Bi-allelic LETM1 variants perturb mitochondrial ion homeostasis leading to a clinical spectrum with predominant nervous system involvement.

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.

Wolf-Hirschhorn syndrome candidate 1 (Whsc1) methyltransferase signals via a Pitx2-miR-23/24 axis to effect tooth development.

Wolf-Hirschhorn syndrome (WHS) is a developmental disorder attributed to a partial deletion on the short arm of chromosome 4. WHS patients suffer from oral manifestations including cleft lip and palate, hypodontia, and taurodontism. WHS candidate 1 (WHSC1) gene is a H3K36-specific methyltransferase that is deleted in every reported case of WHS. Mutation in this gene also results in tooth anomalies in patients. However, the correlation between genetic abnormalities and the tooth anomalies has remained controversial. In our study, we aimed to clarify the role of WHSC1 in tooth development. We profiled the Whsc1 expression pattern during mouse incisor and molar development by immunofluorescence staining and found Whsc1 expression is reduced as tooth development proceeds. Using real-time quantitative reverse transcription PCR, Western blot, chromatin immunoprecipitation, and luciferase assays, we determined that Whsc1 and Pitx2, the initial transcription factor involved in tooth development, positively and reciprocally regulate each other through their gene promoters. miRNAs are known to regulate gene expression posttranscriptionally during development. We previously reported miR-23a/b and miR-24-1/2 were highly expressed in the mature tooth germ. Interestingly, we demonstrate here that these two miRs directly target Whsc1 and repress its expression. Additionally, this miR cluster is also negatively regulated by Pitx2. We show the expression of these two miRs and Whsc1 are inversely correlated during mouse mandibular development. Taken together, our results provide new insights into the potential role of Whsc1 in regulating tooth development and a possible molecular mechanism underlying the dental defects in WHS.

Non-random sampling measures the occurrence but not the strength of a textbook trophic cascade.

Although sampling the five tallest young aspen in a stand is useful for detecting the occurrence of any aspen recruitment, this technique overestimates the population response of aspen to wolf reintroduction. Our original conclusion that random sampling described a trophic cascade that was weaker than the one described by non-random sampling is unchanged.

Foraging rates from metabarcoding: Predators have reduced functional responses in wild, diverse prey communities.

Functional responses describe foraging rates across prey densities and underlie many fundamental ecological processes. Most functional response knowledge comes from simplified lab experiments, but we do not know whether these experiments accurately represent foraging in nature. In addition, the difficulty of conducting multispecies functional response experiments means that it is unclear whether interaction strengths are weakened in the presence of multiple prey types. We developed a novel method to estimate wild predators' foraging rates from metabarcoding data and use this method to present functional responses for wild wolf spiders foraging on 27 prey families. These field functional responses were considerably reduced compared to lab functional responses. We further find that foraging is sometimes increased in the presence of other prey types, contrary to expectations. Our novel method for estimating field foraging rates will allow researchers to determine functional responses for wild predators and address long-standing questions about foraging in nature.

Tracing Eastern Wolf Origins From Whole-Genome Data in Context of Extensive Hybridization.

Southeastern Canada is inhabited by an amalgam of hybridizing wolf-like canids, raising fundamental questions regarding their taxonomy, origins, and timing of hybridization events. Eastern wolves (Canis lycaon), specifically, have been the subject of significant controversy, being viewed as either a distinct taxonomic entity of conservation concern or a recent hybrid of coyotes (C. latrans) and grey wolves (C. lupus). Mitochondrial DNA analyses show some evidence of eastern wolves being North American evolved canids. In contrast, nuclear genome studies indicate eastern wolves are best described as a hybrid entity, but with unclear timing of hybridization events. To test hypotheses related to these competing findings we sequenced whole genomes of 25 individuals, representative of extant Canadian wolf-like canid types of known origin and levels of contemporary hybridization. Here we present data describing eastern wolves as a distinct taxonomic entity that evolved separately from grey wolves for the past ∼67,000 years with an admixture event with coyotes ∼37,000 years ago. We show that Great Lakes wolves originated as a product of admixture between grey wolves and eastern wolves after the last glaciation (∼8,000 years ago) while eastern coyotes originated as a product of admixture between "western" coyotes and eastern wolves during the last century. Eastern wolf nuclear genomes appear shaped by historical and contemporary gene flow with grey wolves and coyotes, yet evolutionary uniqueness remains among eastern wolves currently inhabiting a restricted range in southeastern Canada.

Long-term Small Population Size, Deleterious Variation, and Altitude Adaptation in the Ethiopian Wolf, a Severely Endangered Canid.

Ethiopian wolves, a canid species endemic to the Ethiopian Highlands, have been steadily declining in numbers for decades. Currently, out of 35 extant species, it is now one of the world's most endangered canids. Most conservation efforts have focused on preventing disease, monitoring movements and behavior, and assessing the geographic ranges of sub-populations. Here, we add an essential layer by determining the Ethiopian wolf's demographic and evolutionary history using high-coverage (∼40×) whole-genome sequencing from 10 Ethiopian wolves from the Bale Mountains. We observe exceptionally low diversity and enrichment of weakly deleterious variants in the Ethiopian wolves in comparison with two North American gray wolf populations and four dog breeds. These patterns are consequences of long-term small population size, rather than recent inbreeding. We infer the demographic history of the Ethiopian wolf and find it to be concordant with historic records and previous genetic analyses, suggesting Ethiopian wolves experienced a series of both ancient and recent bottlenecks, resulting in a census population size of fewer than 500 individuals and an estimated effective population size of approximately 100 individuals. Additionally, long-term small population size may have limited the accumulation of strongly deleterious recessive mutations. Finally, as the Ethiopian wolves have inhabited high-altitude areas for thousands of years, we searched for evidence of high-altitude adaptation, finding evidence of positive selection at a transcription factor in a hypoxia-response pathway [CREB-binding protein (CREBBP)]. Our findings are pertinent to continuing conservation efforts and understanding how demography influences the persistence of deleterious variation in small populations.

Demographic history shapes North American gray wolf genomic diversity and informs species' conservation.

Effective population size estimates are critical information needed for evolutionary predictions and conservation decisions. This is particularly true for species with social factors that restrict access to breeding or experience repeated fluctuations in population size across generations. We investigated the genomic estimates of effective population size along with diversity, subdivision, and inbreeding from 162,109 minimally filtered and 81,595 statistically neutral and unlinked SNPs genotyped in 437 grey wolf samples from North America collected between 1986 and 2021. We found genetic structure across North America, represented by three distinct demographic histories of western, central, and eastern regions of the continent. Further, grey wolves in the northern Rocky Mountains have lower genomic diversity than wolves of the western Great Lakes and have declined over time. Effective population size estimates revealed the historical signatures of continental efforts of predator extermination, despite a quarter century of recovery efforts. We are the first to provide molecular estimates of effective population size across distinct grey wolf populations in North America, which ranged between Ne ~ 275 and 3050 since early 1980s. We provide data that inform managers regarding the status and importance of effective population size estimates for grey wolf conservation, which are on average 5.2-9.3% of census estimates for this species. We show that while grey wolves fall above minimum effective population sizes needed to avoid extinction due to inbreeding depression in the short term, they are below sizes predicted to be necessary to avoid long-term risk of extinction.

The vomeronasal system of the wolf (Canis lupus signatus): The singularities of a wild canid.

Wolves, akin to their fellow canids, extensively employ chemical signals for various aspects of communication, including territory maintenance, reproductive synchronisation and social hierarchy signalling. Pheromone-mediated chemical communication operates unconsciously among individuals, serving as an innate sensory modality that regulates both their physiology and behaviour. Despite its crucial role in the life of the wolf, there is a lacuna in comprehensive research on the neuroanatomical and physiological underpinnings of chemical communication within this species. This study investigates the vomeronasal system (VNS) of the Iberian wolf, simultaneously probing potential alterations brought about by dog domestication. Our findings demonstrate the presence of a fully functional VNS, vital for pheromone-mediated communication, in the Iberian wolf. While macroscopic similarities between the VNS of the wolf and the domestic dog are discernible, notable microscopic differences emerge. These distinctions include the presence of neuronal clusters associated with the sensory epithelium of the vomeronasal organ (VNO) and a heightened degree of differentiation of the accessory olfactory bulb (AOB). Immunohistochemical analyses reveal the expression of the two primary families of vomeronasal receptors (V1R and V2R) within the VNO. However, only the V1R family is expressed in the AOB. These findings not only yield profound insights into the VNS of the wolf but also hint at how domestication might have altered neural configurations that underpin species-specific behaviours. This understanding holds implications for the development of innovative strategies, such as the application of semiochemicals for wolf population management, aligning with contemporary conservation goals.

Postherpetic granulomatous dermatitis occurring in the setting of immune checkpoint inhibitor therapy: A case report.

We report a case of a 72-year-old man presenting with a 2-month history of a persistent, painful rash of the chest, axilla, and back. He had a history of recently resolved varicella zoster virus reactivation in the same distribution of the current rash and metastatic malignant melanoma treated with nivolumab and ipilimumab. The histopathology was consistent with granulomatous dermatitis (GD), and a diagnosis of postherpetic isotopic response manifesting as GD was made. Given the paucity of reported cases of postherpetic GD in the setting of treatment with immune checkpoint inhibitors (ICIs), we discuss the clinicopathologic features of this case and potential mechanisms by which ICIs may contribute to the development of granulomatous disease.

Conservation genomics of wolves: The global impact of RK Wayne's research.

RK Wayne has arguably been the most influential geneticist of canids, famously promoting the conservation of wolves in his homeland, the United States. His influence has been felt in other countries and regions outside the contiguous United States, where he inspired others, also including former graduate students and research fellows of his, to use modern molecular techniques to examine the evolutionary biology of canids to inform the conservation and management of wolves. In this review, we focus on the implications of Wayne's work on wolves outside the United States. He envisioned a clear future for wolf conservation research, involving the study of wolves' ecological and genetic diversity, and the description of ecotypes requiring conservation. He also documented widespread hybridization among canids and introgression of DNA from domestic dogs to wolves, a process that started dozens of thousands of years ago. His work therefore calls for innovative studies, such as examining the potential fitness benefits of introgression. Inspired by his results, for example, on the purging of deleterious alleles in small populations, wolf researchers should use novel molecular tools to challenge other conservation genetics paradigms. Overall, RK Wayne's work constitutes a call for answers, which as scientists or citizens concerned with conservation matters, we are obliged to address, as we contribute to monitoring and maintaining biodiversity during our period of dramatic transformations of the biosphere.

An improved method for diagnosis of Parkinson's disease using deep learning models enhanced with metaheuristic algorithm.

Parkinson's disease (PD) is challenging for clinicians to accurately diagnose in the early stages. Quantitative measures of brain health can be obtained safely and non-invasively using medical imaging techniques like magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT). For accurate diagnosis of PD, powerful machine learning and deep learning models as well as the effectiveness of medical imaging tools for assessing neurological health are required. This study proposes four deep learning models with a hybrid model for the early detection of PD. For the simulation study, two standard datasets are chosen. Further to improve the performance of the models, grey wolf optimization (GWO) is used to automatically fine-tune the hyperparameters of the models. The GWO-VGG16, GWO-DenseNet, GWO-DenseNet + LSTM, GWO-InceptionV3 and GWO-VGG16 + InceptionV3 are applied to the T1,T2-weighted and SPECT DaTscan datasets. All the models performed well and obtained near or above 99% accuracy. The highest accuracy of 99.94% and AUC of 99.99% is achieved by the hybrid model (GWO-VGG16 + InceptionV3) for T1,T2-weighted dataset and 100% accuracy and 99.92% AUC is recorded for GWO-VGG16 + InceptionV3 models using SPECT DaTscan dataset.

A panel of tetranucleotide STR markers as an alternative approach to forensic DNA identification of wolf and dog.

Commercial panels of microsatellite (STR) loci are focused on the use of DNA of the domestic dog (Canis lupus familiaris) and are often inapplicable for genotyping the DNA of the gray wolf (Canis lupus lupus). We propose a CPlex test system, including one hexa- and 12 tetranucleotide autosomal STR loci, as well as two sex loci, that is equally efficient in DNA identification of biological samples of the wolf and the dog. Analysis of molecular variance between samples revealed significant differentiation values (FST = 0.0784, p < 0.001), which allows to use the panel to differentiate wolf and dog samples. Population subdivision coefficients (θ-values) were calculated for each of the 13 STR loci of the developed test system. It was shown that the values of the genotype frequency for dogs and wolves, without and with considering the θ-value, differ by three orders of magnitude (for dogs 8.9 × 10-16 and 2.1 × 10-14 and for wolves 1.9 × 10-15 and 4.5 × 10-14, respectively). The use of population subdivision coefficients will allow to identify the most reliable results of an expert identification study and the power of exclusion provided by the STR loci of the CPlex test system makes it possible to achieve a reliable level of evidence in forensic DNA analysis of both wolves and dogs. The test system has been validated for use in forensic identification of the dog and wolf based on biological traces found at crime scenes, as well as for individual identification and establishing biological relationship of animals of these species.

Human Genetics of Hypoplastic Left Heart Syndrome.

Hypoplastic left heart syndrome (HLHS) is a severe congenital cardiovascular malformation characterized by hypoplasia of the left ventricle, aorta, and other structures on the left side of the heart. The pathologic definition includes atresia or stenosis of both the aortic and mitral valves. Despite considerable progress in clinical and surgical management of HLHS, mortality and morbidity remain concerns. One barrier to progress in HLHS management is poor understanding of its cause. Several lines of evidence point to genetic origins of HLHS. First, some HLHS cases have been associated with cytogenetic abnormalities (e.g., Turner syndrome). Second, studies of family clustering of HLHS and related cardiovascular malformations have determined HLHS is heritable. Third, genomic regions that encode genes influencing the inheritance of HLHS have been identified. Taken together, these diverse studies provide strong evidence for genetic origins of HLHS and related cardiac phenotypes. However, using simple Mendelian inheritance models, identification of single genetic variants that "cause" HLHS has remained elusive, and in most cases, the genetic cause remains unknown. These results suggest that HLHS inheritance is complex rather than simple. The implication of this conclusion is that researchers must move beyond the expectation that a single disease-causing variant can be found. Utilization of complex models to analyze high-throughput genetic data requires careful consideration of study design.

Epigenetics.

Epigenetics is the study of heritable changes to the genome and gene expression patterns that are not caused by direct changes to the DNA sequence. Examples of these changes include posttranslational modifications to DNA-bound histone proteins, DNA methylation, and remodeling of nuclear architecture. Collectively, epigenetic changes provide a layer of regulation that affects transcriptional activity of genes while leaving DNA sequences unaltered. Sequence variants or mutations affecting enzymes responsible for modifying or sensing epigenetic marks have been identified in patients with congenital heart disease (CHD), and small-molecule inhibitors of epigenetic complexes have shown promise as therapies for adult heart diseases. Additionally, transgenic mice harboring mutations or deletions of genes encoding epigenetic enzymes recapitulate aspects of human cardiac disease. Taken together, these findings suggest that the evolving field of epigenetics will inform our understanding of congenital and adult cardiac disease and offer new therapeutic opportunities.

Human Genetics of Ventricular Septal Defect.

Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copy number variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.

Morphea as an isotopic response to pigmented purpuric dermatosis and lichen striatus.

Morphea is an uncommon inflammatory and fibrosing disorder that has a polymorphous clinical presentation. We report two cases of morphea developing as an isotopic response after a preceding benign skin disease, accompanied by a review of the literature. This case series highlights the importance of return to care recommendations for benign skin conditions such lichen striatus and pigmented purpuric dermatoses due to the rare possibility of subsequent morphea development.

Wolves make roadways safer, generating large economic returns to predator conservation.

Recent studies uncover cascading ecological effects resulting from removing and reintroducing predators into a landscape, but little is known about effects on human lives and property. We quantify the effects of restoring wolf populations by evaluating their influence on deer-vehicle collisions (DVCs) in Wisconsin. We show that, for the average county, wolf entry reduced DVCs by 24%, yielding an economic benefit that is 63 times greater than the costs of verified wolf predation on livestock. Most of the reduction is due to a behavioral response of deer to wolves rather than through a deer population decline from wolf predation. This finding supports ecological research emphasizing the role of predators in creating a "landscape of fear." It suggests wolves control economic damages from overabundant deer in ways that human deer hunters cannot.

Transcriptomic and metabolomic profiles of Pirata subpiraticus in response to copper exposure.

Copper (Cu) contamination represents a persistent and significant form of heavy metal pollution in agricultural ecosystems, posing serious threats to organisms in current society. Spiders serve as crucial biological indicators for assessing the impact of heavy metals-induced toxicity. However, the specific molecular responses of spiders to Cu exposure and the mechanisms involved are not well understood. In our study, the wolf pond spiders, Pirata subpiraticus, were exposed to Cu for 21 d, resulting in a notable decline in survival rates compared with the control (n = 50, p < 0.05). We observed an increased expression of enzymes like glutathione peroxidase and superoxide dismutase (p < 0.05), signaling a strong oxidative stress response crucial for counteracting the harmful effects of reactive oxygen species. This response was corroborated by a rise in malondialdehyde levels (p < 0.05), a marker of lipid peroxidation and oxidative damage. Transcriptomic and metabolomic analyses revealed 2004 differentially expressed genes (DEGs) and 220 metabolites (DEMs). A significant number of these DEGs were involved in the glutathione biosynthetic process and antioxidant activity. A conjoint analysis revealed that under the Cu stress, several important enzymes and metabolites were altered (e.g., cathepsin A, legumain, and lysosomal acid lipase), affecting the activities of key biological processes and components, such as lysosome and insect hormone biosynthesis. Additionally, the protein interaction network analysis showed an up-regulation of processes like the apoptotic process, glutamate synthase activity, and peroxisome, suggesting that spiders activate cellular protective strategies to cope with stress and maintain homeostasis. This study not only deepens our understanding of spider biology in the context of environmental stress but also makes a significant contribution to the field of environmental stress biology.

Nutritional cataracts in a wolf litter treated by phacoemulsification.

OBJECTIVE: To describe the clinical findings in a wolf litter with nutritional cataracts and determine the treatment outcomes after phacoemulsification. PROCEDURE: Bilateral nutritional cataracts were diagnosed in four hand-fed 10-week-old wolves (Canis lupus). The information collected included signalment, physical and ophthalmic examination findings, ocular ultrasonography and electroretinography results, and postoperative outcomes. RESULTS: All four wolves were rejected from the dam and hand raised with a commercial artificial milk replacer from 5 days of age until weaning at 6 weeks of age. At initial presentation, bilateral cataracts were observed in all patients (8/8 eyes), with vision deficits in three of the four wolves. The main ophthalmic anomalies were mature cataracts with lens-induced uveitis (3/8 eyes, two wolves), immature cataracts (1/8 eyes, one wolf), and perinuclear and posterior cortical/subcapsular lens opacities (4/8 eyes, three wolves). Three of the four wolves (six eyes) underwent bilateral one-handed phacoemulsification with intraocular lens implantation. At the last examination 2 months following surgery, all operated eyes (6/6) were visual, Elschnig pearl proliferation was present in 2/6 eyes, and mild posterior capsular opacification was observed in 6/6 eyes. In the nonoperated wolf, the cataracts remained stable and did not affect the visual axis of either eye. CONCLUSIONS: This is the first description of phacoemulsification surgery in wolves. Phacoemulsification is a viable treatment option for captive wolves with nutritional cataracts. Special attention should be paid to hand-raised cubs to ensure that adequate amino acid levels are present in artificial milk.

Clustered Routing Using Chaotic Genetic Algorithm with Grey Wolf Optimization to Enhance Energy Efficiency in Sensor Networks.

As an alternative to flat architectures, clustering architectures are designed to minimize the total energy consumption of sensor networks. Nonetheless, sensor nodes experience increased energy consumption during data transmission, leading to a rapid depletion of energy levels as data are routed towards the base station. Although numerous strategies have been developed to address these challenges and enhance the energy efficiency of networks, the formulation of a clustering-based routing algorithm that achieves both high energy efficiency and increased packet transmission rate for large-scale sensor networks remains an NP-hard problem. Accordingly, the proposed work formulated an energy-efficient clustering mechanism using a chaotic genetic algorithm, and subsequently developed an energy-saving routing system using a bio-inspired grey wolf optimizer algorithm. The proposed chaotic genetic algorithm-grey wolf optimization (CGA-GWO) method is designed to minimize overall energy consumption by selecting energy-aware cluster heads and creating an optimal routing path to reach the base station. The simulation results demonstrate the enhanced functionality of the proposed system when associated with three more relevant systems, considering metrics such as the number of live nodes, average remaining energy level, packet delivery ratio, and overhead associated with cluster formation and routing.