Limb Formation Research Articles

What Are the Factors Associated With Revision Surgery on the Residual Limb and Functional Results in Patients With Posttraumatic Lower Limb Amputations?

Lower limb amputations performed after trauma are associated with a high risk of revision surgery. While the factors influencing revision surgery in the upper limbs have been studied, no studies have analyzed these factors in the lower limbs. Existing explanations for these revision surgeries are unclear, often leaving patients uninformed. Surgeons also lack the tools to explain the factors that influence repeat operations to patients. Therefore, the aim of this study was to provide surgeons with some answers so they can inform their patients undergoing posttraumatic lower limb amputation, whether military or civilian. (1) What was the survivorship of the initial amputation free from any revision surgery? (2) What patient- and injury-related factors were associated with revision amputation? (3) Do these factors influence functional outcomes in these patients? A single-center, retrospective study was conducted between January 2010 and February 2020 on patients who had undergone traumatic lower limb amputation. Between January 2010 and February 2020, 322 patients underwent amputation or were followed up at Percy Military University Hospital. Thirty-one patients had undergone amputation at another center, 178 had undergone amputation for nontraumatic reasons, and 27 patients had only upper limb amputations. Of those remaining, 1 died before 6 months, and 6% (5 of 86) were not fitted with a prosthesis, leaving 99% (85 of 86) for survivorship free from revision analysis and 93% (80 of 86) for functional endpoints analysis in this retrospective study at a median of 6.5 years (IQR 5 to 9) following the index amputation. The median age at the time of amputation was 31 years (IQR 26 to 52), 85% (72 of 85) of patients were men, and 31% (26 of 85) were military personnel. Revision surgery was defined as surgery performed at or after 6 months to ensure that the residual limb was healed and fitted with a prosthesis. Revision procedures performed before 6 months (median 2 [IQR 0 to 7]) were considered as part of the initial residual limb formation surgery. We performed Kaplan-Meier survivorship analysis for the time free from revision amputation from 6 months after amputation. We considered the competitive risk of death using a Fine-Gray model by an ascending stepwise procedure. To answer our third research question, we performed a chart review and assessed patients' use of prostheses and assistive devices and the percentage of patients who returned to work. An ordinal logistic regression was used to analyze the factors influencing functional outcome using an ascending stepwise procedure. A total of 85 patients (94 limbs) were included, of whom 25 (27 limbs) underwent a revision surgery on the residual limb > 6 months after amputation. Kaplan-Meier survival estimates indicated that 5 years after the initial amputation 64% (95% confidence interval 53% to 77%) of the patients remained free from revision surgery on their residual limb. Factors associated with increased odds of revision amputation were smoking (subdistribution HR 2.6 [95% CI 1.2 to 5.8]; p = 0.02) and an age of > 50 years (subdistribution HR 0.3 [95% CI 0.1 to 0.8]; p = 0.01). Ninety-four percent (80 of 85) of patients were fitted with prostheses, and 40% (32 of 80) of patients used material or human assistance for simple activities of daily living. Seventy-one percent of patients (57 of 80) had returned to work. Ordinal logistic regression revealed an association between preinjury sports activity and the absence of need for assistance in daily activities (OR 9 [95% CI 2.9 to 31.8]; p < 0.001). Smoking appeared to be an associated risk factor for residual limb revision surgery in posttraumatic lower limb amputations and is potentially modifiable. Being at least 50 years of age seemed to be associated with a lower incidence of revision surgery, which is probably linked to lower functional demands made on limbs beyond this age. Our study showed a remarkable rate of fitting with a prosthetic device, with an improved functional result if the patient was athletic before the trauma. These results provide more precise information on the care pathway to be considered for each patient. A study with a much larger sample would make it possible to assess the risk factors for the occurrence of each complication involving revision surgery. Level III, therapeutic study.

Formation of the Atlantic Meridional Overturning Circulation lower limb is critically dependent on Atlantic-Arctic mixing

Deep-water formation in the eastern Subpolar North Atlantic Ocean (eSPNA) and Nordic Seas is crucial for maintaining the lower limb of the Atlantic Meridional Overturning Circulation (AMOC), of consequence for global climate. However, it is still uncertain which processes determine the deep-water formation and how much Atlantic and Arctic waters respectively contribute to the lower limb. To address this, here we used Lagrangian trajectories to diagnose a global eddy-resolving ocean model that agrees well with recent observations highlighting the eSPNA as a primary source of the AMOC lower limb. Comprised of 72% Atlantic waters and 28% Arctic waters, the density and depth of the AMOC lower limb is critically dependent on Atlantic-Arctic mixing, primarily in the vicinity of Denmark Strait. In contrast, Atlantic waters gaining density through air-sea interaction along the eastern periphery of Nordic Seas and not entering the Arctic Ocean make a negligible contribution to the lower limb.

Gene Expression Patterns and Phenotypic Associations in Chondrogenesis: Insights into Skeletal Dysplasia Nosology

The differentiation of mesenchymal stem cells (MSCs) into chondrocytes, known as chondrogenesis, is a complex process that plays a fundamental role in cartilage formation and skeletal development. This study elucidates the transcriptional dynamics and phenotypic correlations at various stages of chondrogenesis (early, mid, and late) using RNA-seq data. We focused on the differential expression of transcription factors (TFs) and RNA-binding proteins (RBPs). We identified critical genes during their highest expression periods and generated heatmaps to visualize these temporal patterns. Additionally, we conducted a comprehensive analysis of skeletal dysplasia nosology genes, determining their highest expression periods and phenotypic implications using the DisGeNET database. Our findings reveal that early-stage (D1) gene expression is linked to craniofacial development and limb formation anomalies, primarily involving genes responsible for extracellular matrix (ECM) organization and signal transduction. Mid-stage (D7) genes are associated with cartilage matrix composition and skeletal growth, highlighting roles in chondrocyte proliferation and matrix deposition. Late-stage (D21) genes are implicated in bone mineral density, cartilage integrity, and joint formation, ensuring the maturation and functionality of cartilage tissue. This study provides a detailed analysis of gene expression regulators and their phenotypic correlations during chondrogenesis, offering insights into the molecular mechanisms driving cartilage development and skeletal dysplasias. Understanding these temporal gene expression patterns enhances our knowledge of chondrogenesis and aids in developing targeted therapies for cartilage-related diseases. These findings underscore the significance of time-point analyses in capturing the dynamic regulation of gene expression throughout the differentiation process.

Comparison of kinematics and pedobarography findings between the unaffected foot of patients with unilateral clubfoot and controls.

Idiopathic clubfoot (ICF) involves structural abnormalities in the lower extremities. Approximately half of patients have unilateral ICF, which could be due to differences in limb formation. The contralateral unaffected foot may have subclinical ICF. The objectives were to compare ankle and foot kinematics and pedobarography findings between the unaffected foot of patients with unilateral ICF and controls. Eleven children with unilateral ICF (11 unaffected feet, 11.7 ± 3.8years) and 15 age-matched controls (30 control feet, 11.1 ± 3.0years) were enroled. Five complete gait trials were performed. Data were collected using ten cameras and a twom long Footscan system and compared between groups using the Wilcoxon rank sum test. All children with ICF underwent the selective soft tissue release procedure. The unaffected feet showed limited ranges in inversion-eversion and dorsiflexion-plantar flexion on kinematic analysis. There was a delay in landing time in all regions of the foot during heel rise and propulsion. The peak time was achieved significantly later in the unaffected feet compared to the controls. Although plantar pressure parameters were comparable, unaffected feet showed a larger contact area in the midfoot region. Gait data on the unaffected foot in unilateral ICF patients do not correspond to those of controls despite an indistinguishable clinical appearance. This could be due to the effect of treatment, the process of foot development, or a combination. The relationship between genetics and gait deviation in patients with unilateral ICF needs further evaluation. The unaffected foot should receive similar attention during follow-up.

Morphological abnormalities in Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) treated with eight toxic compounds

Toxicity testing is essential for preventing chemical pollution of the environment. T. castaneum can be used as an alternative model for preliminary toxicity screening, eight chemical compounds having known toxicity were evaluated. For example, mercury (II) chloride is considered toxic at concentrations higher than 0.1 mg/L in drinking water, phenol exposure to concentrations greater than 5 mg/L in drinking water can be dangerous, in the case of toluene exposure to concentrations greater than 200 ppm (parts per million) in the air can be harmful to health, hydrazine can be dangerous at concentrations higher than 1 mg/L in drinking water, and caffeine can cause adverse effects such as nervousness, insomnia, tachycardia, and tremors from exposure to doses higher than 500 - 600 mg in adults. In this case, adult insects were fed a diet of oatmeal supplemented with each separately toxic. The number of offspring insects, size, weight, and observation of abnormalities was evaluated. The eight chemicals assessed compounds only five had a visible effect on insect development, BPA and mercury (II) chloride induced abnormalities in the larval and pupal stages, while the phenol, toluene, and metronidazole, in the pupal stage only. Major anomalies were observed necrosis in larvae appendices, in pupae, papillae for sex differentiation are sclerotic or absent, and abnormality in the formation of head, limbs, wings, and appendages. Mercury (II) chloride was the most toxic due to the affected growth, development and reproduction of the insect.

Knockdown of vitamin D receptor affects early stages of pectoral fin development in zebrafish.

The vitamin D receptor (VDR) signalling has been implicated in vertebrate limb or fin formation. However, the involvement of VDR signalling in the early stages of limb/fin development remains to be elucidated. In this study, the role of VDR signalling in pectoral fin development was investigated in zebrafish embryos. Knockdown of vdr induced the severe impairment of pectoral fin development. The zebrafish larvae lacking vdr exhibited reduced pectoral fins with no skeletal elements. Insitu hybridization revealed depletion of vdr downregulated fibroblast growth factor 24 (fgf24), a marker of early pectoral fin bud mesenchyme, in the presumptive fin field even before fin buds were visible. Moreover, a perturbed expression pattern of bone morphogenetic protein 4 (bmp4), a marker of the pectoral fin fold, was observed in the developing fin buds of zebrafish embryos that lost the vdr function. These findings suggest that VDR signalling is crucial in the early stages of fin development, potentially influencing the process by regulating other signalling molecules such as Fgf24 and Bmp4.

Normal table of embryonic development in the Anji salamander Hynobius amjiensis (Hynobiidae)

We established a normal embryonic development table for the Anji salamander Hynobius amjiensis, a critically endangered tailed amphibian of the family Hynobiidae with a very limited distribution in East China, following the standards set by the early developmental table of vertebrates. Put together 32 embryonic stages for the Anji salamander was defined. The total embryonic period from oviposition to hatching is approximately 30 days at 9 °C. Stages 1–16 represent early development from cleavage to neurulation. Stages 17–32 represent organogenesis documenting later developmental events such as tail, gill, and limb formation, and hatching (Stage 32). We provided a detailed description of the external morphology and color changes of the head, trunk, limbs, tail, external gills, and balancers at various stages from egg-laying to hatching. We also described several cases of abnormal embryonic development. The establishment of the embryonic development table in H. amjiensis contributes to better understanding of the ontogeny in tailed amphibians, distinguishing closely related species, and identifying abnormal embryonic amphibians.

Interleukin-6 supplementation improves bovine conceptus elongation and transcriptomic indicators of developmental competence†.

A high incidence of pregnancy failures occurs in cattle during the second week of pregnancy as blastocysts transition into an elongated conceptus. This work explored whether interleukin-6 supplementation during in vitro embryo production would improve subsequent conceptus development. Bovine embryos were treated with 0 or 100ng/mL recombinant bovine interleukin-6 beginning on day 5 post-fertilization. At day 7.5 post-fertilization, blastocysts were transferred into estrus synchronized beef cows (n = 5 recipients/treatment, 10 embryos/recipient). Seven days after transfer (day 14.5), cows were euthanized to harvest reproductive tracts and collect conceptuses. Individual conceptus lengths and stages were recorded before processing for RNA sequencing. Increases in conceptus recovery, length, and the proportion of tubular and filamentous conceptuses were detected in conceptuses derived from interleukin-6-treated embryos. The interleukin-6 treatment generated 591 differentially expressed genes in conceptuses (n = 9-10/treatment). Gene ontology enrichment analyses revealed changes in transcriptional regulation, DNA-binding, and antiviral actions. Only a few differentially expressed genes were associated with extraembryonic development, but several differentially expressed genes were associated with embryonic regulation of transcription, mesoderm and ectoderm development, organogenesis, limb formation, and somatogenesis. To conclude, this work provides evidence that interleukin-6 treatment before embryo transfer promotes pre-implantation conceptus development and gene expression in ways that resemble the generation of a robust conceptus containing favorable abilities to survive this critical period of pregnancy.

Evaluation of human mononuclear umbilical cord blood cells systemic administration efficiency in the acute period of experimental severe spinal cord injury

Aim. To evaluate the efficiency of systemic (intravenous) application of cryopreserved human umbilical cord blood mononuclear cells (HUCBCs) in animal models of acute contusion spinal cord injury for the restoration of hind limb motor function and formation of posttraumatic cysts using clinically significant examination methods.Materials and methods. Adult female Sprague–Dowley rats were used for the study. Severe acute contusion spinal cord injury model was performed using standard “weight‑drop” method. All samples of cryopreserved HUCBCs concentrate were prestored prior to infusion for 3 to 4 years at –196 °C. Hind limbs motor function was evaluated using open‑field technique and standard BBB testing system. Magnetic resonance scanning was performed using high‑field magnetic resonance CleanScan 7.0 T tomography (Bruker BioSpin, Germany).Results. Intravenous infusions of HUCBCs were performed on Day 1 following acute severe spinal cord injury. Motor function assessment demonstrated significant (p &lt;0.05) improvement of hind limbs motor function (up to 40–50 %) comparing to self‑healing outcomes. Moreover, by the Days 4 and 5 after severe spinal cord injury, the volume of posttraumatic cystic cavity decreases significantly (up to 40 %) (p &lt;0.05).Conclusion. The obtained results demonstrated that cryopreserved HUCBCs can be used as an effective source for cell therapy of acute contusion spinal cord injury.

Blood and bones: Mechanical cues and Hippo signaling drive vascular invasion during limb formation

Although mechanical cues are known to influence the postnatal skeleton, the impact of bone cell mechano-transduction on early skeletal development remains less clear. In this issue of Developmental Cell, Collins etal. (2023) report that YAP/TAZ deletion in osteoblast precursors reduces Cxcl12 expression, leading to defects in bone vascularization.

The adverse side effects of prenatal and postnatal rats' exposure to silver nanoparticles Induced toxicity.

Silver nanotechnology is widely applied in industry and medicine, with an increased likelihood of environmental and food contamination. This study aimed to explore the adverse effects of orally administering silver nanoparticles (AgNPs) to pregnant or lactating female rats on adults and the development of their offspring. Forty female albino rats were used to assess the immediate impacts of AgNPs in two separate experiments. The experimental group received 1 ml of AgNPs, dissolved in deionized water, at doses of 0, 50, and 100 mg/kg of body weight from the 6th to the 15th day of gestation in pregnant albino rats. After a 20-day gestation period, euthanasia was performed on the female rats, followed by a gross examination post-dissection. The feti were preserved in ethyl alcohol and Poin's solution for the identification of skeletal and visceral malformations. It was noticed that feti of dams that received AgNPs showed teratogenicities such as delayed ossification and deletion of bones or ribs. Notably, dams showed necrosis and satellitosis with evidence of behavioral alteration. While rats' pups showed only brain edema and no behavioral changes. AgNPs at a dose of 50 or 100 mg/kg induced teratogenic effect in terms of delayed ossification, abnormal limb formation, and brain edema in rat pups, however, induced necrosis and satellitosis in dam rats. Hence, greater emphasis should be placed on preventing exposure to Ag-NPs, especially among pregnant females.

Variants in EFCAB7 underlie nonsyndromic postaxial polydactyly.

Polydactyly is the most common limb malformation that occurs in 1.6-10.6 per one thousand live births, with incidence varying with ancestry. The underlying gene has been identified for many of the ~100 syndromes that include polydactyly. While for the more common form, nonsydromic polydactyly, eleven candidate genes have been reported. We investigated the underlying genetic cause of autosomal recessive nonsyndromic postaxial polydactyly in four consanguineous Pakistani families. Some family members with postaxial polydactyly also present with syndactyly, camptodactyly, or clinodactyly. Analysis of the exome sequence data revealed two novel homozygous frameshift deletions in EFCAB7: [c.830delG;p.(Gly277Valfs*5)]; in three families and [c.1350_1351delGA;p.(Asn451Phefs*2)] in one family. Sanger sequencing confirmed that these variants segregated with postaxial polydactyly, i.e., family members with postaxial polydactyly were found to be homozygous while unaffected members were heterozygous or wild type. EFCAB7 displays expressions in the skeletal muscle and on the cellular level in cilia. IQCE-EFCAB7 and EVC-EVC2 are part of the heterotetramer EvC complex, which is a positive regulator of the Hedgehog (Hh) pathway, that plays a key role in limb formation. Depletion of either EFCAB7 or IQCE inhibits induction of Gli1, a direct Hh target gene. Variants in IQCE and GLI1 have been shown to cause nonsyndromic postaxial polydactyly, while variants in EVC and EVC2 underlie Ellis van Creveld and Weyers syndromes, which include postaxial polydactyly as a phenotype. This is the first report of the involvement of EFCAB7 in human disease etiology.

TWIST1 Gene Variants Cause Craniosynostosis with Limb Abnormalities in Asian Patients.

The TWIST1 gene codes for a highly conserved transcription factor in a basic helix-loop-helix transcription factors family. The pattern of inheritance is autosomal dominant in Saethre-Chotzen syndrome, Robinow-Sorauf syndrome, and Sweeney-Cox syndrome. Major features of these syndromes include coronal synostosis, vision problems, and deafness, and facial features include hypertelorism, low-set ears, arched eyebrows, beaked nose, maxillary hypoplasia, and other dysmorphisms including broad great toes, clinodactyly, brachydactyly, and cutaneous syndactyly. TWIST1 (bHLH) transcription factor regulates the formation of head and limbs in the embryo. We describe three families affected with craniosynostosis in whom a sporadic TWIST1 variant was identified on whole exome sequencing, chromosomal microarray, and Sanger sequencing.

Distribution of Bamboo Witches’ Broom Disease in Various Bamboo Species in the Philippines and Molecular Identification of 'Candidatus Phytoplasma luffae'-related Strain 16SrVIII

Bamboo is used for making structures, furniture, handicrafts, and ropes, as well as a source of food in the Philippines. One of the emerging diseases of bamboo in the country is the bamboo witches’ broom (BWB), which has been occasionally noted in three genera of bamboo – including Dendrocalamus, Gigantochloa, and Schizostachyum from various provinces in the Philippines (Ilocos Norte, Laguna, Batangas, Quezon, Agusan del Sur, Bukidnon, and South Cotabato) since the 1990s. However, studies and information about BWB in the country remain lacking and largely unexplored. In this study, we report a similar disease affecting Dendrocalamus and Gigantochloa bamboo species from Bohol and Davao and – for the first time – in Dendrocalamus merrillianus ("bayog") and Bambusa spinosa ("kawayan-tinik") from Isabela and Nueva Vizcaya. As a result of the surveys conducted from 1999–2019, the disease is now identified in six species across four genera of bamboo – namely, Gigantochloa spp. (G. levis and G. atter), Dendrocalamus spp. (D. asper and D. merrillianus), Schizostachyum lumampao, and Bambusa spinosa recorded in 11 provinces in the Philippines. The BWB symptoms include clustering of leaves forming a rosette-like structure, leaf proliferation, excessive limb formation from a single node, and shortening of internodes. Nested PCR using the universal primers P1/P7 and R16MF2/R1 targeting the phytoplasma 16S ribosomal RNA gene revealed positive amplification in five symptomatic BWB samples from Isabela, Philippines. Subsequent sequencing (~ 1.3kbp) and phylogenetic analysis using the representative BWB isolates from Isabela revealed &gt; 98.65% genetic similarity and clustering to Candidatus Phytoplasma luffae, which belongs to the 16SrVIII group (Loofah Witches’ Broom Group). This paper determined the distribution of BWB in different species of bamboo in the Philippines, as well as the association of 'Ca. Phytoplasma luffae'-related strain (16SrVIII) to BWB.

Developmental regulation of conserved non-coding element evolution provides insights into limb loss in squamates.

Limb loss shows recurrent phenotypic evolution across squamate lineages. Here, based on three de novo-assembled genomes of limbless lizards from different lineages, we showed that divergence of conserved non-coding elements (CNEs) played an important role in limb development. These CNEs were associated with genes required for limb initiation and outgrowth, and with regulatory signals in the early stage of limb development. Importantly, we identified the extensive existence of insertions and deletions (InDels) in the CNEs, with the numbers ranging from 111 to 756. Most of these CNEs with InDels were lineage-specific in the limbless squamates. Nearby genes of these InDel CNEs were important to early limb formation, such as Tbx4, Fgf10, and Gli3. Based on functional experiments, we found that nucleotide mutations and InDels both affected the regulatory function of the CNEs. Our study provides molecular evidence underlying limb loss in squamate reptiles from a developmental perspective and sheds light on the importance of regulatory element InDels in phenotypic evolution.

Macrophages mobilized by the overexpression of the macrophage-colony stimulating factor promote efficient recovery of the ischemic muscle functionality

AimsNarrowing or occlusion of arteries that supply the limbs can evolve to critical limb ischemia. M-CSF promotes proliferation, differentiation and survival of monocytes and macrophages, and polarization of macrophages to M2-subtype, which are essential elements for vessel formation and tissue repair. Based on these properties of M-CSF, we hypothesize that transfection of M-CSF into ischemic limbs may promote vessel formation and repair of ischemic limbs. Main methodsHindlimb ischemia was surgically induced in 10–12 weeks old Balb/c and gene therapy was performed with intramuscular application of either uP-MCSF or uP plasmids (100 μg). Macrophage and monocyte subpopulations were assessed by flow cytometry and blood flow was monitored by Laser Doppler Perfusion Imaging (LDPI). Thirty days after transfection, we assessed gastrocnemius mass and muscle force, subsequently collecting the muscle for histology. Key findingsWe successfully developed the uP-MCSF plasmid, which increases M-CSF expression in the muscle transiently. Thirty days after uP-MCSF gene therapy in ischemic muscles, the treated group presented: improved muscle force, reduced fibrosis and increased arteriogenesis, although LDPI analysis did not show any significant difference in blood flow among groups. Noteworthy, we observed a temporary increase in MHCIIhighCD206high macrophages after uP-MCSF transfection. SignificanceM-CSF gene therapy improved ischemic muscle functionality by promoting arteriogenesis and decreasing fibrosis, likely through increased MHCIIhighCD206high macrophages and not via classically known M2-macrophages.

The Prrx1eGFP Mouse Labels the Periosteum During Development and a Subpopulation of Osteogenic Periosteal Cells in the Adult.

The identity of the cells that form the periosteum during development is controversial with current dogma suggesting these are derived from a Sox9-positive progenitor. Herein, we characterize a newly created Prrx1eGFP reporter transgenic mouse line during limb formation and postnatally. Interestingly, in the embryo Prrx1eGFP-labeled cells become restricted around the Sox9-positive cartilage anlage without themselves becoming Sox9-positive. In the adult, the Prrx1eGFP transgene live labels a subpopulation of cells within the periosteum that are enriched at specific sites, and this population is diminished in aged mice. The green fluorescent protein (GFP)-labeled subpopulation can be isolated using fluorescence-activated cell sorting (FACS) and represents approximately 8% of all isolated periosteal cells. The GFP-labeled subpopulation is significantly more osteogenic than unlabeled, GFP-negative periosteal cells. In addition, the osteogenic and chondrogenic capacity of periosteal cells in vitro can be extended with the addition of fibroblast growth factor (FGF) to the expansion media. We provide evidence to suggest that osteoblasts contributing to cortical bone formation in the embryo originate from Prrx1eGFP-positive cells within the perichondrium, which possibly piggyback on invading vascular cells and secrete new bone matrix. In summary, the Prrx1eGFP mouse is a powerful tool to visualize and isolate periosteal cells and to quantify their properties in the embryo and adult. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

The effect of pH on embryonal and larval development of the Caucasian Brown Frog, &lt;i&gt;Rana macrocnemis&lt;/i&gt;

Aim. To study the effect of different pH values on the characteristics of the embryonic and larval development of Rana macrocnemis.Material and methods. Fragments from 6–7 clutches of R. macrocnemis collected on the territory of Foothill Dagestan were used in the work. The experiment included two series of experiments: in the 1st series, the entire cycle of embryonic‐larval development of the R. macrocnemis frog was studied, and in the 2nd, only the postembryonic development of R. macrocnemis was studied. Eggs were individually placed in containers with different pH values (4.0, 5.0, and 9.0).Results. The most vulnerable periods in the development of R. macrocnemis are the stages of neurula, limb formation, and metamorphosis. At pH 4.0, oxygen consumption by larvae decreases, accompanied by suppression of feeding behavior and a decrease in the intensity of metabolic processes. In addition, pH 4.0 has a negative effect on the survival of embryos and larvae. At pH 5.0, changes are noted that are mainly of an adaptive nature. Against the background of the accelerated development of embryos at these pH values, a significant decrease in the length and weight of their body is noted. With the transition to larval development, an increase in body size is observed against the background of a small increase in weight. An elongation of the duration of the period from the beginning of the formation of limbs to the end of metamorphosis was also noted.Conclusion. The data obtained can be used in monitoring the level of acidity of the environment and the oxygen content in natural water bodies during the spawning period, which will help clarify the issue of the reduction in the number of R. macrocnemis frogs in Dagestan.

Genome-wide identification, characterization and expression analysis of the BMP family associated with beak-like teeth in Oplegnathus.

Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor beta (TGF-β) family, are critical for the control of developmental processes such as dorsal-ventral axis formation, somite and tooth formation, skeletal development, and limb formation. Despite Oplegnathus having typical healing beak-like teeth and tooth development showing a trend from discrete to healing, the potential role of BMPs in the development of the beak-like teeth is incompletely understood. In the present study, 19 and 16 BMP genes were found in O. fasciatus and O. punctatus, respectively, and divided into the BMP2/4/16, BMP5/6/7/8, BMP9/10, BMP12/13/14, BMP3/15 and BMP11 subfamilies. Similar TGFb and TGF_β gene domains and conserved protein motifs were found in the same subfamily; furthermore, two common tandem repeat genes (BMP9 and BMP3a-1) were identified in both Oplegnathus fasciatus and Oplegnathus punctatus. Selection pressure analysis revealed 13 amino acid sites in the transmembrane region of BMP3, BMP7, and BMP9 proteins of O. fasciatus and O. punctatus, which may be related to the diversity and functional differentiation of genes within the BMP family. The qPCR-based developmental/temporal expression patterns of BMPs showed a trend of high expression at 30 days past hatching (dph), which exactly corresponds to the ossification period of the bones and beak-like teeth in Oplegnathus. Tissue-specific expression was found for the BMP4 gene, which was upregulated in the epithelial and mesenchymal tissues of the beak-like teeth, suggesting that it also plays a regulatory role in the development of the beak-like teeth in O. punctatus. Our investigation not only provides a scientific basis for comprehensively understanding the BMP gene family but also helps screen the key genes responsible for beak-like tooth healing in O. punctatus and sheds light on the developmental regulatory mechanism.

Abstract 3358: Targeting p63 isoforms in cancer to restore their anti-tumorigenic activity

Abstract p63 plays a central role in limb formation and epithelial differentiation. p63, reported to correlate with cancer formation, is rarely mutated in cancers. Different p63 isoforms can have anti-tumorigenic or pro-tumorigenic activity. TA isoforms act as tumor suppressor by promoting cell death, while deltaN isoforms are strong driver for squamous/squamous-like cancers including lung, head and neck, cervical, skin and pancreas. A large number of tumors are addicted to deltaN isoforms. Furthermore, cancer associated p53R175H mutant gains ability to interact with TAp63 isoforms and inhibit their anti-tumorigenic function, while indirectly promoting tumorigenic function of deltaN isoforms. However, the exact mechanism of p53R175H-p63 interaction is unclear. The changes in the balance of p63 isoform activity are likely to be crucial to understanding the transition between normal cell and tumor formation. Here, we modelled p53R175H-p63 complex using homology modelling, flexible/unbiased docking and molecular dynamics simulations. Moreover, we used our models to design peptides to disrupt p53R175H-p63 interaction and restore anti-tumorigenic function of TAp63 isoforms. Also, we studied deltaN isoform oligomerization and designed peptides to inhibit their oligomerization to reduce their tumorigenic activity. We showed that some of our peptides promoted cell death in a p63 enriched cancel cell line. We characterized our peptides performing kinetic binding assays to validate binding of our peptides to their designated targets. In this study, our computational and experimental analyses presented a detailed model for the p53R175H-p63 interaction. We also provided a framework for potential therapeutic peptides to outweigh the balance in the advantage of anti-tumorigenic p63 activity. Citation Format: E. Sila Ozdemir, Michelle Gomes, Jared Fischer. Targeting p63 isoforms in cancer to restore their anti-tumorigenic activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3358.