Regulatory DNA Sequences Research Articles

Deep molecular learning of transcriptional control of a synthetic CRE enhancer and its variants

Massively parallel reporter assay measures transcriptional activities of various cis-regulatory modules (CRMs) in a single experiment. We developed a thermodynamic computational model framework that calculates quantitative levels of gene expression directly from regulatory DNA sequences. Using the framework, we investigated the molecular mechanisms of cis-regulatory mutations of a synthetic enhancer that cause abnormal gene expression. We found that, in a human cell line, competitive binding between family transcription factors (TFs) with slightly different binding preferences significantly increases the accuracy of recapitulating the transcriptional effects of thousands of single- or multi-mutations. We also discovered that even if various harmful mutations occurred in an activator binding site, CRM could stably maintain or even increase gene expression through a certain form of competitive binding between family TFs. These findings enhance understanding the effect of SNPs and indels on CRMs and would help building robust custom-designed CRMs for biologics production and gene therapy.

Can Bergmann’s Rule and the Thermal Melanism Hypothesis explain the variation in colour and size observed in the wild bee Eoanthidium insulare (Apoidea: Megachilidae) across its Palaearctic range?

To analyse geographic variation patterns and assess the applicability of biogeographical rules in insects, the West Palaearctic anthidiine bee Eoanthidium insulare serves as an excellent model. This species showcases a highly variable black-and-yellow colour pattern, with a distribution spanning over 4400 km from the Adriatic Sea to the Pamir Mountains and extending over 1660 km in a south-north direction. We examined 214 specimens from all geographic regions of its distribution and identified a significant increase in both body size and the proportion of black in the integument from south to north. This phenomenon may be attributed to a more efficient exploitation of solar energy through a larger integument surface area (Bergmann’s Rule) and enhanced heat retention due to lower skin reflectance (Thermal Melanism Hypothesis). These two processes, therefore, complement each other. However, despite the cooler climates at higher altitudes, we found no correlations between body size and altitude. Furthermore, while we observed an increase in the proportion of black in the integument from east to west, there was no significant increase in body size. Notably, no correlation between body size and body colouration was identified, suggesting that size and colour adaptation are independent processes. Given that the various colour and size patterns were identified as clinal rather than discontinuous, and the intraspecific genetic distance across the distribution area was remarkably low (0.00138), featuring only four distinct haplotypes of the mitochondrial COI region DNA sequence, we suggest that the subspecies E. i. persicolum and E. i. lemesium be regarded as synonymous with the nominate subspecies (syn. nov.). These findings suggest that, although Bergmann’s Rule and the Thermal Melanism Hypothesis can account for certain aspects of geographic variation in the body size and colouration of E. insulare, their effects may be influenced or superimposed by factors such as genetic constitution.

Functional divergence of Arabidopsis REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 and 2 in repression of flowering.

Photoperiodic plants coordinate the timing of flowering with seasonal light cues, thereby optimizing their sexual reproductive success. The WD40-repeat protein REPRESSOR OF UV-B PHOTOMORPHOGENESIS 2 (RUP2) functions as a potent repressor of UV RESISTANCE LOCUS 8 (UVR8) photoreceptor-mediated UV-B induction of flowering under noninductive, short-day conditions in Arabidopsis (Arabidopsis thaliana); however, in contrast, the closely related RUP1 seems to play no major role. Here, analysis of chimeric ProRUP1:RUP2 and ProRUP2:RUP1 expression lines suggested that the distinct functions of RUP1 and RUP2 in repressing flowering are due to differences in both their coding and regulatory DNA sequences. Artificial altered expression using tissue-specific promoters indicated that RUP2 functions in repressing flowering when expressed in mesophyll and phloem companion cells, whereas RUP1 functions only when expressed in phloem companion cells. Endogenous RUP1 expression in vascular tissue was quantified as lower than that of RUP2, likely underlying the functional difference between RUP1 and RUP2 in repressing flowering. Taken together, our findings highlight the importance of phloem vasculature expression of RUP2 in repressing flowering under short days and identify a basis for the functional divergence of Arabidopsis RUP1 and RUP2 in regulating flowering time.

Inclusion of up to 20% Black Soldier Fly larvae meal in broiler chicken diet has a minor effect on caecal microbiota.

The Black Soldier Fly larvae (BSFL) are a source of nutrients and bioactive compounds in broiler diets. Some components of the BSFL may serve as a prebiotic or may impact the intestinal microbiota of the broilers by other modes of action, which in turn can affect the health and performance of broilers. Here, we investigate the impact of up to 20% BSFL in broiler diets on the diversity and composition of the broiler's microbiota. Four hundred broilers were fed five iso-nutritious experimental diets with increasing levels of BSFL meal reaching 0%, 5%, 10%, 15%, 20% in the finisher diets. Eight caecal content samples coming from each of the eight replicates per treatment were collected at two time points (day 21 and day 42) for DNA extraction and sequencing of the V3-V4 regions using Illumina MiSeq 2 × 300 bp pair-end sequencing with 341f and 805r primers. Analysis of variance and Spearman's correlation were performed, while QIIME2, DADA2, and Calypso were used for data analysis. When broilers were 21 days of age, the abundance of two groups of sequence variants representing Enterococcus and unclassified Christensenellaceae was significantly lower (p-value = 0.048 and p-value = 0.025, respectively) in the 20% BSFL group compared to the 0% BSFL group. There was no relevant alteration in the microbiota diversity at that stage. On day 42, the Spearman correlation analysis demonstrated that the sequence variants representing the genus Coprococcus showed a negative relationship with the BSFL inclusion levels (p-value = 0.043). The sequence variants representing the genus Roseburia and Dehalobacterium demonstrated a positive relationship with the BSFL dietary inclusion (p-value = 0.0069 and p-value = 0.0034, respectively). There was a reduction in the dissimilarity index (ANOSIM) caused by the 20% BSFL dietary inclusion. The addition of up to 20% BSFL in broiler diets did not affect the overall caeca microbiota diversity or composition at day 21. On day 42, there was a reduction in the beta diversity caused by the 20% BSFL dietary inclusion. The abundance of the bacterial group Roseburia was increased by the BSFL dietary inclusion, and it may be beneficial to broiler immunity and performance.

A Single-laboratory Performance Evaluation of MALDI-TOF MS in Rapid Identification of Staphylococcus aureus, Cronobacter sakazakii, Vibrio parahaemolyticus, and Some Closely Related Bacterial Species of Public Health Importance.

Staphylococcus is a genus of Gram-positive bacteria, known to cause food poisoning and gastrointestinal illness in humans. Additionally, the emergence of methicillin-resistant S. aureus (MRSA) strains have caused major healthcare burden worldwide. Cronobacter is a group of Gram-negative bacteria, that can survive in extreme dry conditions. Cronobacter sakazakii is known to contaminate the powdered infant formula and cause life-threatening infections in neonates. Vibrio is a genus of human-pathogenic Gram-negative bacteria that can cause foodborne illness by consuming undercooked or raw seafood. Vibrio parahaemolyticus can cause serious gastrointestinal disease in humans. Thus, rapid identification of Staphylococcus spp., Cronobacter spp. and Vibrio spp. is crucial for the source tracking of contaminated food, and to measure the transmission dynamics of these bacterial pathogens causing foodborne diseases and outbreaks. This single-laboratory performance evaluation study used the VITEK MS system to evaluate the potential of MALDI-TOF MS technology for rapid identification of S. aureus-like, C. sakazakii-like, and V. parahaemolyticus-like, isolates of public health importance. A total of 226 isolates recovered from various food, environmental surveillance samples and other sources were identified by bioMérieux VITEK 2 and VITEK MS systems as Staphylococcus spp., Cronobacter spp., and Vibrio spp. Five American Type Culture Collection (ATCC) reference Gram-positive and Gram-negative bacterial isolates were also tested to complete the study. In addition, for some Staphylococcus spp. isolates whole genome sequencing (WGS), and DNA sequencing of 16S rRNA partial region was also performed for species identification. The VITEK MS system was able to provide species identification to all 96 isolates of Staphylococcus spp. and to all 29 isolates of Vibrio spp. examined with a high confidence value (99.9%). Similarly, species identification was observed for the majority of spots (245 of 303) for the 101 Cronobacter spp. isolates (∼82.0%) with a high confidence value (99.9%), and genus level identification was noticed for the rest of the Cronobacter spp. isolates (18.0%; 58 of the 303 spots) analyzed. Species identification data generated by VITEK 2 system was comparable to data obtained by VITEK MS system. The VITEK MS system is a reliable high-throughput platform that can rapidly identify Staphylococcus, Vibrio, and Cronobacter to the genus level, as well as S. aureus, C. sakazakii, V. parahaemolyticus, and other closely related foodborne isolates, and bacterial isolates from additional sources, in most cases. The VITEK MS system can be used in the rapid genus and species identification of human-pathogenic Staphylococcus spp., Cronobacter spp., and Vibrio spp. isolates.

Sub-tropical benthic/epiphytic dinoflagellates of Aotearoa New Zealand and Rangitāhua Kermadec Islands

Temperatures and temperature anomalies have been increasing in the sub-tropical regions of Aotearoa New Zealand and these changes may impact on harmful algal bloom (HAB) events. Benthic and epiphytic dinoflagellates, particularly the toxin producers, are the focus of this study as it is predicted that under future climate conditions they may produce more toxins or marine animals may become more susceptible to them. The results of past expeditions to Rangitāhua Kermadec Islands and sampling trips to Northland, Aotearoa New Zealand, are summarised and the results of the most recent trips to both regions are presented. The macroalgal habitats of the dinoflagellates are also characterised. Dinoflagellate species not previously identified in Rangitāhua include Coolia canariensis, C. palmyrensis, and C. tropicalis, all identified by DNA sequencing of the large subunit ribosomal RNA region. Gambierdiscus polynesiensis was again isolated and produced 44-methylgambierone and gambierone, and one isolate produced ciguatoxins, the cause of Ciguatera Poisoning. An Ostreopsis tairoto isolate, as analysed by the oxidative cleavage method, produced a palytoxin (PLTX)-like amine oxidation fragment, but when analysed for PLTX-like analogues using a new intact method none were detected indicating an ‘unknown’ PLTX-like compound is produced by this isolate. Isolates of O. cf. siamensis (Ostreopsis sp. 9), collected in Northland, were also analysed using the oxidative cleavage method, with the common PLTX-like amine fragment and the amide fragment corresponding to bishomoPLTX detected in all isolates. Again, the intact method indicated no detections in the isolates, again suggesting an unknown compound was being produced by these isolates. Prorocentrum hoffmannianum isolates produced okadaic acid (OA) and isoDTX-1 and P. lima isolates produced OA, DTX-1, and isoDTX-1. It is expected that new species of potentially harmful, benthic dinoflagellates will continue to be recorded in Aotearoa New Zealand and the results from Rangitāhua provide a guide to the HAB species to expect in sub-tropical Northland as the oceans continue to warm.

Phylogeny, Morphology, Distribution, and Pathogenicity of Seven Calonectria Species from Leaf Blighted Eucalyptus in HaiNan Island, China.

Leaf blight caused by Calonectria species is a major constraint to Eucalyptus trees in China. Calonectria leaf disease on Eucalyptus planted in China was first reported in HaiNan Island in 1985. No systematic investigation or identification of Calonectria species associated with diseased Eucalyptus in HaiNan Island have been performed to date. In order to comprehensively understand the species diversity, distribution, and pathogenicity of Calonectria associated with diseased Eucalyptus trees in HaiNan Island, 400 Calonectria isolates were obtained from 278 diseased Eucalyptus trees planted in 17 sites in five counties/regions. All 400 isolates were identified based on DNA sequence comparisons of the translation elongation factor 1-alpha (tef1), β-tubulin (tub2), calmodulin (cmdA), and histone H3 (his3) gene regions as well as a combination of morphological characteristics. Seven species, namely C. acaciicola (198 isolates, 49.5%), C. pseudoreteaudii (161 isolates, 40.25%), C. reteaudii (29 isolates, 7.25%), C. hawksworthii (6 isolates, 1.5%), C. hongkongensis (4 isolates, 1%), C. auriculiformis (1 isolate, 0.25%) and C. chinensis (1 isolate, 0.25%), were identified. This is the first report of C. acaciicola in China. Calonectria acaciicola, C. pseudoreteaudii, and C. reteaudii belong to the C. reteaudii species complex and were the dominant species collected, accounting for 97% of all the isolates obtained. Overlap in vesicle shape, macroconidia size, and macroconidia septa number existed among these three species in the C. reteaudii species complex. The geographical regions significantly influenced the distribution of C. acaciicola and C. pseudoreteaudii. Representative isolates of C. acaciicola, C. pseudoreteaudii, C. reteaudii and C. hawksworthii produced abundant macroconidia were used in conidial suspension inoculation on Eucalyptus seedlings. Inoculation showed that all four of the tested Calonectria species were highly pathogenic to the two tested Eucalyptus genotypes. The tolerances of two Eucalyptus genotypes were significantly different. This study conducted the first systematic investigation and identification of Calonectria species associated with Eucalyptus leaf blight in HaiNan Island and advanced the concept of selection of disease resistant Eucalyptus genotypes for managing Eucalyptus leaf blight caused by Calonectria species in China.

Structural insights into the transcriptional regulator NalC, a key component of the MexAB-OprM efflux pump system, from Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen with significant public health implications due to its multi-drug resistance (MDR). Among the mechanisms that mediate MDR, the NalC protein, a member of the TetR family of transcriptional regulators, modulates the mexAB-oprM operon, thus facilitating the efflux pump system. The resistance-nodulation-division (RND) family of multidrug efflux pumps plays a crucial role in expelling a broad spectrum of antimicrobial compounds, serving as a key adaptive mechanism. Structural analyses revealed that NalC adopts a modular architecture consisting of distinct domains involved in ligand recognition and transcriptional regulation. The N-terminal domain of NalC contains a DNA-binding helix-turn-helix motif, which interacts with specific DNA sequences in the PA3720-armR operon region. This interaction initiates the transcriptional activation of the efflux pump system. On the other hand, the C-terminal domain of NalC exhibits a highly dynamic structure and is implicated in ligand sensing and signal transduction. Our findings suggest potential binding sites for small molecules that could act as allosteric modulators, thereby providing new avenues for the development of therapeutic strategies targeting MDR Pseudomonas aeruginosa.

Characterization of Florescent Pseudomonads for Biological control Efficacy, Plant Growth Promotion and Antibiotic Tolerance

A collection of fourteen indigenous Fluorescent Pseudomonads (FLPs) was isolated from diverse crop rhizospheres across different regions of Bihar, and their distinctive characteristics were documented. The differentiation between Pseudomonas aeruginosa and Pseudomonas fluorescens was achieved through pigmentation, fluorescence assessment on King's A media, and growth analysis at 42°C. Among the isolates, nine were identified as P. aeruginosa: FLP strains associated with Rice, Turmeric, Mustard-1, Mustard-2, Okra-G, Pea, Barley, Brinjal, and Chickpea. Meanwhile, five isolates - FLP Wheat, FLP Bean, FLP Mango, FLP Brinjal New, and FLP Cauliflower - were classified as P. fluorescens. Confirmation of these classifications was established through DNA sequencing of the 16s RNA region. These FLP isolates were evaluated against prevalent phytopathogens, including Macrophomina phaseolina, Colletotrichum musae, Fusarium oxysporum f. sp. cubense, and Xanthomonas oryzae pv. oryzae. Notable performance was exhibited by FLP Barley (70.55%) against Macrophomina phaseolina, FLP Okra-G (53.82%) against Colletotrichum musae, FLP Rice (38.58%) against Fusarium oxysporum f. sp. cubense, and FLP Okra-G against Xanthomonas oryzae pv. oryzae. In the context of biocontrol efficacy, FLP Turmeric, FLP Pea, FLP Okra-G, and FLP Mustard-1 showcased superior performance in combatting phytopathogens. Additionally, all FLP isolates, except FLP Cauliflower, exhibited phosphate-solubilizing capabilities, with FLP Brinjal demonstrating the highest solubilization (Phosphate Solubilizing Index of 2.89). Siderophore production was a common trait among the isolates. Compatibility with antibiotics, namely Streptocycline at concentrations of 0.025% and 0.10%, as well as Copper oxychloride at 0.25%, 0.30%, and 0.50%, was assessed. Only FLP Cauliflower exhibited compatibility with both antibiotics across all concentrations. Notably, FLP Rice, FLP Turmeric, and FLP Barley demonstrated positive antibiotic compatibility alongside effective biocontrol potential. In summation, this study unveils the distinct attributes and potential applications of indigenous Fluorescent Pseudomonads (FLPs) isolated from the rhizospheres of diverse crops in Bihar. The identified isolates exhibit promising traits such as disease control, phosphate solubilization, siderophore production, and antibiotic compatibility, thus presenting valuable options for sustainable agricultural practices and disease management.

Resolving relationships despite past hybridization in Aliciella subsection Subnuda (Polemoniaceae)

AbstractPhylogenetics is crucial in the study of evolutionary processes and events transpiring in the course of species diversification. Phylogenetic studies within kingdom Plantae often reveal hybridization and introgression. Here, we study a subsection rife with historic hybridization and discuss the impacts of such processes on evolutionary trajectories. Aliciella subsection Subnuda comprises seven species of herbaceous plants occurring in Utah, the Navajo Nation, and the Four Corners region of North America. Previous molecular and morphological work left relationships in the subsection unresolved. Here, we use comparative DNA sequencing of nuclear ITS and chloroplast DNA regions and genome‐wide RAD‐seq data to clarify phylogenetic relationships and examine the role of hybridization in the subsection. We construct haplotype and nucleotype networks from chloroplast and nuclear ITS sequence matrices and compare nuclear and chloroplast phylogenies to identify multiple putative chloroplast capture events. The RAD‐seq maximum likelihood phylogeny and multispecies coalescent species tree robustly resolve relationships between six species‐level clades. We use STRUCTURE and HyDe on the RAD‐seq data to evaluate the influence of hybridization within the subsection. The HyDe results suggest that hybridization has occurred among all species in the subsection at some point in their history. Cytonuclear discordance reveals historic chloroplast capture, and we discuss potential causes of the observed discordance. Our study robustly resolves relationships in Aliciella subsection Subnuda and provides a framework for discussing its speciation despite a history of hybridization and introgression.

The master regulator for entry into sporulation in Bacillus subtilis becomes a mother cell-specific transcription factor for forespore engulfment.

The Spo0A transcription factor is activated by phosphorylation in starving Bacillus subtilis cells. The activated Spo0A (Spo0A~P) regulates genes controlling entry into sporulation and appears to control mother-cell-specific gene expression after asymmetric division, but the latter remains elusive. Here, we found that Spo0A~P directly binds to three conserved DNA sequences (0A1-3) in the promoter region of the mother cell-specific lytic transglycosylase gene spoIID, which is transcribed by σE -RNA polymerase (RNAP) and negatively controlled by the SpoIIID transcription factor and required for forespore engulfment. Systematic mutagenesis of the 0A boxes revealed that the 0A1 and 0A2 boxes located upstream of the promoter positively control the transcription of spoIID. In contrast, the 0A3 box located downstream of the promoter negatively controls the transcription of spoIID. The mutated SpoIIID binding site located between the -35 and -10 promoter elements causes increased expression of spoIID and reduced sporulation. When the mutations of 0A1, 0A2, and IIID sites are combined, sporulation is restored. Collectively, our data suggest that the mother cell-specific spoIID expression is precisely controlled by the coordination of three factors, Spo0A~P, SpoIIID, and σE -RNAP, for proper sporulation. The conservation of this mechanism across spore-forming species was discussed.

Polycomb Recruiters Inside and Outside of the Repressed Domains.

The establishment and stable inheritance of individual patterns of gene expression in different cell types are required for the development of multicellular organisms. The important epigenetic regulators are the Polycomb group (PcG) and Trithorax group (TrxG) proteins, which control the silenced and active states of genes, respectively. In Drosophila, the PcG/TrxG group proteins are recruited to the DNA regulatory sequences termed the Polycomb response elements (PREs). The PREs are composed of the binding sites for different DNA-binding proteins, the so-called PcG recruiters. Currently, the role of the PcG recruiters in the targeting of the PcG proteins to PREs is well documented. However, there are examples where the PcG recruiters are also implicated in the active transcription and in the TrxG function. In addition, there is increasing evidence that the genome-wide PcG recruiters interact with the chromatin outside of the PREs and overlap with the proteins of differing regulatory classes. Recent studies of the interactomes of the PcG recruiters significantly expanded our understanding that they have numerous interactors besides the PcG proteins and that their functions extend beyond the regulation of the PRE repressive activity. Here, we summarize current data about the functions of the PcG recruiters.

ToxR activates the Vibrio cholerae virulence genes by tethering DNA to the membrane through versatile binding to multiple sites

ToxR, a Vibrio cholerae transmembrane one-component signal transduction factor, lies within a regulatory cascade that results in the expression of ToxT, toxin coregulated pilus, and cholera toxin. While ToxR has been extensively studied for its ability to activate or repress various genes in V. cholerae, here we present the crystal structures of the ToxR cytoplasmic domain bound to DNA at the toxT and ompU promoters. The structures confirm some predicted interactions, yet reveal other unexpected promoter interactions with implications for other potential regulatory roles for ToxR. We show that ToxR is a versatile virulence regulator that recognizes diverse and extensive, eukaryotic-like regulatory DNA sequences, that relies more on DNA structural elements than specific sequences for binding. Using this topological DNA recognition mechanism, ToxR can bind both in tandem and in a twofold inverted-repeat-driven manner. Its regulatory action is based on coordinated multiple binding to promoter regions near the transcription start site, which can remove the repressing H-NS proteins and prepares the DNA for optimal interaction with the RNA polymerase.

Sheep serve as a reservoir of trichophyton mentagrophytes genotype V infection.

Human infections by Trichophyton mentagrophytes occur mainly due to contact with diseased animals. In Iran, T. mentagrophytes genotype V is the most prevalent variant of the fungus. We aimed to determine animal reservoir of T. mentagrophytes genotype V infection. The study was done on a total of 577 dermatophyte strains obtained from animals with signs of dermatophytosis and human patients. The list of extensively sampled animals included sheep, cows, cats and dogs. For human cases, epidemiological data were collected. All dermatophyte isolates from animals along with 70 human isolates morphologically similar to T. verrucosum and T. mentagrophytes genotype V were identified by rDNA internal transcribed spacer region restriction fragment length polymorphism analysis and DNA sequencing. A total of 334 animal dermatophyte strains were identified as Microsporum canis, T. mentagrophytes genotype V, T. verrucosum, Nannizzia gypsea, T. mentagrophytes genotype II*, T. mentagrophytes genotype VII, T. quinckeanum and N. fulva, whereas all human isolates were identified as T. mentagrophytes genotype V and originated exclusively from skin and scalp infections. Almost all T. mentagrophytes genotype V isolates were cultured from sheep, but epidemiological data on animal-to-human transmission of T. mentagrophytes genotype V infection were limited and we found evidence in favor of interhuman transmission. In Iran, sheep maintain T. mentagrophytes genotype V population and therefore serve as animal reservoir of respective infections. The role of sheep as the source of human dermatophytosis due to T. mentagrophytes genotype V isolates is yet to be proven.

Tyramide Signal Amplification: New Opportunities for DNA <i>In Situ</i> Hybridization

DNA in situ hybridization (DNA-ISH) is a widely used method in molecular cytogenetics that allows the localization of specific DNA sequences in particular regions of chromosomes. Implementation of DNA-ISH requires the use of DNA probes, which can be commercial or developed for specific research purposes as non-commercial (homemade) DNA probes. One of the significant drawbacks of non-commercial probes is the difficulty in obtaining a high signal intensity with a small DNA probe size. Therefore, developing approaches to enhance non-commercial DNA probes is an important task in modern molecular cytogenetics. To directly visualize small DNA sequences on a chromosome, the tyramide signal amplification (TSA) method is used. The TSA system is based on the formation of a covalent bond between electron-rich protein fragments in the sample and tyramide molecules linked to a hapten (in chromogenic in situ hybridization) or a fluorophore (in fluorescent in situ hybridization). This is achieved by converting tyramide molecules into free-radical intermediate compounds under the action of horseradish peroxidase (HRP), followed by deposition of precipitated molecules nearby. As a result, a low-intensity signal is amplified. Thus, TSA is a good complement to the DNA-ISH method, thanks to its high sensitivity and ability to detect small genomic imbalances, and can therefore become a valuable tool for diagnosing chromosomal rearrangements in clinical practice.

Antimicrobial and antioxidant potentials of an endophytic cunninghamella sp. isolated from the leaves of Chrysophyllum albidum

Background: Several studies have identified endophytic fungi associated with Nigerian plants as potential sources of new drug discovery. Aim: This study was carried out to investigate the antimicrobial and antioxidant properties of secondary metabolites isolated from an endophytic Cunninghamella sp. associated with leaves of Chrysophyllum albidum, a Nigerian indigenous plant. Method: The plant material was collected in Agulu, Anambra State, South-East Nigeria. Endophytic fungal isolation and identification, as well as fermentation and extraction of fungal secondary metabolites, were carried out using standard methods. The antimicrobial and antioxidant activities of the fungal extract were evaluated using the agar well diffusion method and the 1,1-diphenyl-2-picryl-hydraxyl (DPPH) free radical assay, respectively. Result: An endophytic fungus was isolated from the leaves of C. albidum. Following a molecular identification protocol of DNA amplification and sequencing of the fungal ITS region, the fungus was identified as Cunninghamella sp. The fungal extract exhibited antimicrobial activity against the test bacteria and fungus. At concentrations ranging from 2.5 - 20 mg/mL, the extract produced inhibition zone diameters (IZD) ranging from 2 - 11 mm against Pseudomonas aeruginosa. Antibacterial activity was also observed against Staphylococcus aureus at concentrations ranging from 10 - 20 mg/mL, with IZD ranging from 3 - 6 mm. Antifungal activity was observed against Candida albicans at concentrations ranging from 1.25 - 20 mg/mL, with IZD ranging from 2 - 7 mm. The fungal extract demonstrated significant antioxidant activity in the DPPH assay when compared to the standard control, quercetin. The extract showed a percentage inhibition range of 95 - 98% at concentrations ranging from 20 to 100 µg/mL. Conclusion: The findings of this study highlight the potential of endophytic Cunninghamella sp. and other endophytic microorganisms of indigenous Nigerian plants as sources of bioactive compounds with antimicrobial and antioxidant properties for pharmaceutical and industrial applications.

Spergularia hanoverensis (Caryophyllaceae): Validation and Recircumscription of a Misinterpreted Species from South Africa.

The name "Spergularia hanoverensis Simon" has been misapplied to an endemic taxon confined to inland semidesert ecosystems in central-western South Africa. It is commonly accepted as a small annual species occurring in saline habitats in a wide elevation range, but its identity still remains obscure. In the context of taxonomic and phylogenetic research on the African species of Spergularia, we found that the name was never validly published. After revision of herbarium material housed in South African herbaria, a voucher collected from Hanover was found at PRE bearing some labels handwritten by E. Simon that suggest it might be an intended type for the name. Additional herbarium material and wild populations from the Karoo region were identified that matched the samples in that voucher, and taxonomic research was conducted to clarify their identity. Among other characters, those Karoo plants show a woody dense compact habit, woody perennial at base; stems prostrate to ascendent; leaves entirely glabrous, somewhat glaucous; large white-hyaline conspicuous stipules; inflorescence glanduliferous, many-flowered subdichasial cyme, with minute bracts; flowers small, with white petals approximately equalling sepals in length, stamens 7-8, and styles free from base; capsule small, with seeds dimorphic, unwinged to broadly winged, with testa always densely tuberculate. Molecular analyses of plastid (trnL-trnF region) and nuclear ribosomal (5.8S-ITS2 region) DNA sequence data support morphological differentiation of the Karoo plants, for which the name S. hanoverensis is here effectively published. A full morphological description and data on ecology, habitat, distribution, and taxonomic and phylogenetic relationships of S. hanoverensis are compared to other members of the "South African group", namely S. glandulosa, S. namaquensis, and S. quartzicola, from which the new species considerably differs. The adaptative significance of dimorphic seeds of S. hanoverensis is briefly commented on in the context of the species habitat preference. An identification key is presented for the South African related taxa.

OCCURRENCES OF WHEAT CURL MITE ACERIA TOSICHELLA KEIFER 1969 (ERIOPHYIDAE) AND THE ASSOCIATED VIRUSES, (WSMV, HPWMoV, TriMV) IN IRAQ

This study was aimed to investigate the significant wheat yield losses in northern and central Iraq. The cause of these losses was examining the presence of wheat curl mite Aceria tosichella and three viruses vectored by this species – Wheat streak mosaic virus (WSMV), High Plains wheat mosaic virus (HPWMoV), and Triticum mosaic virus (TriMV). Mite specimens were collected from Erbil, Mosul, and Baghdad over three years (2020-2022) and identified using morphological characterization and DNA sequencing of the ITS region. Virus presence was determined using Double Antibody Sandwich-Enzyme-Linked Immunosorbent Assay (DAS-ELISA). Mites were identified conclusively as A. tosichella, providing the first record of this species in Iraq. All three viruses were found to be present, with WSMV having the largest presence as it was detected in 99.56%, then HPWMoV (79.5%) of mite colonies. This is the first record of HPWMoV anywhere in Eurasia besides Ukraine and its widespread presence in Iraq in all three sampled provinces. TriMV was detected only in Erbil. To date this virus has been recorded only in North America and its limited presence in Iraq. The confirmed presence of A. tosichella and three vectored-viruses in Iraq are all new records and critical information for reducing wheat yield losses in this country.

Crucial DNA at crux of insect wing size evolution

Closely related species are usually remarkably similar. But take a closer look, and subtle differences begin to emerge. Some may have longer limbs or wider bodies, and some fruit fly species have dinky wings while the wings of relatives are relatively large. ‘The diversity of wing size relative to whole body size is remarkable in insects, but it is currently unclear how wing-to-body proportions have changed through evolution’, says Marcos Nahmad from the Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav-IPN), Mexico. The proteins that control the development of wings are essentially the same across almost all fruit fly species, regardless of their physical differences, so how do these differences come about?Regions of DNA that control how the regulatory proteins are produced could tweak the way in which a limb develops. One member of the fruit fly family, Drosophila virilis, has petite wings compared with D. melanogaster and other species, which all have relatively large wings compared with their bodies. Knowing this, Nahmad and Timothy Evans (University of Arkansas, USA) wondered if a region of regulatory DNA found in the gene for a protein known as ‘vestigial’ – which controls wing growth – could be responsible for D. virilis’s smaller wings. If so, could D. melanogaster grow D. virilis-sized wings if Keity Farfán-Pira and Teresa Martinez-Cuevas (from Cinvestav-IPN) replaced the key section of D. melanogaster DNA with regulatory DNA from D. virilis?First, Farfán-Pira confirmed that D. virilis fruit flies develop smaller wings for their body size, and then she began the painstaking task of transferring a 1200 bp segment of D. virilis DNA from the section of DNA preceding the fly's vestigial gene into the correct place in the DNA of D. melanogaster eggs. In addition, the team added a gene that would turn the modified flies’ eyes fluorescent red, to be sure that the offspring produced by the D. melanogaster eggs carried the crucial D. virilis DNA.Eventually, after months of patience in the laboratory creating the genetically modified D. melanogaster eggs and then waiting for them to develop into flies, Farfán-Pira measured the wing and leg lengths of the insects and the team was astonished that the wings of the D. melanogaster carrying D. virilis DNA were far smaller for their body size than those of regular D. melanogaster. ‘We were shocked’, Nahmad admits. Inserting the D. virilis vestigial regulatory DNA sequence into the D. melanogaster vestigial gene was sufficient to make the flies’ wings smaller, like the daintier wings of D. virilis.But what does this discovery mean for how members of the insect family, ranging from flamboyant butterflies to stubby-winged bees, evolved such extensive wing differences given that they all use the same cellular machinery to coordinate wing development? The team suspects that the crucial section of vestigial gene DNA fine-tunes the distribution of the cell signalling proteins that regulate wing growth through the larvae's imaginal disk structure – which develops into the wings – to regulate wing size and allow the insect to develop either small or large wings, depending on their family history. The team concludes that some significant change in the key vestigial DNA segment occurred after the split between the D. melanogaster and D. virilis families, resulting in one branch of the family with relatively large wings while the other evolved their comparatively small wings, which persist to this day.

Report of terbinafine resistant Trichophyton spp. in Italy: Clinical presentations, molecular identification, antifungal susceptibility testing and mutations in the squalene epoxidase gene.

Numerous reports of resistance to terbinafine in Trichophyton spp. from all over the world are arousing justified attention and concern. Point mutations in the gene that encodes the squalene epoxidase (SQLE) enzyme are responsible for these therapeutic resistances. Primary objective of the study was to describe first isolates of Trichophyton spp. resistant to terbinafine among the patients treated between September 2019 and June 2022 at the Dermatology Units of Ospedale Maggiore Policlinico and San Bortolo Hospital. Secondary objective was to study the resistance mechanism. Patients with confirmed Trichophyton spp. infection has been treated with systemic and topical terbinafine. Patients were then re-evaluated 12 weeks after the therapy. Patients with incomplete or absent response to terbinafine underwent a new skin scraping for direct mycological examination, new identification of dermatophyte species from culture and MALDI-TOF, molecular species identification, antifungal susceptibility testing and molecular analysis of SQLE gene. We identified five patients without clinical response to treatment with terbinafine. The DNA sequencing of the ITS region identified one Trichophyton rubrum and four Trichophyton indotineae. The T. rubrum strain showed minimum inhibitory concentration (MIC) (90% growth inhibition) of 4 mg/L for terbinafine. The four T. indotineae strains showed a MICs range of 0.25-4 mg/L for terbinafine. The analysis of the SQLE gene in the T. rubrum strain showed a nucleotide substitution generating a missense mutation (L393F). The SQLE gene sequencing in the T. indotineae strains showed a nucleotide substitution generating a missense mutation (F397L) in two strains, a nucleotide substitution L393S in one strain and a nucleotide substitution F415C in another strain. We report the first cases of terbinafine-resistant Trichophyton isolates in the Italian population. Solid antifungal management programs will be needed to promote more responsible use of antimycotics and preserve their therapeutic efficacy to control antifungal resistance.