeniapterinae).

The new Neotropical genera Amapeza and Nigripeza are described for seven new species and three named species previously treated as part of the genus Grallipeza Rondani. Amapeza includes A. amazonica (Enderlein) new combination, A. affinis (Hennig) new combination, A. hyaloptera (Hendel) new combination and the new species A. camelina A. plicata, and A. tricincta. Nigripeza includes N. obscura (Hennig) new combination and the new species N. cali, N. octa and N. spangleri.

A revision of the Australian species of Howickia Richards (Diptera: Sphaeroceridae: Limosininae).

Howickia Richards, 1951, the largest genus of Sphaeroceridae in Australia, is revised to include 43 species, including all wingless Australian species of the family. The following new species are described: H. acicula (TAS), H. auricoma (VIC), H. biantenna (TAS), H. cruspica (VIC), H. erythrocephala (NSW), H. flagella (TAS), H. grandisterna (TAS), H. holti (NSW), H. irwini (NSW), H. loligo (TAS), H. notechis (TAS), H. nuda (NSW), H. pertusa (TAS), H. pinnula (TAS), H. pseudovittata (NSW, QLD), H. robustaseta (NSW), H. tricolor (NSW), H. truemani (TAS), H. wamini (NSW, QLD, TAS, WA), and H. xanthocephala (QLD, NSW). The genera Apterobiroina Papp, 1979, Australimosina Papp, 2008, Bentrovata Richards, 1973, Monteithiana Richards, 1973, Popondetta Richards, 1973, and Otwayia Richards, 1973 are treated as new synonyms of Howickia. Howickia is redefined and placed in the context of a key to genera of Sphaeroceridae in Australia, the Australian species of Howickia are keyed, and a morphological phylogeny is given for the Australian species. The following new combinations are given: Howickia australis (Papp, 1979) comb. nov., Howickia cynthia (Richards, 1973) comb. nov., Howickia dealata (Richards, 1973) comb. nov., Howickia flavipes (Papp, 2021) comb. nov., Howickia flaviterga (Richards, 1973) comb. nov., Howickia flavithorax (Papp, 2021) comb. nov., Howickia regalis (Richards, 1973) comb. nov., Howickia sabina (Richards, 1973) comb. nov., Howickia truncata (Papp, 2021) comb. nov., Paralimosina nasalis (Richards, 1973) comb. nov. (formerly in Pseudocollinella Duda, 1924), Pseudopterogramma fenestrata (Richards, 1973) comb. nov. (originally as Leptocera (Limosina) Macquart, 1835, transferred to Biroina Richards, 1973 by Marshall in Rohácek et al. (2001)), and Pleuroseta dorrigonis (Richards, 1973) comb. nov. (formerly in Biroina). A new name, Howickia neoregalis nom. nov. is proposed for H. regalis Marshall, 2014, junior homonym of H. regalis (Richards, 1973). The following genera are newly recorded from Australia: Chaetopodella Duda, 1920, Paralimosina Papp, 1973, Pachytarsella Richards, 1963, Parapterogramma Papp, 2008, Philocoprella Richards, 1929, Piliterga Papp, 2008, Pterogrammoides Papp, 1972, Spelobia Spuler, 1924, and Spinilimosina Rohácek, 1983.

Vertical stratification of insect abundance and species richness in an Amazonian tropical forest.

Tropical forests are among the most biodiverse biomes on the planet. Nevertheless, quantifying the abundance and species richness within megadiverse groups is a significant challenge. We designed a study to address this challenge by documenting the variability of the insect fauna across a vertical canopy gradient in a Central Amazonian tropical forest. Insects were sampled over two weeks using 6-m Gressitt-style Malaise traps set at five heights (0 m-32 m-8 m intervals) on a metal tower in a tropical forest north of Manaus, Brazil. The traps contained 37,778 specimens of 18 orders of insects. Using simulation approaches and nonparametric analyses, we interpreted the abundance and richness of insects along this gradient. Diptera, Hymenoptera, and Coleoptera had their greatest abundance at the ground level, whereas Lepidoptera and Hemiptera were more abundant in the upper levels of the canopy. We identified species of 38 of the 56 families of Diptera, finding that 527 out of 856 species (61.6%) were not sampled at the ground level. Mycetophilidae, Tipulidae, and Phoridae were significantly more diverse and/or abundant at the ground level, while Tachinidae, Dolichopodidae, and Lauxaniidae were more diverse or abundant at upper levels. Our study suggests the need for a careful discussion of strategies of tropical forest conservation based on a much more complete understanding of the three-dimensional distribution of its insect diversity.

Stipulosina new genus (Diptera: Sphaeroceridae: Limosininae) from bamboo (Chusquea scandens) stipules in Ecuador.

The new genus Stipulosina (Sphaeroceridae: Limosininae) is described for four new species, S. amphibarba n. sp. (type species), S. barbistylus n. sp., S. ceroptistylus n. sp. and S. curvistylus n. sp., collected from bamboo stipules in the Ecuadorian Andes in 2002 and 2019. All four species are associated with stipules of the Andean bamboo Chusquea scandens Kunth but not with those of sympatric bamboo species.

The In Vitro Production of prFMN for Reconstitution of UbiD Enzymes.

Prenylated flavin (prFMN) is a modified FMN cofactor, the isoalloxazine is extended by an additional six membered nonaromatic ring. The modification confers azomethine ylide characteristics on the oxidised prFMN, allowing it to support the reversible nonoxidative decarboxylation of unsaturated acids by the UbiD family of decarboxylases. In absence of a chemical synthesis route for prFMN, enzymatic production by the flavin prenyltransferase, UbiX, is required for in vitro reconstitution of prFMN-dependent enzymes. Here we provide an overview of the methods for producing prFMN in vitro using the flavin prenyltransferase UbiX, and the subsequent reconstitution and activation of UbiD enzymes.

Synopsis of Nothybidae (Diptera: Brachycera) from China, with redescription of Nothybus absens Lonsdale amp; Marshall.

The Nothybidae of China are reviewed, including Nothybus absens Lonsdale Marshall, 2016 which was previously known only from photographs, is here redescribed on the basis of male and female specimens. A key to Chinese species of Nothybidae is provided.

Systematic relationships of the Taeniapterini (Diptera: Micropezidae, Taeniapterinae).

A molecular phylogeny for the Taeniapterinae is presented based on mitochondrial (cytochrome c oxidase subunit I, COI), ribosomal (16S rRNA and 28S rRNA) and nuclear (EF-1 and CAD) genes of 48 specimens including 40 species. Maximum likelihood and Bayesian inference were used to analyze the total concatenated dataset of 8769 bp. The results confirm that tribal classifications in Taeniapterinae are artificial and support the separation of Paragrallomyia Hendel and Taeniaptera sensu Ferro Marshall (2020).