Bioactivity screening and molecular identification of Anchusa milleri L. sunflower crud extract for antioxidant, antiviral, antimicrobial, and anticancer properties.

The current study evaluates hot water A. milleri sunflower extracts at 40 °C temperature (AMSE40) for their antibacterial, anti-biofilm, antiviral and anticancer activities. AMSE40 exhibited excellent antioxidant activity with an IC50 of 1.17 mg/mL (ascorbic acid) and was found to be rich in phytochemical compounds such as alkaloids, flavonoids, phenolics, saponins, and tannins, with concentrations of 1.23%, 351.60 mg/g, 152.50 mg/g, 0.98%, and 146.35 mg/g, respectively. AMSE40 showed strong antibacterial and anti-biofilm activity against four multidrug-resistant isolates, comprising E. faecalis, S. aureus, P. aeruginosa, and K. pneumonia with minimum bactericidal concentrations (25 mg/mL) and minimum inhibitory concentrations (12.5 mg/mL) for all isolates. The AMSE40 (62.5 µg/mL) showed antiviral efficacy against CoxB4 (9.1%) and HSV-1 (34.4%). Additionally, AMSE40 induced DNA fragmentation in liver cell lines, indicating cell death. The cytotoxic concentration of AMSE40 had a mild impact on Vero (283.2 µg) and HepG2 cells (76.4 µg). A. milleri has the potential to serve as a natural and eco-friendly source for innovative pharmaceutical and medical applications.

Experimental and computational assessment of Antiparkinson Medication effects on meiofauna: Case study of Benserazide and Trihexyphenidyl.

Two concentrations (6.25 and 1.25 mg/L) were used for two Parkinson's disease medications, Benserazide, and Trihexyphenidyl, to test their effects on the meiobenthic nematofauna. It is predicted that these highly hydrosoluble drugs will end up in marine environments. The results showed that both medications when added alone, induced (i) important changes in the numbers and (ii) taxonomic composition. The impact of Benserazide and Trihexyphenidyl was also reflected in the (iii) functional traits of nematofauna, with the most affected categories following exposure being the trophic group 1B, the clavate tails, the circular amphids, the c-p2 life history, and the body length of 1-2 mm. These results were supported by the molecular interactions of the studied drugs with both GLD-3 and SDP proteins of Caenorhabditis elegans. (iv) The mixtures of both drugs did not show any changes in the nematode communities, suggesting that no synergistic or antagonistic interactions exist between them.

Probing the interaction of Paenibacillus larvae bacteriophage as a biological agent to control the american foulbrood disease in honeybee.

American foulbrood (AFB) is a harmful honeybee disease primarily caused by Paenibacillus larvae. The study aims to isolate and identify the AFB causative agent P. larvae and their specific phages to use as a new biological method for AFB disease control. Eight apiaries were inspected for AFB infections. Symptoms of diseased brood comb, were odd brood cells with soft brown decayed brood amongst healthy brood, were identified in the field and demonstrated the prevalence of AFB in every apiary. Three P. larvae isolates were identified using traditional techniques using a 452-bp PCR amplicon specific to the bacterial 16SrRNA gene and was compared between Paenibacillus isolates. Additionally, specific phages of P. larvae strains were applied to examine their efficiency in reducing the infection rate under the apiary condition. The infection rate was reduced to approximately 94.6 to 100 % through the application of a phage mixture, as opposed to 20 to 85.7 % when each phage was administered individually or 78.6 to 88.9 % when antibiotic treatment was implemented. Histological studies on phage-treated bee larvae revealed some cells regaining normal shape, with prominent nuclei and microvilli. The gastrointestinal tract showed normal longitudinal and circular muscles, unlike bee larvae treated with bacterial strains with abnormal and destroyed tissues, as shown by the basement membrane surrounding the mid-gut epithelium. Phage techniques exhibited promise in resolving the issue of AFB in honeybees due to their ease of application, comparatively lower cost, and practicality for beekeepers in terms of laboratory preparation.

Green synthesis and bioefficacy screening of new insect growth regulators as eco-friendly insecticides against the cotton mealybug Phenacoccus solenopsis.

Overcoming or reducing the majority of difficulties caused by the use of common pesticides requires the use of developed, secure, unique and selective organic compounds. Due to their clear mechanism of action on pests and lower poisonousness towards vertebrates than conventional insecticides, juvenile hormone analogues as an example of insect growth regulators appear promising. Thus, a unique set of pure insect growth regulators has been synthesized. The structure of these synthesized compounds, which were related to the most well-known insect growth regulator insecticides, was confirmed by elemental and contemporary spectroscopic investigations (IR, UV, 1 H-NMR, 13 C NMR, and Dept 135 spectrum). Under laboratory conditions, the effectiveness of a chemically newly synthesized products was tested against the cotton mealybug, Phenacoccus solenopsis, and compared with Fenoxycarb as a reference insecticide. Compound 7 was discovered to be more effective than the other synthetic compounds, with LC50 values of 0.907 mg/L for adult female P. solenopsis and 0.377 mg/L for third instar nymphs. Furthermore, this results concluded that the adult female's stage of P. solenopsis was less sensitive to the checked treatments as matched to the third instar nymphs.

Coinfection of the gut with protozoal and metazoal parasites in broiler and laying chickens.

The chicken business faces substantial economic losses due to the risk of parasitic coinfection. Because the current study aimed to investigate enteric parasitic coinfections problems among the suspected examined chicken farms, samples were collected during the field investigation from suspected freshly dead birds, clinically diseased, apparently healthy, and litter samples for further laboratory parasitological, histopathological, and immunological examinations. Variable mortalities with various clinical indicators, such as ruffled feathers, weight loss, diarrhea of various colors, and a decline in egg production, occurred on the farms under investigation. In addition, the treatment protocols of each of the farms that were evaluated were documented and the m-RNA levels of some cytokines and apoptotic genes among the infected poultry have been assessed. The prevalence rate of parasitic coinfection in the current study was found to be 8/120 (6.66%). Parasitological analysis of the samples revealed that they belonged to distinct species of Eimeria, cestodes, and Ascaridia galli. When deposited, A. galli eggs were nonembryonated and ellipsoidal, but cestodes eggs possessed a thin, translucent membrane that was subspherical. Eimeria spp. oocysts in layer chickens were identified as Eimeria acervulina and Eimeria maxima in broiler chickens. Our findings proved that coinfection significantly upregulated the IL-1ß, BAX, and Cas-3 genes. Conversely, the IL-10, BCL-2, and AKT mRNA levels were downregulated, indicating that nematode triggered apoptosis. The existence of parasite coinfection was verified by histological investigation of the various intestinal segments obtained from affected flocks. A. galli and cestodes obstructed the intestinal lumen, causing different histological alternations in the intestinal mucosa. Additionally, the lamina propria revealed different developmental stages of Eimeria spp. It was determined that parasite coinfection poses a significant risk to the poultry industry. It was recommended that stringent sanitary measures management methods, together with appropriate treatment and preventative procedures, be employed in order to resolve such issues.

Expanding the therapeutic arsenal against cancer: a computational investigation of hybrid xanthone derivatives as selective Topoisomerase 2α ATPase inhibitors.

The DNA topoisomerase II (topo II) enzyme plays an important role in the replication, recombination, and repair of DNA. Despite their widespread applications in cancer therapy, new, selective, and potent topo II inhibitors with better pharmaceutical profiles are needed to handle drug resistance and severe adverse effects. In this respect, an array of 36 new anticancer compounds was designed based on a Xanthone core tethered to multifunctional Pyridine-amines and Imidazole scaffold via alkyl chain linkers. An integrated in silico approach was used to understand the structural basis and mechanism of inhibition of the hybrid xanthone derivatives. In this study, we established an initial virtual screening workflow based on pharmacophore mapping, docking, and cancer target association to validate the target selection process. Next, a simulation-based docking was conducted along with pharmacokinetic analysis to filter out the five best compounds (7, 10, 25, 27, and 30) having binding energies within the range of -60.45 to -40.97 kcal/mol. The screened compounds were further subjected to molecular dynamics simulation for 200 ns followed by MM-GBSA and ligand properties analysis to assess the stability and binding affinity to hTOP2α. The top-ranking hits 3,7-bis(3-(2-aminopyridin-3-ylhydroxy)propoxy)-1-hydroxy-9H-xanthen-9-one (ligand 7) and 3,8-bis(3-(2-aminopyridin-3-ylhydroxy)propoxy)-1-hydroxy-9H-xanthen-9-one (ligand 25) were found to have no toxicity, optimum pharmacokinetic and, DFT properties and stable intermolecular interactions with the active site of hTopo IIα protein. In conclusion, further in vitro and in vivo experimental validation of the identified lead molecules is warranted for the discovery of new human Topoisomerase 2 alpha inhibitors.Communicated by Ramaswamy H. Sarma.

Persea americana extract protects intestinal tissue from Eimeria papillata-induced murine Infection.

Coccidiosis is the most prevalent disease-causing widespread economic loss among farm and domestic animals. Currently, several drugs are available for the control of this disease but resistance has been confirmed for all of them. There is an urgent need, therefore, for the identification of new sources as alternative treatments to control coccidiosis. The present work aimed to study the effect of the Persea americana extract (PAE) as an anti-coccidial, anti-oxidant, and anti-apoptotic modulator during murine intestinal Eimeria papillata infection. A total of 25 male mice were divided into five groups, as follows: Group1: Non-infected-non-treated (negative control), Group2: Non-infected-treated group with PAE (500 mg/kg b.w). Group3: Infected-non-treated (positive control), Group4: Infected-treated group with PAE (500 mg/kg b.w.), and Group5: Infected-treated group with Amprolium (120 mg/kg b.w.). Groups (3-5) were orally inoculated with 1 × 103 sporulated E. papillata oocysts. After 60 min of infection, groups (4 and 5) were treated for 5 consecutive days with the recommended doses of PAE and amprolium. The fact that PAE has an anti-coccidial efficacy against intestinal E. papillata infection in mice has been clarified by the reduction of fecal oocyst output on the 5th day post-infection by about 85.41%. Moreover, there is a significant reduction in the size of each parasite stage in the jejunal tissues of the infected-treated group with PAE. PAE counteracted the E. papillata-induced loss of glutathione peroxidase (GPx), superoxide dismutase (SOD), and total antioxidant capacity (TCA). E. papillata infection also induced an increase in the apoptotic cells expressed by caspase-3 which modulated after PAE treatment. Moreover, the mRNA expression of the goblet cell response gene, mucin (MUC2), was upregulated from 0.50 to 1.20-fold after treatment with PAE. Based on our results, PAE is a promising medicinal plant with anti-coccidial, anti-oxidant, and anti-apoptotic activities and could be used as a food additive.

Identification of novel dual acting ligands targeting the adenosine A2A and serotonin 5-HT1A receptors.

GPCRs are a family of transmembrane receptors that are profoundly linked to various neurological disorders, among which is Parkinson's disease (PD). PD is the second most ubiquitous neurological disorder after Alzheimer's disease, characterized by the depletion of dopamine in the central nervous system due to the impairment of dopaminergic neurons, leading to involuntary movements or dyskinesia. The current standard of care for PD is Levodopa, a dopamine precursor, yet the chronic use of this agent can exacerbate motor symptoms. Recent studies have investigated the effects of combining A2AR antagonist and 5-HT1A agonist on dyskinesia and motor complications in animal models of PD. It has been proved that the drug combination has significantly improved involuntary movements while maintaining motor activity, highlighting as a result new lines of therapy for PD treatments, through the regulation of both receptors. Using a combination of ligand-based pharmacophore modelling, virtual screening, and molecular dynamics simulation, this study intends on identifying potential dual-target compounds from IBScreen. Results showed that the selected models displayed good enrichment metrics with a near perfect receiver operator characteristic (ROC) and Area under the accumulation curve (AUAC) values, signifying that the models are both specific and sensitive. Molecular docking and ADMET analysis revealed that STOCK2N-00171 could be potentially active against A2AR and 5-HT1A. Post-MD analysis confirmed that the ligand exhibits a stable behavior throughout the simulation while maintaining crucial interactions. These results imply that STOCK2N-00171 can serve as a blueprint for the design of novel and effective dual-acting ligands targeting A2AR and 5-HT1A.Communicated by Ramaswamy H. Sarma.

In-silico screening of Acacia pennata and Bridelia retusa reveals pinocembrin-7-O-ß-D-glucopyranoside as a promising ß-lactamase inhibitor to combat antibiotic resistance.

The ß-lactamase of Pseudomonas aeruginosa is known to degrade ß-lactam antibiotics such as penicillins, cephalosporins, monobactams, and carbapenems. With the discovery of an extended-spectrum ß-lactamase in a clinical isolate of P. aeruginosa, the bacterium has become multi-drug resistant. In this study, we aim to identify new ß-lactamase inhibitors by virtually screening a total of 43 phytocompounds from two Indian medicinal plants. In the molecular docking studies, pinocembrin-7-O-ß-D-glucopyranoside (P7G) (-9.6 kcal/mol) from Acacia pennata and ellagic acid (EA) (-9.2 kcal/mol) from Bridelia retusa had lower binding energy than moxalactam (-8.4 kcal/mol). P7G and EA formed 5 (Ser62, Asn125, Asn163, Thr209, and Ser230) and 4 (Lys65, Ser123, Asn125, and Glu159) conventional hydrogens bonds with the active site residues. 100 ns MD simulations revealed that moxalactam and P7G (but not EA) were able to form a stable complex. The binding free energy calculations further revealed that P7G (-59.6526 kcal/mol) formed the most stable complex with ß-lactamase when compared to moxalactam (-46.5669 kcal/mol) and EA (-28.4505 kcal/mol). The HOMO-LUMO and other DFT parameters support the stability and chemical reactivity of P7G at the active site of ß-lactamase. P7G passed all the toxicity tests and bioavailability tests indicating that it possesses drug-likeness. Among the studied compounds, we identified P7G of A. pennata as the most promising phytocompound to combat antibiotic resistance by potentially inhibiting the ß-lactamase of P. aeruginosa.Communicated by Ramaswamy H. Sarma.

Spectroscopic investigation of phase transformation of calcium oxalate dehydrates (renal calculi) using acidic Bryophyllum pinnatom powder.

Urolithiasis is one of most common renal disorders, characterized by the formation of kidney stones (renal calculi) through the crystallization process within the urinary system. The frequently observed renal calculi are calcium oxalate renal calculi and treatment is done by shock wave method or lithotripsy which is harmful for other cells of the internal system. The objective of this work was to evaluate in vitro diagnosis of calcium oxalate kidney stones in the aqueous solution of Bryophyllum pinnatum. The B. pinnatum powder was mixed in apple cider vinegar and lemon juice separately to make solution 1 and 2 respectively. Apple cider vinegar and lemon juice were used as solvents due to their acidic and body compatible nature. Two surgically removed stones was dipped in solution 1 and 2. After two weeks, kidney stone of weight 2.7 g is completely dissolved in solution 2 while a considerable weight reduction of other kidney stone has been observed in solution 1. Fourier transform infrared (FTIR) spectroscopy results show the presence of two strong absorption peaks at 610 and 912 (cm-1) in both solutions after dissolution of urinary stones are related to calcium oxalate dehydrate (COD). Raman spectra further confirm the dissolution of COD in solution having Raman shifts at 504 and 910 (cm-1). Cluster formation and aggregation of particles has been observed in scanning electron microscopy images. This in vitro study proves that a mixture of Bryophyllum pinnatum powder and lemon juice is a best remedy to remove kidney stones.

The multifaceted effects of fluoranthene and polystyrene on the taxonomic composition and associated functional traits of marine meiofauna, by using single and mixture applications.

The current experiment measured the multifaceted effects of polystyrene and fluoranthene, acting alone or in a mixture on marine meiofauna, but with a special focus on nematodes' morphological and functional traits. The results showed changes in the abundances for all tested concentrations of both compounds. The nematode communities exposed to the highest concentrations of fluoranthene (30 ng.g-1 Dry Weight (DW)) and polystyrene (100 mg.kg-1 DW) alone or in a mixture, were significantly less diverse compared to control and were associated with significant changes in the percentage of taxonomic composition and feeding-guilds. The most sensitive taxa to fluoranthene comprised epistratum feeders, whereas the nematodes mostly affected by polystyrene were omnivores-carnivores. A new functional tool, the Index of Sensitivity (IOS), proved to be reliable in depicting the changes that occurred in the taxonomic and functional features of the nematofauna.

Are iron ore microparticles toxic for the European clam Ruditapes decussatus? Response elements from biomarker activities and in silico modeling.

Inevitably, high concentrations of iron, the most widely produced ore globally, can be found in aquatic environments. To assess the toxicity of iron on aquatic organisms, Ruditapes decussatus specimens were subjected to microparticles derived from two types of iron ore (hematite and magnetite) at four different concentrations (0.5, 1, 1.5, and 5 g/L). The findings revealed that both types of iron ore were absorbed by clams in a concentration-dependent manner. Biomarkers analysis demonstrated significant and organ-specific impacts on the health of the clams caused by these microparticles, which was further supported by computational analyses on bioavailability. Within seven days of exposure, changes were observed in the activities of several enzymes, including catalase, acetylcholinesterase, and glutathione S-transferases, as well as in the rate of lipid peroxidation in both the digestive gland and gills. This study provides an environmental perspective on the toxicological effects of iron ore microparticles.

Protective effect of Salvia hispanica (chia seeds) against obesity induced ovarian disorders in rat model.

The main goal of the current report is to assess the protective impacts of chia seeds against obesity-induced ovarian dysfunctions with a trial to elucidate the mechanism of action. Forty rats were divided into 4 groups including lean untreated, lean consuming chia seeds, obese untreated, and rats consumed high-fat diet (HFD) mixed with ground chia seeds for 10 weeks. Anthropometric measures including visceral fat, peri-ovarian fat, ovarian weights, and duration of the estrous cycle were computed. Serum luteinizing (LH), follicular stimulating (FSH), progesterone, estradiol hormones, and tumor necrosis-α (TNF-α) were estimated. Ovarian histopathology and immunohistochemistry (CD31) were performed. Results showed that chia seeds clearly reduced obesity and induced alteration in anthropometric measures with a clear increase in LH and progesterone. Such seeds notably reversed histopathological alteration and reduced TNF-α, and CD31 induced by HFD. Conclusively, chia seeds have a potential protective role against obesity-induced ovarian dysfunction owing to their anti-inflammatory properties.

Dual biocontrol potential of the entomopathogenic fungus Akanthomyces muscarius against Thaumetopoea pityocampa and plant pathogenic fungi.

Akanthomyces spp. species are known for their capacity to biocontrol of certain insects and plant pathogens; however, their ability to biocontrol the pine processionary (Thaumetopoea pityocampa) and certain phytopathogenic fungi belonging to the genera Fusarium and Curvularia have not been studied before. In this study, a strain from Akanthomyces muscarius was isolated from wheat grains and then identified by morphological and molecular tests. The strain was further studied for its capacity to control Thaumetopoea pityocampa larvae through dose-mortality tests, and its ability to control some phytopathogenic fungi strains of the genera Fusarium and Curvularia was studied through direct confrontation tests. Dose-mortality tests at three concentrations of Akanthomyces muscarius against the first instar larvae revealed a mortality of 92.15% after 11 days for the concentration of 2.3 × 106conidia.ml-1, with a median lethal concentration of 7.6 x103 conidia.ml1. Our isolate also showed antifungal activity against these phytopathogenic fungi with inhibition rates ranging from 39.61% to 52.94%. Akanthomyces muscarius proved to be a promising biocontrol agent for plant pests and diseases.

The development of decontamination methods in coastal marine habitats by transplantation of the mussel Mytilus galloprovincialis (Lamarck, 1819): Comparison between in vivo and in situ investigations.

The health status of Mytilus galloprovincialis native from a polluted habitat was surveyed before and after 30 and 60 days of in situ transplantation and in vivo experiments. The results showed a reduction in filtration rate by 24 % and 45 %, respectively, after 60 days of in vivo and in situ experiments compared to the rates at polluted sites. The respiration rate reached a minimum of 0.081 ± 0.05 mg O2.L-1 after 60 days of in situ transplantation. Moreover, the antioxidant activities were changed in a time-dependent manner for both transplantation conditions. The highest superoxide dismutase and catalase activities corresponded to the stressed mussels and declined by 76 % and 54 %, respectively, after 60 days of in situ transplantation. Changes in lipid peroxidation and acetylcholinesterase activity were observed in internal organs following 60 days of transplantation. At this time slot, the lowest content of metals and microplastics was also noticed.

An in-silico investigation based on molecular simulations of novel and potential brain-penetrant GluN2B NMDA receptor antagonists as anti-stroke therapeutic agents.

GluN2B-induced activation of NMDA receptors plays a key function in central nervous system (CNS) disorders, including Parkinson, Alzheimer, and stroke, as it is strongly involved in excitotoxicity, which makes selective NMDA receptor antagonists one of the potential therapeutic agents for the treatment of neurodegenerative diseases, especially stroke. The present study aims to examine a structural family of thirty brain-penetrating GluN2B N-methyl-D-aspartate (NMDA) receptor antagonists, using virtual computer-assisted drug design (CADD) to discover highly candidate drugs for ischemic strokes. Initially, the physicochemical and ADMET pharmacokinetic properties confirmed that C13 and C22 compounds were predicted as non-toxic inhibitors of CYP2D6 and CYP3A4 cytochromes, with human intestinal absorption (HIA) exceeding 90%, and designed to be as efficient central nervous system (CNS) agents due to the highest probability to cross the blood-brain barrier (BBB). Compared to ifenprodil, a co-crystallized ligand complexed with the transport protein encoded as 3QEL.pdb, we have noticed that C13 and C22 chemical compounds were defined by good ADME-Toxicity profiles, meeting Lipinski, Veber, Egan, Ghose, and Muegge rules. The molecular docking results indicated that C22 and C13 ligands react specifically with the amino acid residues of the NMDA receptor subunit GluN1 and GluN2B. These intermolecular interactions produced between the candidate drugs and the targeted protein in the B chain remain stable over 200 nanoseconds of molecular dynamics simulation time. In conclusion, C22 and C13 ligands are highly recommended as anti-stroke therapeutic drugs due to their safety and molecular stability towards NMDA receptors.Communicated by Ramaswamy H. Sarma.

Cucullanus bulbosus (Lane, 1916) Barreto, 1918 (Nematoda, Cucullanidae) from the common ponyfish Leiognathus equulus (Leiognathidae): Morphology and molecular study.

The common ponyfish Leiognathus equulus is a marine fish species with very high commercial value. Little information is available about its parasitic infections. Based on light and scanning electron microscopy, as well as sequencing and analysis of the partial regions of the ITS-1, 18S rRNA, COX1 genes, were employed for the systematic evaluation of a nematode parasite, which it first isolated from L. equulus in Jeddah Province, Red Sea, Saudi Arabia. Results revealed that this nematode parasite closely resembles the previously described Cucullanus bulbosus. Microscopic examination showed that it distinguished from congeners by the unique structure of hemispherical elevation at pseudobuccal capsule level, the ratio of esophagus/body length, spicules size, presence of pre-cloacal sucker, rod-shaped gubernaculum, and the arrangement of caudal papillae in males. Phylogenetic analyses based on ITS-1, 18S rRNA, and COX1 gene regions were constructed to investigate phylogenetic relationships between this parasite species and other related taxa. Results supported that Cucullanus bulbosus resembles a sister of Cucullanus genypteri, Cucullanus pulcherrimus, Cucullanus bourdini, Cucullanus extraneus, and Cucullanus hainanensis by using different genetic markers. This study provides more information about combining morphological and molecular data to identify Cucullanus species with the first natural occurrence in the common ponyfish inhabited in Saudi Arabia.

First record of third-stage Terranova larval type II (Nematoda, Anisakidae) in the common ponyfish Leiognathus equulus Forsskål.

The current study was carried out to investigate the natural occurrence of nematode parasites that infect the common ponyfish Leiognathus equulus from Jeddah, Saudi Arabia. Third-stage nematode larvae were found to be encysted in the peritoneum of the fish studied, with the prevalence of infection being 25%. Light microscopy revealed that this parasite belongs to the Anisakidae family within the genus Terranova by having all the generic characteristic features. Based on the intestinal caecum ratio to the length of the ventriculus being 2:1, the excretory pore with ventral location below the boring tooth, the body ended with a conical tail; the larvae found in the present study were identified as Terranova larval type. To validate its taxonomic position within Anisakidae, this Terranova species' morphological features were combined with the ITS-1 gene's molecular analysis. It demonstrated sequence similarities 94.38-76.57% with taxa of Anisakidae. A preliminary genetic comparison between the present parasite and other ascaridoids placed it as a putative sister taxon to the previously described Terranova species. The first record of the current anisakid larvae in the common ponyfish with a unique genetic sequence for the partial sequence of the ITS-1 gene was observed in this study. Its taxonomic position was confirmed in Anisakidae.