Injecting hope: chitosan hydrogels as bone regeneration innovators.

Spontaneous bone regeneration encounters substantial restrictions in cases of bone defects, demanding external intervention to improve the repair and regeneration procedure. The field of bone tissue engineering (BTE), which embraces a range of disciplines, offers compelling replacements for conventional strategies like autografts, allografts, and xenografts. Among the diverse scaffolding materials utilized in BTE applications, hydrogels have demonstrated great promise as templates for the regeneration of bone owing to their resemblance to the innate extracellular matrix. In spite of the advancement of several biomaterials, chitosan (CS), a natural biopolymer, has garnered significant attention in recent years as a beneficial graft material for producing injectable hydrogels. Injectable hydrogels based on CS formulations provide numerous advantages, including their capacity to absorb and preserve a significant amount of water, their minimally invasive character, the existence of porous structures, and their capability to adapt accurately to irregular defects. Moreover, combining CS with other naturally derived or synthetic polymers and bioactive materials has displayed its effectiveness as a feasible substitute for traditional grafts. We aim to spotlight the composition, production, and physicochemical characteristics and practical utilization of CS-based injectable hydrogels, explicitly focusing on their potential implementations in bone regeneration. We consider this review a fundamental resource and a source of inspiration for future research attempts to pioneer the next era of tissue-engineering scaffold materials.

Biogenic Salvia species synthesized silver nanoparticles with catalytic, sensing, antimicrobial, and antioxidant properties.

Salvia (Lamiaceae family) is used as a brain tonic to improve cognitive function. The species including S. plebeia and S. moorcroftiana are locally used to cure hepatitis, cough, tumours, hemorrhoids, diarrhoea, common cold, flu, and asthma. To the best of authors' knowledge, no previous study has been conducted on synthesis of S. plebeia and S. moorcroftiana silver nanoparticles (SPAgNPs and SMAgNPs). The study was aimed to synthesize AgNPs from the subject species aqueous and ethanol extracts, and assess catalytic potential in degradation of standard and extracted (from yums, candies, and snacks) dyes, nitrophenols, and antibiotics. The study also aimed at AgNPs as probe in sensing metalloids and heavy metal ions including Pb2+, Cu2+, Fe3+, Ni2+, and Zn2+. From the results, it was found that Salvia aqueous extract afforded stable AgNPs in 1:9 and 1:15 (quantity of aqueous extract and silver nitrate solution concentration) whereas ethanol extract yielded AgNPs in 1:10 (quantity of ethanol extract and silver nitrate solution concentration) reacted in sunlight. The size of SPAgNPs and SMAgNPs determined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were 21.7 nm and 19.9 nm, with spherical, cylindrical, and deep hollow morphology. The synthesized AgNPs demonstrated significant potential as catalyst in dyes; Congo red (85 %), methylene blue (75 %), Rhodamine B (<50 %), nitrophenols; ortho-nitrophenol (95-98 %) and para-nitrophenol (95-98 %), dyes extracted from food samples including yums, candies, and snacks. The antibiotics (amoxicillin, doxycycline, levofloxacin) degraded up to 80 %-95 % degradation. Furthermore, the synthesized AgNPs as probe in sensing of Pb2+, Cu2+, and Fe3+ in Kabul river water, due to agglomeration, caused a significant decrease and bathochromic shift of SPR band (430 nm) when analyzed after 30 min. The Pb2+ ions was comparatively more agglomerated and chelated. Thus, the practical applicability of AgNPs in Pb2+ sensing was significant. Based on the results of this research study, the synthesized AgNPs could provide promising efficiency in wastewater treatment containing organic dyes, antibiotics, and heavy metals.

Elucidating the binding mechanism of SARS-CoV-2 NSP6-TBK1 and structure-based designing of phytocompounds inhibitors for instigating the host immune response.

SARS-CoV-2, also referred to as severe acute respiratory syndrome coronavirus 2, is the virus responsible for causing COVID-19, an infectious disease that emerged in Wuhan, China, in December 2019. Among its crucial functions, NSP6 plays a vital role in evading the human immune system by directly interacting with a receptor called TANK-binding kinase (TBK1), leading to the suppression of IFNß production. Consequently, in the present study we used the structural and biophysical approaches to analyze the effect of newly emerged mutations on the binding of NSP6 and TBK1. Among the identified mutations, four (F35G, L37F, L125F, and I162T) were found to significantly destabilize the structure of NSP6. Furthermore, the molecular docking analysis highlighted that the mutant NSP6 displayed its highest binding affinity with TBK1, exhibiting docking scores of -1436.2 for the wildtype and -1723.2, -1788.6, -1510.2, and -1551.7 for the F35G, L37F, L125F, and I162T mutants, respectively. This suggests the potential for an enhanced immune system evasion capability of NSP6. Particularly, the F35G mutation exhibited the strongest binding affinity, supported by a calculated binding free energy of -172.19 kcal/mol. To disrupt the binding between NSP6 and TBK1, we conducted virtual drug screening to develop a novel inhibitor derived from natural products. From this screening, we identified the top 5 hit compounds as the most promising candidates with a docking score of -6.59 kcal/mol, -6.52 kcal/mol, -6.32 kcal/mol, -6.22 kcal/mol, and -6.21 kcal/mol. The molecular dynamic simulation of top 3 hits further verified the dynamic stability of drugs-NSP6 complexes. In conclusion, this study provides valuable insight into the higher infectivity of the SARS-CoV-2 new variants and a strong rationale for the development of novel drugs against NSP6.

Antioxidant, antibacterial, and catalytic performance of biosynthesized silver nanoparticles of Rhus javanica, Rumex hastatus, and Callistemon viminalis.

Rhus javanica (Anacardiaceae) containing abundant glucopyranosidal constituents, is traditionally used to treat gastric and duodenal ulcer, dysentery, and diarrhea. Rumex hastatus (Polygonaceae) widely distributed in Pakistan, has traditional importance in treating wound healing, jaundice, rheumatism, and skin diseases. Callistemon viminalis (Myrtaceae), a rich source of essential oils, saponins, triterpenoids, phloroglucinols, and flavonoids is used in industries, perfumes, nutrition, and cosmetics. Taking the importance of the subject plants, this study is designed to synthesize silver nanoparticles via aqueous extracts of R. javanica (RJAgNPs), R. hastatus (RHAgNPs), and C. viminalis (CVAgNPs). Synthesis, surface, and sizes of silver nanoparticles (AgNPs) were confirmed using spectroscopic techniques including ultraviolet-visible (UV-Vis), Fourier transform-infrared (FT-IR), and scanning electron microscopy (SEM). AgNPs were produced in ratios 1:15, 1:16, and 1:9 and inferred via appearance of a sharp surface plasmon resonance (SPR) absorption peak (400-435 nm), which represented well-defined, stable, and spherical AgNPs. From SEM analysis, the sizes of RJAgNPs, RHAgNPs, and CVAgNPs were found to be 67 nm, 61 nm, and 55 nm, respectively. The synthesized AgNPs exhibited potential free radical scavenging, antibacterial, and catalytic properties in degradation of dyes including Congo red, methylene blue, methyl orange, rhodamine B, ortho and para-nitrophenols, and several food colours. Hence, the subject AgNPs in the current study might display promising role in drug development and remediation of environmental/industrial effluents.

The Sequence and Three-Dimensional Structure Characterization of Snake Venom Phospholipases B.

Snake venom phospholipases B (SVPLBs) are the least studied enzymes. They constitute about 1% of Bothrops crude venoms, however, in other snake venoms, it is present in less than 1%. These enzymes are considered the most potent hemolytic agent in the venom. Currently, no structural information is available about these enzymes from snake venom. To better understand its three-dimensional structure and mechanisms of envenomation, the current work describes the first model-based structure report of this enzyme from Bothrops moojeni venom named as B. moojeni phospholipase B (PLB_Bm). The structure model of PLB_Bm was generated using model building software like I-TESSER, MODELLER 9v19, and Swiss-Model. The build PLB_Bm model was validated using validation tools (PROCHECK, ERRAT, and Verif3D). The analysis of the PLB_Bm modeled structure indicates that it contains 491 amino acid residues that form a well-defined four-layer αßßα sandwich core and has a typical fold of the N-terminal nucleophile aminohydrolase (Ntn-hydrolase). The overall structure of PLB_Bm contains 18 ß-strands and 17 α-helices with many connecting loops. The structure divides into two chains (A and B) after maturation. The A chain is smaller and contains 207 amino acid residues, whereas the B chain is larger and contains 266 amino acid residues. The sequence and structural comparison among homologous snake venom, bacterial, and mammals PLBs indicate that differences in the length and sequence composition may confer variable substrate specificity to these enzymes. Moreover, the surface charge distribution, average volume, and depth of the active site cavity also vary in these enzymes. The present work will provide more information about the structure-function relationship and mechanism of action of these enzymes in snakebite envenomation.

Determining the Structures of the Snake and Spider Toxins by X-Rays.

Snake and spider venom is a complex mixture that contains proteins, peptides, and small organic and inorganic compounds. In contrast to spider venom, snake venom proteins are well known both functionally and structurally. This work describes methods for purification and crystallization of snake and spider venom toxins and their three-dimensional structure determination by X-ray crystallography.

Thrombin-like enzymes from snake venom: Structural characterization and mechanism of action.

Snake venom thrombin-like enzymes (SVTLEs) constitute the major portion (10-24%) of snake venom and these are the second most abundant enzymes present in the crude venom. During envenomation, these enzymes had shown prominently the various pathological effects, such as disturbance in hemostatic system, fibrinogenolysis, fibrinolysis, platelet aggregation, thrombosis, neurologic disorders, activation of coagulation factors, coagulant, procoagulant etc. These enzymes also been used as a therapeutic agent for the treatment of various diseases such as congestive heart failure, ischemic stroke, thrombotic disorders etc. Although the crystal structures of five SVTLEs are available in the Protein Data Bank (PDB), there is no single article present in the literature that has described all of them. The current work describes the structural aspects, structure-based mechanism of action, processing and inhibition of these enzymes. The sequence analysis indicates that these enzymes show a high sequence identity (57-85%) with each other and low sequence identity with trypsin (36-43%), human alpha-thrombin (29-36%) and other snake venom serine proteinases (57-85%). Three-dimensional structural analysis indicates that the loops surrounding the active site are variable both in amino acids composition and length that may convey variable substrate specificity to these enzymes. The surface charge distributions also vary in these enzymes. Docking analysis with suramin shows that this inhibitor preferably binds to the C-terminal region of these enzymes and causes the destabilization of their three-dimensional structure.

Spider's venom phospholipases D: A structural review.

Spider venoms are complex mixtures of proteins, peptides and small organic and inorganic molecules. Among the proteins, phospholipases D (PLDs) present the major portion, and till now they are the most studied enzymes in spider venom. These PLDs have been divided into two classes, I and II, based on their primary and tertiary structure. Currently, crystal structures of both classes of these enzymes are available in the Protein Data Bank (PDB). Their three-dimensional structure is composed of eight α-helices and eight ß-strands forming the ubiquitous fold called triosephosphate isomerase (TIM) barrel. These enzymes use general acid-base catalysis to hydrolyzes their substrate. In this review, we have described the structural features, structure-based mechanisms of catalysis, maturation, and inhibition of these enzymes using the synthetic inhibitor.

Serine proteinases from Bothrops snake venom activates PI3K/Akt mediated angiogenesis.

The discovery of rapid acting and powerful angiogenic proteins are of significant interest in the treatment of various human disorders associated with insufficient angiogenesis such as ischemia, menorrhagia and delayed wound healing. Snake venoms consist of a mixture of bioactive proteins and polypeptides and are rich sources of pharmacologically important molecules. Serine proteinases are one of the abundant proteins present in Bothrops snake venoms and possess multiple biological functions including the regulation of the blood coagulation cascade. In this study, serine proteinases from Bothrops atrox (B. atrox) and Bothrops brazili (B. brazili) that modulate angiogenesis were purified and characterized. Molecular size exclusion chromatography, affinity chromatography followed by ion exchange chromatography of the serine proteinases indicated molecular masses of around 32 kDa. Serine proteinases from both the species exhibited diverse catalytic activities such as the ability to induce amidolytic, fibrinogenolytic, gelatinolytic activities and also coagulated plasma with a minimal coagulation concentration of 2.4 µg/mL. Serine proteinases facilitated the sprouting of human umbilical vein endothelial cells (HUVEC) in three-dimensional culture systems and induced tubule formation in monolayer culture systems. Serine proteinase stimulated Aktser473 and eNOSser1177 phosphorylation in endothelial cells and addition of PI3K inhibitor LY294002 abrogated the effects of serine proteinases on sprout formation of endothelial cells in 3D collagen gels, suggesting that serine proteinase facilitated angiogenesis was mediated by PI3K/eNOS signaling axis. We also show in agarose plug assays using a mouse model, serine proteinases from Bothrops venoms significantly enhanced neovascularization. Our data suggests pro-angiogenic activity by the serine proteinases from B. atrox and B. brazili venom and further studies are warranted to explore the therapeutic applications.

Crystal structure of mature 2S albumin from Moringa oleifera seeds.

2S albumins, the seed storage proteins, are the primary sources of carbon and nitrogen and are involved in plant defense. The mature form of Moringa oleifera (M. oleifera), a chitin binding protein isoform 3-1 (mMo-CBP3-1) a thermostable antifungal, antibacterial, flocculating 2S albumin is widely used for the treatment of water and is potentially interesting for the development of both antifungal drugs and transgenic crops. The crystal structure of mMo-CBP3-1 determined at 1.7 Å resolution demonstrated that it is comprised of two proteolytically processed α-helical chains, stabilized by four disulfide bridges that is stable, resistant to pH changes and has a melting temperature (TM) of approximately 98 °C. The surface arginines and the polyglutamine motif are the key structural factors for the observed flocculating, antibacterial and antifungal activities. This represents the first crystal structure of a 2S albumin and the model of the pro-protein indicates the structural changes that occur upon formation of mMo-CBP3-1 and determines the structural motif and charge distribution patterns for the diverse observed activities.

Crystallization and preliminary X-ray diffraction studies of an L-amino-acid oxidase from Lachesis muta venom.

Snake-venom proteins form multi-component defence systems by the recruitment and rapid evolution of nonvenomous proteins and hence serve as model systems to understand the structural modifications that result in toxicity. L-Amino-acid oxidases (LAAOs) are encountered in a number of snake venoms and have been implicated in the inhibition of platelet aggregation, cytotoxicity, haemolysis, apoptosis and haemorrhage. An L-amino-acid oxidase from Lachesis muta venom has been purified and crystallized. The crystals belonged to space group P21, with unit-cell parameters a=66.05, b=79.41, c=100.52 Å, ß=96.55°. The asymmetric unit contained two molecules and the structure has been determined and partially refined at 3.0 Šresolution.

Crystallization and preliminary X-ray diffraction analysis of a novel sphingomyelinase D from Loxosceles gaucho venom.

Brown spider envenomation results in dermonecrosis, intravascular coagulation, haemolysis and renal failure, mainly owing to the action of sphingomyelinases D (SMases D), which catalyze the hydrolysis of sphingomyelin to produce ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidylcholine to produce lysophosphatidic acid. Here, the heterologous expression, purification, crystallization and preliminary X-ray diffraction analysis of LgRec1, a novel SMase D from Loxosceles gaucho venom, are reported. The crystals belonged to space group P21212, with unit-cell parameters a = 52.98, b = 62.27, c = 84.84 Šand diffracted to a maximum resolution of 2.6 Å.

Rapid purification of serine proteinases from Bothrops alternatus and Bothrops moojeni venoms.

Envenomation by Bothrops species results, among other symptoms, in hemostatic disturbances. These changes can be ascribed to the presence of enzymes, primarily serine proteinases some of which are structurally similar to thrombin and specifically cleave fibrinogen releasing fibrinopeptides. A rapid, three-step, chromatographic procedure was developed to routinely purify serine proteinases from the venoms of Bothrops alternatus and Bothrops moojeni. The serine proteinase from B. alternatus displays an apparent molecular mass of ~32 kDa whereas the two closely related serine proteinases from B. moojeni display apparent molecular masses of ~32 kDa and ~35 kDa in SDS-PAGE gels. The partial sequences indicated that these enzymes share high identity with serine proteinases from the venoms of other Bothrops species. These proteins coagulate plasma and possess fibrinogenolytic activity but lack fibrinolytic activity.

Purification, crystallization and preliminary X-ray diffraction analysis of a class P-III metalloproteinase (BmMP-III) from the venom of Bothrops moojeni.

Snake-venom metalloproteinases (SVMPs) comprise a family of haemostatically active toxins which can cause haemorrhage, coagulopathy, inhibition of platelet aggregation and inflammatory response. These effects are attributed to the proteolytic action of SVMPs on extracellular matrix components, plasma proteins and cell-surface proteins. SVMPs are classified into four classes (P-I to P-IV) based on their domain structures. In order to understand the multiple roles played by the domains of P-III SVMPs, a P-III SVMP (BmMP-III) from the venom of Bothrops moojeni was purified, characterized and crystallized. The crystals belonged to space group I4(1)22, with unit-cell parameters a = b = 108.16, c = 196.09 Å. Initially, flash-cooled crystals diffracted poorly to a resolution of about 10 Å. However, a significant improvement in the diffraction resolution was observed upon annealing and a complete data set was collected to 3.3 Šresolution. The asymmetric unit contained one molecule and the structure was determined and partially refined to an R factor of 34%. Structural comparisons indicated that the cysteine-rich domain can adopt different conformations in relation to the catalytic domain, which may modulate the enzyme activity.