Effect of Phosphate and Ferritin Subunit Composition on the Kinetics, Structure, and Reactivity of the Iron Core in Human Homo- and Heteropolymer Ferritins.

Ferritins are highly conserved supramolecular protein nanostructures that play a key role in iron homeostasis. Thousands of iron atoms can be stored inside their hollow cavity as a hydrated ferric oxyhydroxide mineral. Although phosphate associates with the ferritin iron nanoparticles, the effect of physiological concentrations on the kinetics, structure, and reactivity of ferritin iron cores has not yet been explored. Here, the iron loading and mobilization kinetics were studied in the presence of 1-10 mM phosphate using homopolymer and heteropolymer ferritins having different H to L subunit ratios. In the absence of ferritin, phosphate enhances the rate of ferrous ion oxidation and forms large and soluble polymeric Fe(III)-phosphate species. In the presence of phosphate, Fe(II) oxidation and core formation in ferritin is significantly accelerated with oxidation rates several-fold higher than with phosphate alone. High-angle annular dark-field scanning transmission electron microscopy measurements revealed a strong phosphate effect on both the size and morphology of the iron mineral in H-rich (but not L-rich) ferritins. While iron nanoparticles in L-rich ferritins have spherical shape in the absence and presence of phosphate, iron nanoparticles in H-rich ferritins change from irregular shapes in the absence of phosphate to spherical particles in the presence of phosphate with larger size distribution and smaller particle size. In the presence of phosphate, the kinetics of iron-reductive mobilization from ferritin releases twice as much iron than in its absence. Altogether, our results demonstrate an important role for phosphate, and the ferritin H and L subunit composition toward the kinetics of iron oxidation and removal from ferritin, as well as the structure and reactivity of the iron mineral, and may have an important implication on ferritin iron management in vivo.

Iron Mobilization from Ferritin in Yeast Cell Lysate and Physiological Implications.

Most in vitro iron mobilization studies from ferritin have been performed in aqueous buffered solutions using a variety of reducing substances. The kinetics of iron mobilization from ferritin in a medium that resembles the complex milieu of cells could dramatically differ from those in aqueous solutions, and to our knowledge, no such studies have been performed. Here, we have studied the kinetics of iron release from ferritin in fresh yeast cell lysates and examined the effect of cellular metabolites on this process. Our results show that iron release from ferritin in buffer is extremely slow compared to cell lysate under identical experimental conditions, suggesting that certain cellular metabolites present in yeast cell lysate facilitate the reductive release of ferric iron from the ferritin core. Using filtration membranes with different molecular weight cut-offs (3, 10, 30, 50, and 100 kDa), we demonstrate that a cellular component >50 kDa is implicated in the reductive release of iron. When the cell lysate was washed three times with buffer, or when NADPH was omitted from the solution, a dramatic decrease in iron mobilization rates was observed. The addition of physiological concentrations of free flavins, such as FMN, FAD, and riboflavin showed about a two-fold increase in the amount of released iron. Notably, all iron release kinetics occurred while the solution oxygen level was still high. Altogether, our results indicate that in addition to ferritin proteolysis, there exists an auxiliary iron reductive mechanism that involves long-range electron transfer reactions facilitated by the ferritin shell. The physiological implications of such iron reductive mechanisms are discussed.

Thermodynamic and Kinetic Studies of the Interaction of Nuclear Receptor Coactivator-4 (NCOA4) with Human Ferritin.

Ferritinophagy is a ferritin autophagic degradation process mediated by the selective nuclear receptor coactivator-4 (NCOA4). NCOA4 binds to ferritin and delivers it to nascent autophagosomes, which then merge with the lysosomes for ferritin degradation and iron release. Earlier studies have demonstrated a specific association of NCOA4 with ferritin H-subunits, but not L-subunits. However, neither the thermodynamics of this interaction nor the effect of NCOA4 on iron oxidation, iron mineral core formation, or iron mobilization in ferritin has been explored. Using isothermal titration calorimetry, light absorption spectroscopy, and a soluble fragment (residues 383-522) of human NCOA4 expressed in Escherichia coli, we show that the NCOA4 fragment specifically binds H-rich ferritins with a binding stoichiometry of ∼8 NCOA4 molecules per ferritin shell, and Kd values of ∼0.4 and ∼2 µM for homopolymer H-chain ferritin and heteropolymer H-rich ferritin, respectively. The binding reaction was both enthalpically and entropically favored. Whereas the iron oxidation kinetics were not affected by the presence of NCOA4, iron mobilization from ferritin by two different reducing agents (FMN/NADH and sodium dithionite) showed a strong inhibitory effect that was dependent on the concentration of NCOA4 present in solution. Our results suggest that the binding of NCOA4 to ferritin may interfere in the electron transfer pathway through the ferritin shell and may have important biological implications on cellular iron homeostasis.

Ebstein's anomaly of tricuspid valve with aortic stenosis and coarctation of aorta: Successful single-stage repair of a rare adult congenital heart disease.

Ebstein's anomaly of the tricuspid valve is infrequently associated with left heart anomalies. The association of aortic stenosis in Ebstein's anomaly has been reported to be extremely rare and the association of coarctation of aorta is even rarer especially in adults. The combination of all three of these lesions is virtually unknown without any references in literature. We report here an unusual case of Ebstein's anomaly of the tricuspid valve and severe aortic stenosis with coarctation of aorta in an adult who presented to us with exertional dyspnoea in the third decade and underwent a successful single-stage intracardiac repair.

Expression and characterization of the ferritin binding domain of Nuclear Receptor Coactivator-4 (NCOA4).

Ferritinophagy is the process of autophagic degradation of ferritin that participates in the regulation of cellular iron homeostasis. This process was shown to be mediated by the selective cargo-receptor Nuclear Receptor Coactivator-4 (NCOA4) that binds ferritin and targets it to emerging autophagosome. To characterize some of the biochemical properties of the interaction between the two proteins we cloned and expressed in E. coli the ferritin-binding domain of human NCOA4, fragment 383-522. It was purified and subjected to biochemical analysis. The NCOA4(383-522) fragment was expressed in soluble and dimeric form, and CD spectra indicated low level of secondary structure. The Ferritin binding activity of the fragment was investigated by developing an electrophoretic mobility shift and an ELISA assays. They showed that the NCOA4 fragment binds the H-ferritin with an affinity in the nM range, but not the R23A H-ferritin mutant and the L-ferritin chain, confirming the high specificity for the H-chain. The H-ferritin could bind up to 24 NCOA4(383-522) fragments forming highly stable and insoluble complexes. The binding was partially inhibited only by Fe(II) among the various divalent metal ions analyzed. The iron-dependent, highly-specific formation of the remarkably stable H-ferritin-NCOA4 complex shown in this work may be important for the characterization of the mechanism of ferritinophagy.

Effects of ferritin iron loading, subunit composition, and the NCOA4-iron sulfur cluster on ferritin-NCOA4 interactions: An isothermal titration calorimetry study.

Ferritin is a 24-mer protein nanocage that stores iron and regulates intracellular iron homeostasis. The nuclear receptor coactivator-4 (NCOA4) binds specifically to ferritin H subunits and facilitates the autophagic trafficking of ferritin to the lysosome for degradation and iron release. Using isothermal titration calorimetry (ITC), we studied the thermodynamics of the interactions between ferritin and the soluble fragment of NCOA4 (residues 383-522), focusing on the effects of the recently identified FeS cluster bound to NCOA4, ferritin subunit composition, and ferritin-iron loading. Our findings show that in the presence of the FeS cluster, the binding is driven by a more favorable enthalpy change and a decrease in entropy change, indicating a key role for the FeS cluster in the structural organization and stability of the complex. The ferritin iron core further enhances this association, increasing binding enthalpy and stabilizing the NCOA4-ferritin complex. The ferritin subunit composition primarily affects binding stoichiometry of the reaction based on the number of H subunits in the ferritin H/L oligomer. Our results demonstrate that both the FeS cluster and the ferritin iron core significantly affect the binding thermodynamics of the NCOA4-ferritin interactions, suggesting regulatory roles for the FeS cluster and ferritin iron content in ferritinophagy.

Deciphering the Potentials of Cardamom in Cancer Prevention and Therapy: From Kitchen to Clinic.

Cardamom (cardamum) is a spice produced from the seeds of several Elettaria and Amomum plants of the Zingiberaceae family. Cardamom has been demonstrated to offer numerous benefits, including its antioxidant, antimicrobial, anti-inflammatory, and other metabolic (anti-diabetic) properties, and its potential to reduce cancer risk. Recently, researchers have extracted and tested multiple phytochemicals from cardamom to assess their potential effectiveness against various types of human malignancy. These studies have indicated that cardamom can help overcome drug resistance to standard chemotherapy and protect against chemotherapy-induced toxicity due to its scavenging properties. Furthermore, chemical compounds in cardamom, including limonene, cymene, pinene, linalool, borneol, cardamonin, indole-3-carbinol, and diindolylmethane, primarily target the programmed cell death lignin-1 gene, which is more prevalent in cancer cells than in healthy cells. This review provides the medicinal properties and pharmacological uses of cardamom, its cellular effects, and potential therapeutic uses in cancer prevention and treatment, as well as its use in reducing drug resistance and improving the overall health of cancer patients. Based on previous preclinical studies, cardamom shows significant potential as an anti-cancer agent, but further exploration for clinical use is warranted due to its diverse mechanisms of action.

Risk of Stroke in Real-World US Individuals with Type 2 Diabetes Receiving Semaglutide or a Dipeptidyl Peptidase 4 Inhibitor.

INTRODUCTION: People with type 2 diabetes (T2D) have a higher risk of stroke and worse outcomes than those without T2D. Pooled data from randomized controlled trials indicate that the glucagon-like peptide 1 receptor agonist semaglutide is associated with stroke risk reduction in people with T2D at high cardiovascular risk. We compared real-world stroke risk in people with T2D or T2D plus atherosclerotic cardiovascular disease (ASCVD) initiating either semaglutide or a dipeptidyl peptidase 4 inhibitor (DPP4i). METHODS: Adults (≥ 18 years old) in a US claims database with a claim indicating initiation of either semaglutide or a DPP4i (index date) during the index period (1 January 2018-30 September 2020), a diagnosis code for T2D on or before the index date and at least 12 months' continuous enrolment in the database pre-index were included and propensity score matched 1:1 on baseline demographic and clinical characteristics. The primary outcome was time to first stroke event during follow-up. Healthcare resource utilization was also compared between groups. RESULTS: The analysis included 17,920 matched pairs with T2D and 4234 matched pairs with T2D and ASCVD. The groups were well matched on baseline characteristics. People initiating semaglutide had a lower risk of stroke over short-term follow-up than those initiating a DPP4i (T2D: hazard ratio 0.63 [95% confidence interval 0.41-0.95], p = 0.029; T2D plus ASCVD: 0.45 [0.24-0.86], p = 0.015). Semaglutide was also associated with a lower rate of inpatient, outpatient and emergency room visits compared with a DPP4i. CONCLUSION: This proof-of-concept analysis indicates that semaglutide has the potential to reduce the risk of stroke in people with T2D when prescribed in clinical practice.

Structural basis for the intracellular regulation of ferritin degradation.

The interaction between nuclear receptor coactivator 4 (NCOA4) and the iron storage protein ferritin is a crucial component of cellular iron homeostasis. The binding of NCOA4 to the FTH1 subunits of ferritin initiates ferritinophagy-a ferritin-specific autophagic pathway leading to the release of the iron stored inside ferritin. The dysregulation of NCOA4 is associated with several diseases, including neurodegenerative disorders and cancer, highlighting the NCOA4-ferritin interface as a prime target for drug development. Here, we present the cryo-EM structure of the NCOA4-FTH1 interface, resolving 16 amino acids of NCOA4 that are crucial for the interaction. The characterization of mutants, designed to modulate the NCOA4-FTH1 interaction, is used to validate the significance of the different features of the binding site. Our results explain the role of the large solvent-exposed hydrophobic patch found on the surface of FTH1 and pave the way for the rational development of ferritinophagy modulators.

Obturators: A proposed classification and its associated techniques.

Statement of Problem: Multiple classifications exist for maxillectomy defects. However, none of the existing classifications describes the defects as favorable or unfavorable from the prosthodontist's standpoint. The most common problem with prosthetic treatment in such patients is in getting adequate retention, stability, and support. The size and location of the defect usually influence the amount of impairment and difficulty in prosthetic rehabilitation. Proposed Classification: A series of cases has been studied, and a newer type of maxillary defect is seen with a better presurgical involvement of the prosthodontist. This type of defects is not present in any of the existing classification; hence, a modification is proposed, and its requisite cast partial framework design is also given. Another treatment-based classification is also proposed for easy treatment planning in these cases. A case series of maxillectomy patients with varying types of defects rehabilitated with obturators with different designs, modes of retention, and fabrication procedure in accordance to the newer classification is described. Discussion: Surgical intervention creates communication among the oral cavity, nasal cavity, and maxillary sinus. The obturator prosthesis is commonly used as an effective means for rehabilitating such cases. There is a plethora of classifications available for maxillectomy defects though none of them takes existing dentition into consideration. While remaining dentition and various other favorable and unfavorable factors decide on the final prognosis of the prosthesis. Hence, a newer classification was planned with keeping in mind newer treatment modalities. Conclusions: Prosthodontic rehabilitation with obturator prosthesis design and manufactured by various principles and techniques restores the missing structures and acts as a barrier between the communication among the various cavities and definitely improving their quality of life. Considering the complexities of maxillary anatomy, the various permutations of the maxillectomy defect, the current trends in surgical management with presurgical prosthodontic planning, and various prosthodontic treatment options available, it is imperative that a more objective modification of the current classification described in this article is warranted for and could be more operator friendly in finalizing and communicating of the treatment plan.

Hyperthermostable recombinant human heteropolymer ferritin derived from a novel plasmid design.

Mammalian ferritins are predominantly heteropolymeric species consisting of 2 structurally similar, but functionally and genetically distinct subunit types, called H (Heavy) and L (Light). The two subunits co-assemble in different H and L ratios to form 24-mer shell-like protein nanocages where thousands of iron atoms can be mineralized inside a hollow cavity. Here, we use differential scanning calorimetry (DSC) to study ferritin stability and understand how various combinations of H and L subunits confer aspects of protein structure-function relationships. Using a recently engineered plasmid design that enables the synthesis of complex ferritin nanostructures with specific H to L subunit ratios, we show that homopolymer L and heteropolymer L-rich ferritins have a remarkable hyperthermostability (Tm = 115 ± 1°C) compared to their H-ferritin homologues (Tm = 93 ± 1°C). Our data reveal a significant linear correlation between protein thermal stability and the number of L subunits present on the ferritin shell. A strong and unexpected iron-induced protein thermal destabilization effect (ΔTm up to 20°C) is observed. To our knowledge, this is the first report of recombinant human homo- and hetero-polymer ferritins that exhibit surprisingly high dissociation temperatures, the highest among all known ferritin species, including many known hyperthermophilic proteins and enzymes. This extreme thermostability of our L and L-rich ferritins may have great potential for biotechnological applications.

Ferritin nanocages as efficient nanocarriers and promising platforms for COVID-19 and other vaccines development.

BACKGROUND: The development of safe and effective vaccines against SARS-CoV-2 and other viruses with high antigenic drift is of crucial importance to public health. Ferritin is a well characterized and ubiquitous iron storage protein that has emerged not only as a useful nanoreactor and nanocarrier, but more recently as an efficient platform for vaccine development. SCOPE OF REVIEW: This review discusses ferritin structure-function properties, self-assembly, and novel bioengineering strategies such as interior cavity and exterior surface modifications for cargo encapsulation and delivery. It also discusses the use of ferritin as a scaffold for biomedical applications, especially for vaccine development against influenza, Epstein-Barr, HIV, hepatitis-C, Lyme disease, and respiratory viruses such as SARS-CoV-2. The use of ferritin for the synthesis of mosaic vaccines to deliver a cocktail of antigens that elicit broad immune protection against different viral variants is also explored. MAJOR CONCLUSIONS: The remarkable stability, biocompatibility, surface functionalization, and self-assembly properties of ferritin nanoparticles make them very attractive platforms for a wide range of biomedical applications, including the development of vaccines. Strong immune responses have been observed in pre-clinical studies against a wide range of pathogens and have led to the exploration of ferritin nanoparticles-based vaccines in multiple phase I clinical trials. GENERAL SIGNIFICANCE: The broad protective antibody response of ferritin nanoparticles-based vaccines demonstrates the usefulness of ferritin as a highly promising and effective approaches for vaccine development.

Ferritin microheterogeneity, subunit composition, functional, and physiological implications.

Ferritin is a ubiquitous intracellular iron storage protein that plays a crucial role in iron homeostasis. Animal tissue ferritins consist of multiple isoforms (or isoferritins) with different proportions of H and L subunits that contribute to their structural and compositional heterogeneity, and thus physiological functions. Using size exclusion and anion exchange chromatography, capillary isoelectric focusing (cIEF), and SDS-capillary gel electrophoresis (SDS-CGE), we reveal for the first time a significant variation in ferritin subunit composition and isoelectric points, in both recombinant and native ferritins extracted from animal organs. Our results indicate that subunits composition is the main determinant of the mean pI of recombinant ferritin heteropolymers, and that ferritin microheterogeneity is a common property of both natural and recombinant proteins and appears to be an intrinsic feature of the cellular machinery during ferritin expression, regulation, post-translational modifications, and post-subunits assembly. The functional significance and physiological implications of ferritin heterogeneity in terms of iron metabolism, response to oxidative stress, tissue-specific functions, and pathological processes are discussed.

An idiosyncratic reaction of unilateral common peroneal nerve palsy associated with below desired therapeutic range of tacrolimus level in a patient postheart transplantation.

Tacrolimus (TAC) is a very effective medication in routine use after solid organ transplantation. The potential, but infrequently reported neurological adverse effect of TAC is peripheral neuropathy (PN). This has rarely been reported in heart transplant patients. To the best of our knowledge, the data regarding mononeuropathy of common peroneal nerve presented with foot drop due to low whole blood trough TAC level are very limited in the early days postheart transplantation. An idiosyncratic reaction might be suspected in the early postoperative period, when the whole blood trough levels of TAC fall below or within the desired therapeutic range associated with any adverse events after ruling out other causes. We report a 21-year-old patient, who underwent heart transplantation after a suitable donor was identified, and presented with a new-onset right side foot drop on the 10th postoperative day. According to the WHO-Uppsala Monitoring Center causality assessment scale, the likely culprit agent is TAC. Rapid and progressive improvement of foot drop occurred after stopping it and changed over to cyclosporine.

Bridging form and function: A bilateral auricular prosthesis.

Unfortunate loss or absence of an ear has a far-reaching impact on an individual psyche. Auricular defects are seen commonly due to trauma, congenital abnormalities, and malignancies which result in disfigurment of the pinna. Rehabilitation of an auricular defect with a custom-made auricular prosthesis improves social acceptance and self-confidence in an individual. Auricular defects present reconstructive challenges, especially if they are bilateral. Surgical reconstruction provides effective results for defects; however, for some patients, surgical intervention is contraindicated. This case report describes an innovative technique to rehabilitate patients with auricular defects with mixed hearing loss and bilateral microtia using a multidisciplinary approach. The patient was provided with a functional auricular prosthesis. The prime purpose of the treatment rendered was to restore the lost auricular structure to the patient's satisfaction comfortably and cost-effectively. An early rehabilitation promotes physical as well as psychological healing of the patient.

Micromagnetic and morphological characterization of heteropolymer human ferritin cores.

The physical properties of in vitro iron-reconstituted and genetically engineered human heteropolymer ferritins were investigated. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), electron energy-loss spectroscopy (EELS), and 57Fe Mössbauer spectroscopy were employed to ascertain (1) the microstructural, electronic, and micromagnetic properties of the nanosized iron cores, and (2) the effect of the H and L ferritin subunit ratios on these properties. Mössbauer spectroscopic signatures indicate that all iron within the core is in the high spin ferric state. Variable temperature Mössbauer spectroscopy for H-rich (H21/L3) and L-rich (H2/L22) ferritins reconstituted at 1000 57Fe/protein indicates superparamagnetic behavior with blocking temperatures of 19 K and 28 K, while HAADF-STEM measurements give average core diameters of (3.7 ± 0.6) nm and (5.9 ± 1.0) nm, respectively. Most significantly, H-rich proteins reveal elongated, dumbbell, and crescent-shaped cores, while L-rich proteins present spherical cores, pointing to a correlation between core shape and protein shell composition. Assuming an attempt time for spin reversal of τ 0 = 10-11 s, the Néel-Brown formula for spin-relaxation time predicts effective magnetic anisotropy energy densities of 6.83 × 104 J m-3 and 2.75 × 104 J m-3 for H-rich and L-rich proteins, respectively, due to differences in surface and shape contributions to magnetic anisotropy in the two heteropolymers. The observed differences in shape, size, and effective magnetic anisotropies of the derived biomineral cores are discussed in terms of the iron nucleation sites within the interior surface of the heteropolymer shells for H-rich and L-rich proteins. Overall, our results imply that site-directed nucleation and core growth within the protein cavity play a determinant role in the resulting core morphology. Our findings have relevance to iron biomineralization processes in nature and the growth of designer's magnetic nanoparticles within recombinant apoferritin nano-templates for nanotechnology.

p53/p21 pathway activation contributes to the ependymal fate decision downstream of GemC1.

Multiciliated ependymal cells and adult neural stem cells are components of the adult neurogenic niche, essential for brain homeostasis. These cells share a common glial cell lineage regulated by the Geminin family members Geminin and GemC1/Mcidas. Ependymal precursors require GemC1/Mcidas expression to massively amplify centrioles and become multiciliated cells. Here, we show that GemC1-dependent differentiation is initiated in actively cycling radial glial cells, in which a DNA damage response, including DNA replication-associated damage and dysfunctional telomeres, is induced, without affecting cell survival. Genotoxic stress is not sufficient by itself to induce ependymal cell differentiation, although the absence of p53 or p21 in progenitors hinders differentiation by maintaining cell division. Activation of the p53-p21 pathway downstream of GemC1 leads to cell-cycle slowdown/arrest, which permits timely onset of ependymal cell differentiation in progenitor cells.

Mandibular guidance prosthesis: Conventional and innovative approach: A case series.

Surgical resection of the lower jaw due to the presence of a benign or malignant tumor is the most frequent cause of mandibular deviation. Location and extent of the tumor decide the surgical modality of mandibulectomy to be performed. The clinician must wait for an adequate span of time for completion of the healing and acceptance of the osseous graft before considering a definitive prosthesis. During the inceptive healing period, prosthodontic intervention is of utmost priority for preventing the mandibular deviation. A corrective appliance termed "guide flange prosthesis (GFP)" is indicated to limit this clinical manifestation. The basic intention of rehabilitation is to train the mandibular muscles and to re-establish an acceptable occlusal relationship so that the patient can adequately control the opening and closing movements. This case series describes early prosthodontic management of three patients who had undergone hemimandibulectomy, with different techniques of fabrication of a GFP. The three techniques described consist of two conventional methods of fabrication while the third technique is a new innovative approach.

Body Packer Syndrome.

"Body packers" are persons who voluntarily or through coercion, swallow or insert drug-filled packets into body cavity, generally in an attempt to smuggle them across secure borders. The drugs most often involved in body packing are heroin and cocaine. Body packers can present in the emergency department as a result of ruptured drug packets, bowel obstruction, or for medicolegal purposes. Suspected cases are diagnosed with X-ray and computed tomography scan of the abdomen. Symptomatic patients require urgent removal of packets. We present a case of foreign national male in whom a drug packet got ruptured and 49 other packets were retrieved with help of laxatives and manual evacuation.

A Novel Approach for the Synthesis of Human Heteropolymer Ferritins of Different H to L Subunit Ratios.

Mammalian ferritins are predominantly heteropolymeric species consisting of 24 structurally similar, but functionally different subunit types, named H and L, that co-assemble in different proportions. Despite their discovery more than 8 decades ago, recombinant human heteropolymer ferritins have never been synthesized, owing to the lack of a good expression system. Here, we describe for the first time a unique approach that uses a novel plasmid design that enables the synthesis of these complex ferritin nanostructures. Our study reveals an original system that can be easily tuned by altering the concentrations of two inducers, allowing the synthesis of a full spectrum of heteropolymer ferritins, from H-rich to L-rich ferritins and any combinations in-between (isoferritins). The H to L subunit composition of purified ferritin heteropolymers was analyzed by SDS-PAGE and capillary gel electrophoresis, and their iron handling properties characterized by light absorption spectroscopy. Our novel approach allows future investigations of the structural and functional differences of isoferritin populations, which remain largely obscure. This is particularly exciting since a change in the ferritin H- to L-subunit ratio could potentially lead to new iron core morphologies for various applications in bio-nanotechnologies.