Small Subunit Research Articles

Impact of yeast extract and basal salts medium on 1,4-dioxane biodegradation rates and the microorganisms involved in carbon uptake from 1,4-dioxane

Conventional physical and chemical treatment technologies for 1,4-dioxane can be ineffective and consequently attention has focused on bioremediation. Towards this, the current research investigated the impact of basal salts medium (BSM) and yeast extract on 1,4-dioxane biodegradation rates in microcosms with different soil or sediment (agricultural soil, wetland sediment, sediment from an impacted site). Phylotypes responsible for carbon uptake from 1,4-dioxane were determined using stable isotope probing (SIP), both with and without BSM and yeast extract. Further, putative functional genes were investigated using 1) soluble di-iron monooxygenase (SDIMO) based amplicon sequencing, 2) qPCR targeting propane monooxygenase (large subunit, prmA) and 3) a predictive approach (PICRUSt2). The addition of BSM and yeast extract significantly enhanced 1,4-dioxane removal rates the agricultural soil and impacted site sediment microcosms. The phylotypes associated with carbon uptake varied across treatments and inocula. Gemmatimonas was important in the heavy SIP fractions of the wetland sediment microcosms. Unclassified Solirubacteraceae, Solirubrobacter, Pseudonocardia and RB4 were dominant in the heavy SIP fractions of the agricultural soil microcosms. The heavy SIP fractions of the impacted site microcosms were dominated by only two phylotypes, unclassified Burkholderiaceae and oc3299. SDIMO based amplicon sequencing detected three genes previously associated with 1,4-dioxane. The predicted functional gene analysis suggested the importance of propane monooxygenases associated with Solirubrobacter and Pseudonocardia. Overall, more microorganisms were involved in carbon uptake from 1,4-dioxane in both the wetland and agricultural soil microcosms compared to the impacted site sediment microcosms. Many of these microorganisms have not previously been associated with 1,4-dioxane removal.

Two species of the green algae Volvox sect. Volvox from the Japanese ancient lake, Lake Biwa.

Volvox sect. Volvox is a group of green algae with unique morphological features (thick cytoplasmic bridges between somatic cells and spiny zygote walls) and a worldwide distribution. Despite research interest in the diversity of organisms in ancient lakes, Volvox sect. Volvox from ancient lakes worldwide has not been identified to the species level. Here, we established clonal cultures of two species of this group originating from Lake Biwa, an ancient lake in Japan, and performed identification based on morphological and molecular data. One was identified as Volvox kirkiorum based on the nuclear ribosomal DNA internal spacer region (ITS) sequence, bisexual (monoicous or monoecious) spheroids, and zygote morphology. The other showed genetic separation from related species based on the secondary structure of the ITS and results of phylogenetic analysis of a combined data set from the nuclear actin gene, ITS, and two plastid genes (large subunit of RuBisCO and photosystem II CP43 apoprotein gene); it represented a new phylogenetic lineage within Volvox sect. Volvox, suggesting possible endemism in Lake Biwa. This species produced bisexual spheroids with different zygote morphology and zygote number from other species with bisexual spheroids in Volvox sect. Volvox. Therefore, Volvox biwakoensis Nozaki et H. Yamaguchi sp. nov. is described herein. This is the first endemic species of the genus Volvox described from an ancient lake.

Modular Structure and Polymerization Status of GABAA Receptors Illustrated with EM Analysis and AlphaFold2 Prediction.

Type-A γ-aminobutyric acid (GABAA) receptors are channel proteins crucial to mediating neuronal balance in the central nervous system (CNS). The structure of GABAA receptors allows for multiple binding sites and is key to drug development. Yet the formation mechanism of the receptor's distinctive pentameric structure is still unknown. This study aims to investigate the role of three predominant subunits of the human GABAA receptor in the formation of protein pentamers. Through purifying and refolding the protein fragments of the GABAA receptor α1, β2, and γ2 subunits, the particle structures were visualised with negative staining electron microscopy (EM). To aid the analysis, AlphaFold2 was used to compare the structures. Results show that α1 and β2 subunit fragments successfully formed homo-oligomers, particularly homopentameric structures, while the predominant heteropentameric GABAA receptor was also replicated through the combination of the three subunits. However, homopentameric structures were not observed with the γ2 subunit proteins. A comparison of the AlphaFold2 predictions and the previously obtained cryo-EM structures presents new insights into the subunits' modular structure and polymerization status. By performing experimental and computational studies, a deeper understanding of the complex structure of GABAA receptors is provided. Hopefully, this study can pave the way to developing novel therapeutics for neuropsychiatric diseases.

First report of Pseudopithomyces palmicola causing leaf spot on tobacco in China.

In July 2023, a new leaf spot disease emerged on tobacco leaves in Meitan County, Guizhou Province, China (27°20'18" - 28°12'30"N, 107°15'36" - 107°41'08"E, average altitude 972 meters). Initially, the symptoms showed raised yellow-brown spots; subsequently, the lesions expanded and became broken and perforated, leading to a significant loss of economic value, the prevalence rate exceeded 30%. For isolation, two tissue fragments (0.2 × 0.2 cm) of symptomatic leaves were sterilized in 75% ethanol for 30 s, 3% NaClO for 2 min, and were washed 3 times in sterilized distilled water, and were subsequently inoculated on potato dextrose agar (PDA), and incubated at 28°C for 9 days in the dark. The two strains CW16 and CW28 were isolated using the single hyphae method (Nouri et al. 2023). Both strains formed pale to yellow white colonies on PDA. Conidia had three constricted transverse septa and 1 to 2 longitudinal septa in the central cells, with thick and hyaline conidiophores and mostly globose, pale brown conidia with slightly constricted septa, their average size were measured as 13.4-22.4×8.358-13.347 μm (n = 50). Genomic DNA was extracted from the isolated strains CW16 and CW28. The internal transcribed spacer regions 1 and 2 as well as 5.8S nuclear ribosomal RNA (ITS), large subunit nrRNA (LSU), and partial DNA-directed RNA polymerase II second largest subunit (RPB2) genes were amplified using primers (Cui et al. 2023). The sequences had been deposited in GenBank under accession numbers ITS: PP024201, PP024205; LSU: PP024207, PP024209; RPB2: PP060480, PP060481. The sequences analysis revealed a high similarity of 99.74 to 100% between strains CW16 and CW28 with P. palmicola isolate KM42 (ITS OQ875842, LSU OQ875844, RPB2 OQ883943) in GenBank. Using BLAST for homology matching, two isolates (CW16, CW28) and with the sequences of the ten type isolates from GenBank, phylogenetic analysis was conducted using the Maximum Likelihood method in MEGA (11.0) software based on ITS, LSU and RPB2 sequences, which showed that strains CW16, CW28 clustered in the same score as the Pseudopithomyces palmicola, confirming the morphological and molecular characteristics identification. The pathogenicity tests were conducted on healthy tobacco plants with 4-5 leaves (Fig. S1B), the isolated strains, CW16 and CW28, were used to inoculate the healthy tobacco leaves, while blank PDA was used as a control. All plants were maintained in a greenhouse at 28°C with a relative humidity of 90%. After 9 days, necrotic spots were observed on all tobacco leaves inoculated with CW16 and CW28 fungal plugs, while the blank PDA-inoculated tobacco leaves showed no symptoms. Based on morphological and molecular characteristics, the same pathogen P. palmicola was identified from the inoculated leaves, fulfilling Koch's postulates. This study represents the first reported of tobacco leaf spot caused by P. palmicola in China and provides a theoretical basis for future prevention and control measures.

Large-scale map of RNA-binding protein interactomes across the mRNA life cycle

mRNAs interact with RNA-binding proteins (RBPs) throughout their processing and maturation. While efforts have assigned RBPs to RNA substrates, less exploration has leveraged protein-protein interactions (PPIs) to study proteins in mRNA life-cycle stages. We generated an RNA-aware, RBP-centric PPI map across the mRNA life cycle in human cells by immunopurification-mass spectrometry (IP-MS) of ∼100 endogenous RBPs with and without RNase, augmented by size exclusion chromatography-mass spectrometry (SEC-MS). We identify 8,742 known and 20,802 unreported interactions between 1,125 proteins and determine that 73% of the IP-MS-identified interactions are RNA regulated. Our interactome links many proteins, some with unknown functions, to specific mRNA life-cycle stages, with nearly half associated with multiple stages. We demonstrate the value of this resource by characterizing the splicing and export functions of enhancer of rudimentary homolog (ERH), and by showing that small nuclear ribonucleoprotein U5 subunit 200 (SNRNP200) interacts with stress granule proteins and binds cytoplasmic RNA differently during stress.

Occurrence rate and species and subtypes of Cryptosporidium spp. in pet dogs in Yunnan Province, China

BackgroundCryptosporidium spp. is a ubiquitous, globally distributed intestinal protozoan infecting humans and at least 260 animal hosts. Due to close human contact with pet dogs and identification of zoonotic Cryptosporidium species and subtypes in these animals, dog health is not only a veterinarian issue but also a public health issue. This study aimed to understand occurrence and genetic characterization at both genotype and subtype levels in pet dogs in Yunnan Province, China.ResultsA total of 589 fresh fecal specimens were collected from adult pet dogs in the rural areas of eight cities/autonomous prefectures of Yunnan Province, China. 16 fecal specimens were positive for Cryptosporidium spp. by polymerase chain reaction (PCR) amplification and sequence analysis of the small subunit ribosomal RNA (SSU rRNA) gene, with an average occurrence rate of 2.7% (16/589) being observed. Three zoonotic Cryptosporidium species were identified: C. parvum (n = 7), C. suis (n = 5) and C. canis (n = 4). At the 60-kDa glycoprotein (gp60) locus, only three C. parvum and two C. canis specimens were successfully amplified and sequenced, with subtype IIaA17G2R1 (n = 3) and subtypes XXa4 (n = 1) and XXa5 (n = 1) being identified, respectively.ConclusionsThe present finding of three zoonotic Cryptosporidium species in dogs implied that dogs infected with Cryptosporidium spp. may pose a threat to human health. C. suis was identified in dogs in this study for the first time, expanding the host range of this species. Identification of C. parvum subtype IIaA17G2R1 and C. canis subtypes XXa4 and XXa5 will be helpful to explore the source attribution of infection/contamination and assess the transmission dynamics of C. parvum and C. canis in the investigated areas in the future.

Addition to the genus Harmajaea (Agaricales, Pseudoclitocybaceae): a new and a known species from China

A new species, Harmajaea xizangense, discovered in Tibet, China, is illustrated and described based on morphological and molecular data. Morphologically, H. xizangense is mainly characterized by its small pileus, relatively larger and longer spores, and unique geographical distribution. Phylogenetic analyses were performed based on nucleotide sequences of four molecular loci: the internal transcribed spacer (ITS), the nuclear ribosomal large subunit (nrLSU), the DNA-directed RNA polymerase II subunit 2 (RPB2), and the translation elongation factor (TEF1-α). The results indicated that the specimens belonged to the genus Harmajaea, occupying an isolated position in the phylogenetic trees. Furthermore, three collections of H. harperi are introduced for the first time from Liaoning Province of China. This study provides full descriptions, color illustrations, and phylogenetic trees of the two Harmajaea species.

Host Tropism and Structural Biology of ABC Toxin Complexes.

ABC toxin complexes are a class of protein toxin translocases comprised of a multimeric assembly of protein subunits. Each subunit displays a unique composition, contributing to the formation of a syringe-like nano-machine with natural cargo carrying, targeting, and translocation capabilities. Many of these toxins are insecticidal, drawing increasing interest in agriculture for use as biological pesticides. The A subunit (TcA) is the largest subunit of the complex and contains domains associated with membrane permeation and targeting. The B and C subunits, TcB and TcC, respectively, package into a cocoon-like structure that contains a toxic peptide and are coupled to TcA to form a continuous channel upon final assembly. In this review, we outline the current understanding and gaps in the knowledge pertaining to ABC toxins, highlighting seven published structures of TcAs and how these structures have led to a better understanding of the mechanism of host tropism and toxin translocation. We also highlight similarities and differences between homologues that contribute to variations in host specificity and conformational change. Lastly, we review the biotechnological potential of ABC toxins as both pesticides and cargo-carrying shuttles that enable the transport of peptides into cells.

Mapping of oxidative modifications on the alpha-keto glutarate dehydrogenase complex induced by singlet oxygen: Effects on structure and activity

The large multi-subunit mitochondrial alpha-keto glutarate dehydrogenase (KGDH) complex plays a key, rate-determining, role in the tricarboxylic acid (Krebs) cycle, catalyzing the conversion of alpha-keto glutarate to succinyl-CoA. This complex is both a source and target of oxidants, but the sites of modification and association with structural changes and activity loss are poorly understood. We report here oxidative modifications induced by Rose Bengal (RB) in the presence of O2, a source of singlet oxygen (1O2). A rapid loss of activity was detected, with this being dependent on light exposure, illumination time, and the presence of RB and O2. Activity loss was enhanced by D2O (consistent with 1O2 involvement), but diminished by both pre- and (to a lesser extent) post-illumination addition of lipoic acid and lipoamide. Aggregates containing all three KGDH subunits were detected on photooxidation. LC-MS experiments provided evidence for oxidation at 45 sites, including specific Met, His, Trp, Tyr residues and the lipoyllysine active-site cofactor. Products include mono- and di-oxygenated species, and kynurenine from Trp. Mapping of the modifications to the 3-D structure showed that these are localized to both the inner channel and the external surface, consistent with reactions of free 1O2, however the sites and extent of modification do not correlate with their solvent accessibility. These products are generated concurrently with loss of activity, indicative of strong links between these events. These data provide evidence for the impairment of KGDH activity by 1O2 via the oxidation of specific residues on the protein subunits of the complex.

First Report of Powdery Mildew on Vitis bryoniifolia Caused by Erysiphe necator in China.

Grapes, belonging to the genus Vitis, are one of the world's most economically valuable fruit crops, which are widely used as the source of wine, raisins, and juice. The genus Vitis contains about 60 species mainly distributed in Asia, Europe, and North America to northern South America (Wan et al. 2013). In May 2022, severe powdery mildew symptoms were found on wild Vitis bryoniifolia plants at Guizhou Normal University, Guiyang, China. The incidence observed was approximately 85% among 50 V. bryoniifolia plants. Infected leaves appear white powdery patches, eventually leading to chlorosis to necrosis. Mycelia occurred on adaxial and abaxial leaf surfaces, petioles and young shoots. Upon microscopic observation hyphae were hyaline and 3.5-6 μm wide. Hyphal appressoria were solitary and lobed to multilobed. Conidiophores were erect, straight to somewhat flexuous and 80-130 µm long (n = 30). Foot cells were subcylindrical to curved-sinuous at the base, followed by 2-4 cells. Conidia formed singly (occasionally 2-6 in a chain) and were ellipsoid to ovoid in shape with dimensions of 22.5-38 × 12-19.5 µm (n = 50). No fibrosin bodies were observed on conidia. Based on these morphological characteristics, the powdery mildew fungus strongly resembled Erysiphe necator (Braun and Takamasu 2000; Zheng et al. 1987) that has been shown to cause powdery mildew on Vitis (Gadoury et al. 2012; Gaforio et al. 2011; Qiu et al. 2015). To confirm the identification, the ribosomal DNA internal transcribed spacer (ITS) and the ribosomal large subunit (LSU) region were amplified and sequenced using the ITS1/ITS4 primer pair (White et al. 1990) and the NL1/NL4 primer pair (Ziemiecki et al. 1990), respectively. The spliced 1250-bp ITS-LSU sequence (GenBank accession no. PP188565) shared 99.84-100% identity with ITS-LSU sequences of E. necator (LC028995, LC028996, ON073862, LC777882, and OM033353). Based on the phylogenetic analysis of the combined ITS-LSU dataset of Erysiphe species (Takamasu et al. 2015), PP188565 was grouped in a clade with E. necator strains MUMH530, MUMHs141, and VPRI19719. To perform pathogenicity analysis, leaves of three healthy, potted 1-year-old V. bryoniifolia plants were inoculated by gently pressing with diseased leaves. Three non-inoculated healthy plants served as controls. All plants were incubated in a greenhouse at 25 ± 2°C with 80% relative humidity. Powdery mildew symptoms, similar to field conditions were observed on inoculated plants 13 days after inoculation, whereas control plants remained symptomless. Fungus reisolated from inoculated V. bryoniifolia plants was morphologically identical to that on originally diseased plants, and the spliced ITS-LSU sequence of reisolated fungus shared 100% identity with PP188565, fulfilling Koch's postulates. Based on the morphological and molecular characterizations, the powdery mildew fungus was identified as E. necator. To our knowledge, this is the first report of powdery mildew caused by E. necator on V. bryoniifolia in China. This work further expands the host range of Erysiphe necator on Vitis species.

A cross-sectional analysis identifies a low prevalence of Plasmodium ovale species infections in symptomatic and asymptomatic individuals in Kilifi county, Kenya.

Background The focus on P. falciparum diagnosis has led to an underestimation of the global burden of malaria resulting from neglected Plasmodium species. However, there is still scarce data on the prevalence of P. ovale species (spp) globally. To address this knowledge gap, data collected from cross-sectional studies in Kilifi county were used to: 1) determine the prevalence of P. ovale spp infections; and 2) determine the sensitivity of different diagnostic assays in detecting P. ovale spp infections. Methods A total of 531 individuals were sampled across three study sites in Kilifi County, Kenya between 2009 and 2020. Blood smears were prepared from peripheral blood and screened for Plasmodium parasite stages using light microscopy. Molecular screening involved DNA extraction of dried blood spots and blood in ethylenediaminetetraacetic acid, polymerase chain reaction (PCR) using primers targeting the 18 small ribosomal subunit and sequencing. Results Microscopy screening revealed that the most prevalent species was P. falciparum (32.0%) followed by P. malariae (9.0%) and then P. ovale spp( 1.5%). PCR screening identified additional P. ovale spp positives cases. Overall PCR results indicate that43 (8.1%) out of the 531 individuals harbored P. ovale spp infection with the highest prevalence reported in the tertiary health facility, (14.6%, 95% CI 8-23.6%), followed by the primary health facility (8.3%, 95% CI 5.4-11.9%), and the community from a cross-sectional blood survey, (3.6%, 95% CI 1.2-8.2%). Microscopy screening for P. ovale spp had a low sensitivity of 7% (95% CI 1-19-30%) and a high specificity of 99% (95% CI 98-100%). Sequencing results confirmed the presence of P.ovale curtisi. Conclusions This study provides baseline data for P.ovale spp surveillance in Kilifi County, primarily using PCR to improve diagnosis. These results suggest that malaria elimination and eradication efforts should not only concentrate on P. falciparum but should embrace a holistic approach towards elimination of all Plasmodium spp.

Monitoring SARS-CoV-2 IgA, IgM and IgG antibodies in dried blood and saliva samples using antibody proximity extension assays (AbPEA)

Using a modified proximity extension assay, total and immunoglobulin (Ig) class-specific anti-SARS-CoV-2 antibodies were sensitively and conveniently detected directly from ø1.2 mm discs cut from dried blood and saliva spots (DBS and DSS) without the need for elution. For total Ig detection, antigen probes were prepared by conjugating recombinant spike protein subunit 1 (S1-RBD) to a pair of oligonucleotides. To detect isotype-specific antibody reactivity, one antigen probe was replaced with oligonucleotide-conjugated antibodies specific for antibody isotypes. Binding of pairs of oligonucleotide-conjugated probes to antibodies in patient samples brings oligonucleotides in proximity. An added DNA polymerase uses a transient hybridization between the oligonucleotides to prime synthesis of a DNA strand, which serves as a DNA amplicon that is quantified by real-time PCR. The S1-RBD-specific IgG, IgM, and IgA antibodies in DBS samples collected over the course of a first and second vaccination exhibited kinetics consistent with previous reports. Both DBS and DSS collected from 42 individuals in the autumn of 2023 showed significant level of total S1-RBD antibodies with a correlation of R = 0.70. However, levels in DSS were generally 10 to 100-fold lower than in DBS. Anti-S1-RBD IgG and IgA in DSS demonstrated a correlation of R = 0.6.

The DNA damage response and RNA Polymerase II regulator Def1 has posttranscriptional functions in the cytoplasm.

Yeast Def1 mediates RNA polymerase II degradation and transcription elongation during stress. Def1 is predominantly cytoplasmic, and DNA damage signals cause its proteolytic processing, liberating its N-terminus to enter the nucleus. Cytoplasmic functions for this abundant protein have not been identified. Proximity-labeling (BioID) experiments indicate that Def1 binds to an array of proteins involved in posttranscriptional control and translation of mRNAs. Deleting DEF1 reduces both mRNA synthesis and decay rates, indicating transcript buffering in the mutant. Directly tethering Def1 to a reporter mRNA suppressed expression, suggesting that Def1 directly regulates mRNAs. Surprisingly, we found that Def1 interacts with polyribosomes, which requires its ubiquitin-binding domain located in its N-terminus. The binding of Def1 to ribosomes requires the ubiquitylation of eS7a (Rsp7A) in the small subunit by the Not4 protein in the Ccr4-Not complex. Not4 ubiquitylation of the ribosome regulates translation quality control and co-translational mRNA decay. The polyglutamine-rich unstructured C-terminus of Def1 is required for its interaction with decay and translation factors, suggesting that Def1 acts as a ubiquitin-dependent scaffold to link translation status to mRNA decay. Thus, we have identified a novel function for this transcription and DNA damage response factor in posttranscriptional regulation in the cytoplasm and establish Def1 as a master regulator of gene expression, functioning during transcription, mRNA decay, and translation.

Current Challenges of Vaccination in Fish Health Management.

Vaccination is an essential method of immunological preventive care required for the health management of all animals, including fish. More particularly, immunization is necessary for in-land aquaculture to manage diseases in fish broodstocks and healthy seed production. According to the latest statistics in 2020, 90.3 million tons of capture fishery production was achieved from the aquaculture sector. Out of the above, 78.8 million tons were from marine water aquaculture sectors, and 11.5 million tons were from inland water aquaculture sectors. About a 4% decline in fish production was achieved in 2020 in comparison to 2018 from inland aquaculture sectors. On the other hand, the digestive protein content, healthy fats, and nutritional values of fish products are comparatively more affordable than in other meat sources. In 2014, about 10% of aquatic cultured animals were lost (costing global annual losses > USD 10 billion) due to infectious diseases. Therefore, vaccination in fish, especially in broodstocks, is one of the essential approaches to stop such losses in the aquaculture sector. Fish vaccines consist of whole-killed pathogens, protein subunits, recombinant proteins, DNA, or live-attenuated vaccines. Challenges persist in the adaption of vaccination in the aquaculture sector, the route of administration, the use of effective adjuvants, and, most importantly, the lack of effective results. The use of autogenous vaccines; vaccination via intramuscular, intraperitoneal, or oral routes; and, most importantly, adding vaccines in feed using top dressing methods or as a constituent in fish feed are now emerging. These methods will lower the risk of using antibiotics in cultured water by reducing environmental contamination.

Two new species of Fusarium in the F. incarnatum-equiseti species complex from Oryza sativa in Iran.

Identification of Fusarium species associated with diseases symptoms in plants is an important step toward understanding the ecology of plant-fungus associations. In this study, four Fusarium isolates were obtained from root rot of Oryza sativa L. in Izeh (southwest of Iran) and identified based on phylogenetic analyses combined with morphology. Phylogenetic analyses based on combined translation elongation factor 1-α, calmodulin, RNA polymerase II second largest subunit, and Beta-tubulin (tub2) sequence data delimited two new species, namely F. khuzestanicum and F. oryzicola spp. nov., from previously known species of Fusarium within F. incarnatum-equiseti species complex (FIESC). Morphologically, F. khuzestanicum produces the macroconidia with distinctly notched to foot-shaped basal cells, while basal cells in the macroconidia of F. oryzicola are more extended and distinctly elongated foot shape. Furthermore, these two new species are distinguished by the size of their sporodochial phialides and macroconidia. The results of the present show that the FIESC species complex represent more cryptic species.

IPSC-Derived LCHADD Retinal Pigment Epithelial Cells Are Susceptible to Lipid Peroxidation and Rescued by Transfection of a Wildtype AAV-HADHA Vector.

Progressive choroid and retinal pigment epithelial (RPE) degeneration causing vision loss is a unique characteristic of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), a fatty acid oxidation disorder caused by a common c.1528G>C pathogenic variant in HADHA, the α subunit of the mitochondrial trifunctional protein (TFP). We established and characterized an induced pluripotent stem cell (iPSC)-derived RPE cell model from cultured skin fibroblasts of patients with LCHADD and tested whether addition of wildtype (WT) HAHDA could rescue the phenotypes identified in LCHADD-RPE. We constructed an rAAV expression vector containing 3' 3xFLAG-tagged human HADHA cDNA under the transcriptional control of the cytomegalovirus (CMV) enhancer-chicken beta actin (CAG) promoter (CAG-HADHA-3XFLAG). LCHADD-RPE were cultured, matured, and transduced with either AAV-GFP (control) or AAV-HADHA-3XFLAG. LCHADD-RPE express TFP subunits and accumulate 3-hydroxy-acylcarnitines, cannot oxidize palmitate, and release fewer ketones than WT-RPE. When LCHADD-RPE are exposed to docosahexaenoic acid (DHA), they have increased oxidative stress, lipid peroxidation, decreased viability, and are rescued by antioxidant agents potentially explaining the pathologic mechanism of RPE loss in LCHADD. Transduced LCHADD-RPE expressing a WT copy of TFPα incorporated TFPα-FLAG into the TFP complex in the mitochondria and accumulated significantly less 3-hydroxy-acylcarnitines, released more ketones in response to palmitate, and were more resistant to oxidative stress following DHA exposure than control. iPSC-derived LCHADD-RPE are susceptible to lipid peroxidation mediated cell death and are rescued by exogenous HADHA delivered with rAAV. These results are promising for AAV-HADHA gene addition therapy as a possible treatment for chorioretinopathy in patients with LCHADD.

A Broad Spectrum Lasso Peptide Antibiotic Targeting the Bacterial Ribosome.

Lasso peptides, biologically active molecules with a distinct structurally constrained knotted fold, are natural products belonging to the class of ribosomally-synthesized and posttranslationally modified peptides (RiPPs). Lasso peptides act upon several bacterial targets, but none have been reported to inhibit the ribosome, one of the main antibiotic targets in the bacterial cell. Here, we report the identification and characterization of the lasso peptide antibiotic, lariocidin (LAR), and its internally cyclized derivative, lariocidin B (LAR-B), produced by Paenabacillussp. M2, with broad-spectrum activity against many bacterial pathogens. We show that lariocidins inhibit bacterial growth by binding to the ribosome and interfering with protein synthesis. Structural, genetic, and biochemical data show that lariocidins bind at a unique site in the small ribosomal subunit, where they interact with the 16S rRNA and aminoacyl-tRNA, inhibiting translocation and inducing miscoding. LAR is unaffected by common resistance mechanisms, has a low propensity for generating spontaneous resistance, shows no human cell toxicity, and has potent in vivo activity in a mouse model of Acinetobacter baumannii infection. Our finding of the first ribosome-targeting lasso peptides uncovers new routes toward discovering alternative protein synthesis inhibitors and offers a new chemical scaffold for developing much-needed antibacterial drugs.

EMS-induced missense mutation in TaCHLI-7D affects leaf color and yield-related traits in wheat.

Mutations in TaCHLI impact chlorophyll levels and yield-related traits in wheat. Natural variations in TaCHLI-7A/B influence plant productivity, offering potential for molecular breeding. Chlorophyll is essential for plant growth and productivity. The CHLI subunit of the magnesium chelatase protein plays a key role inserting magnesium into protoporphyrin IX during chlorophyll biosynthesis. Here, we identify a novel wheat mutant chlorophyll (chl) that exhibits yellow-green leaves, reduced chlorophyll levels, and increased carotenoid content, leading to an overall decline in yield-related traits. Map-based cloning reveals that the chl phenotype is caused by a point mutation (Asp186Asn) in the TaCHLI-7D gene, which encodes subunit I of magnesium chelatase. Furthermore, the three TaCHLI mutants: chl-7b-1 (Pro82Ser), chl-7b-2 (Ala291Thr), and chl-7d-1 (Gly357Glu), also showed significant reductions in chlorophyll content and yield-related traits. However, TaCHLI-7D overexpression in rice significantly decreased thousand kernel weight, yield per plant, and germination. Additionally, natural variations in TaCHLI-7A/B are significantly associated with flag leaf, spike exsertion length, and yield per plant. Notably, the favorable haplotype, TaCHLI-7B-HapII, which displayed higher thousand kernel weight and yield per plant, is positively selected in wheat breeding. Our study provides insights on the regulatory molecular mechanisms underpinning leaf color and chlorophyll biosynthesis, and highlights TaCHLI functions, which provide useful molecular markers and genetic resources for wheat breeding.

CALHM2 is a mitochondrial protein import channel that regulates fatty acid metabolism.

For mitochondrial metabolism to occur in the matrix, multiple proteins must be imported across the two (inner and outer) mitochondrial membranes. Classically, two protein import channels, TIM/TOM, are known to perform this function, but whether other protein import channels exist is not known. Here, using super-resolution microscopy, proteomics, and electrophysiological techniques, we identify CALHM2 as the import channel for the ECHA subunit of the mitochondrial trifunctional protein (mTFP), which catalyzes β-oxidation of fatty acids in the mitochondrial matrix. We find that CALHM2 sits specifically at the inner mitochondrial and cristae membranes and is critical for membrane morphology. Depletion of CALHM2 leads to a mislocalization of ECHA outside of the mitochondria leading to severe cellular metabolic defects. These defects include cytosolic accumulation of fatty acids, depletion of tricarboxylic acid cycle enzymes and intermediates, and reduced cellular respiration. Our data identify CALHM2 as an essential protein import channel that is critical for fatty acid- and glucose-dependent aerobic metabolism.

The First Introduction of an Exogenous 5' Untranslated Region for Control of Plastid Transgene Expression in Chlamydomonas reinhardtii.

The utilization of heterologous 5' untranslated regions (5'UTRs) for expressing foreign proteins in the chloroplast of Chlamydomonas reinhardtii (C. reinhardtii) has posed a persistent challenge over the years. This challenge stems from the lack of a defined and comprehensive set of translational cis-elements responsible for stability, ribosome binding, and translation initiation, which are mediated by trans-acting factors native to C. reinhardtii. In the current study, we aimed to address this bottleneck by employing the 5'UTR from gene 10 of the T7 bacteriophage (T7g10 5'UTR), fused to the promoter of C. reinhardtii small subunit ribosomal RNA (rrnS), to facilitate the translation of a reporter gene, YFP. Using a chimeric construct, the YFP mRNA was efficiently translated utilizing the heterologous T7g10 5'UTR. Furthermore, the accumulation of YFP protein under the control of the T7g10 5'UTR was approximately one third of that observed under the control of the endogenous psaA promoter/5'UTR in the C. reinhardtii chloroplast. The results of computational analyses demonstrated that the T7g10 5'UTR sequence shares common elements with the endogenous 5'UTRs of the chloroplast genes. Moreover, the findings of the current study highlighted the potential of employing bacteriophage 5'UTRs for the foreign protein accumulation from the chloroplast genome of C. reinhardtii.