RESUMEN

Organism condition plays an important role in sexual selection. Sexual ornaments and displays can be condition-dependent, reflecting either underlying genetic quality, experience of environmental stressors, or both. As such, the phenotypic expression of such traits, and the resulting patterns of mate choice acting on them, may be shaped by intrinsic genetic quality and the environment. Moreover, condition may also influence the choosing individual in mate choice, influencing their ability to invest in mate discrimination, or changing what traits of the chosen, including resources, are most preferred. Here we consider sexual selection and condition in the seed bug Lygaeus simulans, a species characterised by strong post-copulatory sexual selection, but rather limited pre-copulatory discrimination. We manipulated short-term condition in both males and females by restricting access to water for 24 h. Water is particularly important in these bugs, given their feeding ecology and physiology. We found that water-deprived males proved less likely to mate, while copulation duration with water-deprived females was significantly reduced. Given the importance of copulation duration for the successful transfer of sperm by males to females, the data suggest cryptic male choice acting against water-deprived females. These data add to those suggesting that cryptic male choice for fecund females plays an important role in sexual selection in this species. More generally, our results support the widespread importance of condition in terms of mating dynamics and sexual selection.

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Several reports have presented that balanced chromosomal rearrangements (BCRs) carriers with normal phenotypes may be carriers of complex rearrangements. However, the incidence and PGT clinical outcomes of cryptic complex chromosome rearrangements (CCCRs) in individuals with BCRs is remain unknown. We recruited a cohort of 1,264 individuals with BCR carriers from 2016 to 2021 at the Reproductive and Genetic Hospital of CITIC Xiangya. Peripheral blood was collected for karyotyping and genomic DNA extraction and the PGT-SR clinical outcomes of CCCRs carriers were analyzed and compared with those of BCR carriers. Our findings revealed that 3.6% (45/1,264) of BCR carriers had CCCRs, involving 3-25 breakpoints on 1-3 chromosomes. Furthermore, when mate-pair sequencing was employed, 63.3% (19/30) of CCCR carriers were found to have chromosome rearrangements that were different from those identified by the MicroSeq technique. And the transferable embryo rate of CCCR carriers with 3 chromosomes was significantly lower than that of CCCR carriers with only 1-2 chromosomes. In this research, we revealed that some of the BCR carriers were actually CCCR carriers, and the prognosis of PGT in CCCR carriers with one or two chromosomes is better than that of CCCR carriers with three chromosomes.

Asunto(s)

Aberraciones Cromosómicas , Humanos , Femenino , Masculino , Adulto , Translocación Genética , Cariotipificación , Heterocigoto , Embarazo

RESUMEN

Egg-laying is one of the key aspects of female reproductive behavior in insects. Egg-laying has been studied since the dawn of Drosophila melanogaster as a model organism. The female's internal state, hormones, and external factors, such as nutrition, light, and social environment, affect egg-laying output. However, only recently, neurobiological features of egg-laying behavior have been studied in detail. fruitless and doublesex, two key players in the sex determination pathway, have become focal points in identifying neurons of reproductive significance in both central and peripheral nervous systems. The reproductive tract and external terminalia house sensory neurons that carry the sensory information of egg maturation, mating and egg-laying. These sensory signals include the presence of male accessory gland products and mechanical stimuli. The abdominal neuromere houses neurons that receive information from the reproductive tract, including sex peptide abdominal ganglion neurons (SAGs), and send their information to the brain. In the brain, neuronal groups like aDNs and pC1 clusters modulate egg-laying decision-making, and other neurons like oviINs and oviDNs are necessary for egg-laying itself. Lastly, motor neurons involved in egg-laying, which are mostly octopaminergic, reside in the abdominal neuromere and orchestrate the muscle movements required for laying the egg. Egg-laying neuronal control is important in various evolutionary processes like cryptic female choice, and using different Drosophila species can provide intriguing avenues for the future of the field.

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Clostridium butyricum has emerged as a promising candidate for both industrial and medical biotechnologies, underscoring the key pursuit of stable gene overexpression in engineering C. butyricum. Unlike antibiotic-selective vectors, native-cryptic plasmids can be utilized for antibiotic-free expression systems in bacteria but have not been effectively exploited in C. butyricum to date. This study focuses on leveraging these plasmids, pCB101 and pCB102, in C. butyricum DSM10702 for stable gene overexpression without antibiotic selection via efficient gene integration using the SacB-based allelic exchange method. Integration of reporter IFP2.0 and glucuronidase generated sustained near-infrared fluorescence and robust enzyme activity across successive subcultures. Furthermore, successful secretion of a cellulase, Cel9M, and the human interleukin 10 from pCB102 highlights native-cryptic plasmids' potential in conferring stable gene products for industrial and medical applications in C. butyricum. This work appears to be the first study to harness the Clostridium native-cryptic plasmid for stable gene overexpression without antibiotics, thereby advancing the biotechnological prospects of C. butyricum.

Asunto(s)

Clostridium butyricum , Plásmidos , Clostridium butyricum/genética , Plásmidos/genética , Humanos , Expresión Génica , Biotecnología/métodos , Glucuronidasa/genética , Glucuronidasa/metabolismo , Celulasa/genética , Celulasa/metabolismo , Genes Reporteros , Microbiología Industrial/métodos , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos

RESUMEN

EGF-CFC proteins are obligate coreceptors for Nodal signaling and are thus required for gastrulation and left-right patterning. Species with multiple family members show evidence of specialization. For example, mouse Cripto is required for gastrulation, whereas Cryptic is involved in left-right patterning. However, the members of the family across model organisms have little sequence conservation beyond the EGF-CFC domain, posing challenges for determining their evolutionary history and functional conservation. In this study we outline the evolutionary history of the EGF-CFC family of proteins. We traced the EGF-CFC gene family from a single gene in the deuterostome ancestor through its expansion and functional specialization in tetrapods, and subsequent gene loss and translocation in eutherian mammals. Mouse Cripto and Cryptic, zebrafish Tdgf1, and all three Xenopus EGF-CFC genes (Tdgf1, Tdgf1.2 and Cripto.3) and are all descendants of the ancestral Tdgf1 gene. We propose that subsequent to the family expansion in tetrapods, Tdgf1B (Xenopus Tdgf1.2) acquired specialization in the left-right patterning cascade, and after its translocation in eutherians to a different chromosomal location, Cfc1/Cryptic has maintained that specialization.

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Although the barred sand burrower, Limnichthys fasciatus, is widely distributed throughout the western Pacific, including Japan, Taiwan, and Australia, its morphology and genetics are poorly known. We discovered four cryptic species of Limnichthys from the western Pacific based on mtDNA cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (rRNA) sequences. Genetic distances showed remarkably large differences (12.7%-24.3% in COI and 7.9%-19.6% in 16S rRNA) between true L. fasciatus (type locality: southeastern Australia) and the others. A relaxed clock model with optimized selected substitution models showed that their deep divergence began in the middle Miocene epoch and subsequently diverged into the current cryptic species in the Plio-Pleistocene. A eurythermal common ancestor may have evolved independently in each region due to geographical events and paleoclimatic fluctuations, which made it possible for L. fasciatus complex to be an anti-equatorial species. Despite their deep genetic divergence, they showed marked phenotypic similarity, suggesting that they have experienced similar selective pressures related to their specific behavior.

RESUMEN

The genome duplication program is affected by multiple factors in vivo, including developmental cues, genotoxic stress, and aging. Here, we monitored DNA replication initiation dynamics in regenerating livers of young and old mice after partial hepatectomy to investigate the impact of aging. In young mice, the origin firing sites were well defined; the majority were located 10-50 kb upstream or downstream of expressed genes, and their position on the genome was conserved in human cells. Old mice displayed the same replication initiation sites, but origin firing was inefficient and accompanied by a replication stress response. Inhibitors of the ATR checkpoint kinase fully restored origin firing efficiency in the old mice but at the expense of an inflammatory response and without significantly enhancing the fraction of hepatocytes entering the cell cycle. These findings unveil aging-dependent replication stress and a crucial role of ATR in mitigating the stress-associated inflammation, a hallmark of aging.

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One yet unresolved question in the study of mating system evolution is the occurrence of mating failure, when individuals go through their lives without successfully mating. This includes the failure to produce offspring even following copulation, for instance due to insemination or fertilisation failure. Copulations are costly in a variety of ways, but also a fundamental route to fitness in sexual species, and so we should expect that engaging in copulations that generate no offspring should be strongly selected against. Nonetheless, it has become apparent that mating failure is quite common in nature. Here we consider post-copulatory sexual selection in Lygaeus simulans seed bugs to test the hypothesis that the high levels of mating failure found in this species (approximately 40%-60%) are caused by cryptic male choice (i.e. males choosing not to inseminate a female during copulation). In our first experiment, we found that mating failure depended on female size, but not male size, with smaller females experiencing mating failure more frequently. Mechanistically this is likely to be due to copulation duration, as shorter copulations were more likely to lead to mating failure. Likewise, copulations with smaller females were shorter. In our second and third experiments, rates of mating failure decreased when pairs were allowed to repeatedly interact with the same partner over longer durations (hours through to days), implying that mating failure is not primarily caused by infertility or chronic mechanical failure. Instead, our results strongly suggest cryptic male choice as the cause of mating failure in this species.

RESUMEN

Soybean red leaf blotch (RLB), caused by the fungus Coniothyrium glycines, represents a foliar disease of soybean that is thus far restricted to Africa. The fungus is listed as a Select Agent by the Federal Select Agent Program because it could pose a severe threat to plant health were it to establish in the United States. Previous work uncovered tremendous molecular diversity at the internal transcribed spacer region, suggesting that there may be multiple species causing RLB. To determine whether multiple species cause RLB, we reconstructed the phylogeny of C. glycines and taxonomic allies using sequence data from four genes. We included 33 C. glycines isolates collected from six African countries and determined that all isolates form a well-supported, monophyletic lineage. Within this lineage there are at least six well-supported clades that largely correspond to geography, with one clade exclusively composed of isolates from Ethiopia, another exclusively composed of isolates from Uganda, and four composed of isolates from southern Africa. However, we did not detect any concordance for these clades between the four genes, indicating that all isolates included in this analysis are representative of a single species. Isolates in the Ethiopia clade are morphologically distinct from isolates in the other clades, as they produce larger sclerotia and smaller pycnida and more sclerotia in planta. Additionally, ancestral range estimations suggest that the C. glycines lineage emerged in southern Africa. These results show that there is significantly more genetic and morphological diversity than was initially suspected with this high-consequence fungal plant pathogen.

RESUMEN

Cotton leaf curl Multan virus (CLCuMuV), a serious viral disease causative agent in cotton plants in South Asia, is transmitted by the Bemisia tabaci cryptic species complex in a persistent circulative manner. A previous study indicated that Asia II-7 whiteflies could transmit CLCuMuV, while Mediterranean (MED) whiteflies failed to transmit CLCuMuV. However, little is known about the genes involved in this process. In this study, Asia II-7 and MED B. tabaci were utilized to determine transcriptomic responses after 48 h of acquisition access periods (AAPs). Result of Illumina sequencing revealed that, 14,213 and 8,986 differentially expressed genes (DEGs) were identified. Furthermore, DEGs related to the immune system and metabolism of Asia II-7 and MED in response to CLCuMuV-infected plants were identified and analyzed using Gene Ontologies (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), and the number of related DEGs in MED was lower than that of Asia II-7. The most abundant groups of DEGs between both viruliferous and aviruliferous whitefly species were the zf-C2H2 family of transcription factors (TFs). Notably, in comparison to viruliferous MED, Asia II-7 exhibited more DEGs related to cathepsin biosynthesis. Overall, this study provides the basic information for investigating the molecular mechanism of how begomoviruses affect B. tabaci metabolism and immune response either as vector cryptic species or non-vector species.

RESUMEN

Clostridioides difficile is an intestinal pathogen of humans and animals. In community-associated infections, the environment is suggested to play a significant role in overall transmission routes. Although the prevalence of C. difficile in freshwater and soil has been widely studied, little is known about its presence in sediments. In this study, we tested 15 sediment samples collected from various freshwater sources. C. difficile was isolated from all sampled sites, yielding a total of 171 strains grouped into 26 ribotypes, with 001/072 and 014/020 being the most prevalent. Genome sequencing of 37 isolates from 17 PCR ribotypes confirmed the presence of highly related strains in the geographically distant and unlinked water samples. Eight divergent PCR ribotypes from clades C-II and C-III were found in six samples. In each sample, the unbound fraction (supernatant after sediment wash) and bound fraction (sonicated sediment sample) were subjected to enrichment. Sonication was only slightly better than washing in terms of sample positivity (14 positive samples with sonication and 11 with washing). However, sonication substantially increased the diversity of the PCR ribotypes obtained (23 in sonicated samples vs nine in washed samples). In conclusion, sediments are a rich source for investigating the diversity of environmental C. difficile, including isolates from divergent lineages. Selection of the isolation method can significantly impact the diversity of captured PCR ribotypes.IMPORTANCEClostridioides difficile, a pathogenic bacterium that can cause intestinal infections in humans and animals, thrives in the gut but also disperses widely through spores found in the environment. Clinical and environmental strains often overlap with common PCR ribotypes, which are consistently isolated worldwide. Environmental studies have mostly focused on water and soil, but sediments have been very poorly studied. In this study, we investigated the presence of C. difficile in various freshwater sediments and evaluated the effectiveness of two different isolation approaches on positivity rates and strain diversity. C. difficile was found to be highly prevalent in sediments, with an isolation rate of 100%. Sonication proved to be more effective than simple washing for capturing a greater diversity of PCR ribotypes. Overall, this study underscores the widespread presence of C. difficile in freshwater sediments and emphasizes the importance of continued surveillance and monitoring to understand its ecology and transmission dynamics.

RESUMEN

In this work, we describe a new species of the genus Oreonectes, Oreonectes yuedongensis sp. nov., collected from the Lianhua Mountains in eastern Guangdong, China. Phylogenetic trees constructed based on the mitochondrial cytochrome b (Cyt b) gene showed that this new species represents an independent evolutionary lineage, with uncorrected genetic distances (Kimura 2-parameter model) from congeners ranging from 5.1% to 8.3%. In addition, nuclear DNA analysis indicated O. yuedongensis as an independent lineage separate from its closely related species. Morphologically, the new species can be distinguished from other six species in the genus Oreonectes by a combination of serial characters. The description of this new species suggests that it is necessary to reassess the biodiversity of Oreonectes platycephalus as a complex, especially in the middle reaches of the Pearl River near the border between Guangdong and Guangxi. Morphological and genetic evidence supports O. yenlingi as a synonym of O. platycephalus.

RESUMEN

BACKGROUND: Infections resulting from multidrug-resistant Enterobacterales (MDR-E) pose a growing global threat, presenting challenges in treatment and contributing significantly to morbidity and mortality rates. The main objective of this study was to characterize phenotypically and genetically extended-spectrum ß-lactamase- and carbapenemase- producing Enterobacterales (ESBLE and CPE respectively) isolated from clinical samples in the West Bank, Palestine. METHODS: A cross sectional study was conducted in October 2023 on clinical bacterial isolates collected from five governmental hospitals in the West Bank, Palestine. The isolates obtained from the microbiology laboratories of the participating hospitals, underwent identification and antibiotic susceptibility testing (AST) using the VITEK® 2 Compact system. ESBL production was determined by the Vitek2 Compact system. A modified carbapenem inactivation method (mCIM) was employed to identify carbapenemase-producing Enterobacterales (CPE). Resistance genes were detected by real-time PCR. RESULTS: Out of the total 1380 collected isolates, we randomly selected 600 isolates for analysis. Our analysis indicated that 287 (47.83%) were extended-spectrum beta-lactamase producers (ESBLE), and 102 (17%) as carbapenem-resistant Enterobacterales (CRE) isolates. A total of 424 isolates (70.67%) were identified as multidrug-resistant Enterobacterales (MDRE). The most prevalent ESBL species were K. pneumoniae (n = 124; 43.2%), E. coli (n = 119; 41.5%) and E. cloacae (n = 31; 10.8%). Among the CRE isolates, 85 (83.33%) were carbapenemase-producing Enterobacterales (CPE). The most frequent CRE species were K. pneumoniae (n = 63; 61.7%), E. coli (n = 25; 24.5%) and E. cloacae (n = 13; 12.8%). Additionally, 47 (7.83%) isolates exhibited resistance to colistin (CT), with 38 (37.62%) being CT-resistant CRE and 9 (3.14%) being CT-resistant ESBLE while sensitive to carbapenems. We noticed that 11 isolates (6 Klebsiella pneumoniae and 5 Enterobacter cloacae complex) demonstrated sensitivity to carbapenems by phenotype but carried silent CPE genes (1 blaOXA48, and 6 blaNDM, 4 blaOXA48, blaNDM). ESBL-producing Enterobacterales strains exhibited varied resistance patterns across different antibiotic classes. E. coli isolates showed notable 48% resistance to trimethoprim/sulfamethoxazole. K. pneumoniae isolates displayed a significant resistance to trimethoprim/sulfamethoxazole, nitrofurantoin, and fosfomycin (54%, 90%, and 70% respectively). E. cloacae isolates showed complete resistance to nitrofurantoin and fosfomycin. P. mirabilis isolates exhibited high resistance against fluoroquinolones (83%), and complete resistance to trimethoprim/sulfamethoxazole, nitrofurantoin and fosfomycin. CONCLUSION: This study showed the high burden of the ESBLE and CRE among the samples collected from the participating hospitals. The most common species were K. pneumoniae and E. coli. There was a high prevalence of blaCTXm. Adopting both conventional and molecular techniques is essential for better surveillance of the emergence and spread of antimicrobial-resistant Enterobacterales infections in Palestine.

Asunto(s)

Antibacterianos , Proteínas Bacterianas , Farmacorresistencia Bacteriana Múltiple , Infecciones por Enterobacteriaceae , Enterobacteriaceae , Pruebas de Sensibilidad Microbiana , beta-Lactamasas , Humanos , beta-Lactamasas/genética , Estudios Transversales , Infecciones por Enterobacteriaceae/microbiología , Infecciones por Enterobacteriaceae/epidemiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Antibacterianos/farmacología , Medio Oriente/epidemiología , Femenino , Adulto , Enterobacteriaceae/genética , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/aislamiento & purificación , Enterobacteriaceae/enzimología , Masculino , Persona de Mediana Edad , Fenotipo , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Enterobacteriaceae Resistentes a los Carbapenémicos/aislamiento & purificación , Enterobacteriaceae Resistentes a los Carbapenémicos/efectos de los fármacos , Adulto Joven , Adolescente , Anciano , Niño , Carbapenémicos/farmacología , Preescolar

RESUMEN

Plasticity is found in all domains of life and is particularly relevant when populations experience variable environmental conditions. Traditionally, evolutionary models of plasticity are non-mechanistic: they typically view reactions norms as the target of selection, without considering the underlying genetics explicitly. Consequently, there have been difficulties in understanding the emergence of plasticity, and in explaining its limits and costs. In this paper, we offer a novel mechanistic approximation for the emergence and evolution of plasticity. We simulate random "epigenetic mutations" in the genotype-phenotype mapping, of the kind enabled by DNA-methylations/demethylations. The frequency of epigenetic mutations at loci affecting the phenotype is sensitive to organism stress (trait-environment mismatch), but is also genetically determined and evolvable. Thus, the "random motion" of epigenetic markers enables developmental learning-like behaviors that can improve adaptation within the limits imposed by the genotypes. However, with random motion being "goal-less," this mechanism is also vulnerable to developmental noise leading to maladaptation. Our individual-based simulations show that epigenetic mutations can hide alleles that are temporarily unfavorable, thus enabling cryptic genetic variation. These alleles can be advantageous at later times, under regimes of environmental change, in spite of the accumulation of genetic loads. Simulations also demonstrate that plasticity is favored by natural selection in constant environments, but more under periodic environmental change. Plasticity also evolves under directional environmental change as long as the pace of change is not too fast and costs are low.

RESUMEN

While the presence of morphologically cryptic species is increasingly recognized, we still lack a useful understanding of what causes and maintains co-occurring cryptic species and its consequences for the ecology, evolution, and conservation of communities. We sampled 724 Pocillopora corals from five habitat zones (the fringing reef, back reef, and fore reef at 5, 10, and 20 m) at four sites around the island of Moorea, French Polynesia. Using validated genetic markers, we identified six sympatric species of Pocillopora, most of which cannot be reliably identified based on morphology: P. meandrina (42.9%), P. tuahiniensis (25.1%), P. verrucosa (12.2%), P. acuta (10.4%), P. grandis (7.73%), and P. cf. effusa (2.76%). For 423 colonies (58% of the genetically identified hosts), we also used psbA ncr or ITS2 markers to identify symbiont species (Symbiodiniaceae). The relative abundance of Pocillopora species differed across habitats within the reef. Sister taxa P. verrucosa and P. tuahiniensis had similar niche breadths and hosted the same specialist symbiont species (mostly Cladocopium pacificum) but the former was more common in the back reef and the latter more common deeper on the fore reef. In contrast, sister taxa P. meandrina and P. grandis had the highest niche breadths and overlaps and tended to host the same specialist symbiont species (mostly C. latusorum). Pocillopora acuta had the narrowest niche breadth and hosted the generalist, and more thermally tolerant, Durusdinium gynnii. Overall, there was a positive correlation between reef habitat niche breadth and symbiont niche breadth-Pocillopora species with a broader habitat niche also had a broader symbiont niche. Our results show how fine-scale variation within reefs plays an important role in the generation and coexistence of cryptic species. The results also have important implications for how niche differences affect community resilience, and for the success of coral restoration practices, in ways not previously appreciated.

RESUMEN

The HIV-1 protease is critical for the process of viral maturation and as such, it is one of the most well characterized proteins in the Protein Data Bank. There is some evidence to suggest that the HIV-1 protease is capable of accommodating small molecule fragments at several locations on its surface outside of the active site. However, some pockets on the surface of proteins remain unformed in the apo structure and are termed "cryptic sites." To date, no cryptic sites have been identified in the structure of HIV-1 protease. Here, we characterize a novel cryptic cantilever pocket on the surface of the HIV-1 protease through mixed-solvent molecular dynamics simulations using several probes. Interestingly, we noted that several homologous retroviral proteases exhibit evolutionarily conserved dynamics in the cantilever region and possess a conserved pocket in the cantilever region. Immobilization of the cantilever region of the HIV-1 protease via disulfide cross-linking resulted in curling-in of the flap tips and the propensity for the protease to adopt a semi-open flap conformation. Structure-based analysis and fragment-based screening of the cryptic cantilever pocket suggested that the pocket may be capable of accommodating ligand structures. Furthermore, molecular dynamics simulations of a top scoring fragment bound to the cryptic pocket illustrated altered flap dynamics of the fragment-bound enzyme. Together, these results suggest that the mobility of the cantilever region plays a key role in the global dynamics of retroviral proteases. Therefore, the cryptic cantilever pocket of the HIV-1 protease may represent an interesting target for future in vitro studies.

RESUMEN

The Chiricahua Mountains in southeastern Arizona are renowned for their exceptional biodiversity and high levels of endemism. Morphological, genomic, behavioral, and distributional data were used to report the discovery of a remarkable new tarantula species from this range. Aphonopelmajacobii sp. nov. inhabits high-elevation mixed conifer forests in these mountains, but also co-occurs and shares its breeding period with A.chiricahua-a related member of the Marxi species group-in mid-elevation Madrean evergreen oak and pine-oak woodlands. This marks the first documented case of syntopy between two montane endemics in the Madrean Archipelago and adds to our knowledge of this threatened region's unmatched tarantula diversity in the United States. An emended diagnosis and redescription for A.chiricahua are also provided based on several newly acquired and accurately identified specimens. Phylogenetic analyses of mitochondrial and genomic-scale data reveal that A.jacobii sp. nov. is more closely related to A.marxi, a species primarily distributed on the Colorado Plateau, than to A.chiricahua or the other Madrean Sky Island taxa. These data provide the evolutionary framework for better understanding the region's complex biogeographic history (e.g., biotic assembly of the Chiricahua Mountains) and conservation of these spiders.

RESUMEN

Biallelic variants in USH2A are associated with retinitis pigmentosa (RP) and Type 2 Usher Syndrome (USH2), leading to impaired vision and, additionally, hearing loss in the latter. Although the introduction of next-generation sequencing into clinical diagnostics has led to a significant uplift in molecular diagnostic rates, many patients remain molecularly unsolved. It is thought that non-coding variants or variants of uncertain significance contribute significantly to this diagnostic gap. This study aims to demonstrate the clinical utility of the reverse transcription-polymerase chain reaction (RT-PCR)-Oxford Nanopore Technology (ONT) sequencing of USH2A mRNA transcripts from nasal epithelial cells to determine the splice-altering effect of candidate variants. Five affected individuals with USH2 or non-syndromic RP who had undergone whole genome sequencing were recruited for further investigation. All individuals had uncertain genotypes in USH2A, including deep intronic rare variants, c.8682-654C>G, c.9055+389G>A, and c.9959-2971C>T; a synonymous variant of uncertain significance, c.2139C>T; p.(Gly713=); and a predicted loss of function duplication spanning an intron/exon boundary, c.3812-3_3837dup p.(Met1280Ter). In silico assessment using SpliceAI provided splice-altering predictions for all candidate variants which were investigated using ONT sequencing. All predictions were found to be accurate; however, in the case of c.3812-3_3837dup, the outcome was a complex cryptic splicing pattern with predominant in-frame exon 18 skipping and a low level of exon 18 inclusion leading to the predicted stop gain. This study detected and functionally characterised simple and complex mis-splicing patterns in USH2A arising from previously unknown deep intronic variants and previously reported variants of uncertain significance, confirming the pathogenicity of the variants.

Asunto(s)

Proteínas de la Matriz Extracelular , Empalme del ARN , Síndromes de Usher , Humanos , Proteínas de la Matriz Extracelular/genética , Síndromes de Usher/genética , Femenino , Masculino , Empalme del ARN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Exones/genética , Mutación/genética , Retinitis Pigmentosa/genética , Adulto , ARN Mensajero/genética , ARN Mensajero/metabolismo , Intrones/genética , Persona de Mediana Edad

RESUMEN

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases with a progressive and fatal course. They are often comorbid and share the same molecular spectrum. Their key pathological features are the formation of the aggregation of TDP-43, an RNA-binding protein, in the cytoplasm and its depletion from the nucleus in the central nervous system. In the nucleus, TDP-43 regulates several aspects of RNA metabolism, ranging from RNA transcription and alternative splicing to RNA transport. Suppressing the aberrant splicing events during RNA processing is one of the significant functions of TDP-43. This function is impaired when TDP-43 becomes depleted from the nucleus. Several critical cryptic splicing targets of TDP-43 have recently emerged, such as STMN2, UNC13A, and others. UNC13A is an important ALS/FTD risk gene, and the genetic variations, single nucleotide polymorphisms, cause disease via the increased susceptibility for cryptic exon inclusion under the TDP-43 dysfunction. Moreover, TDP-43 has an autoregulatory mechanism that regulates the splicing of its mRNA (TARDBP mRNA) in the healthy state. This study provides recent findings on the splicing regulatory function of TDP-43 and discusses the prospects of using these aberrant splicing events as efficient biomarkers.