Sponge Research Articles

Decoupling of strain- and intrastrain-level interactions of microbiomes in a sponge holobiont

Holobionts are highly organized assemblages of eukaryotic hosts, cellular microbial symbionts, and viruses, whose interactions and evolution involve complex biological processes. It is largely unknown which specific determinants drive similarity or individuality in genetic diversity between holobionts. Here, we combine short- and long-read sequencing and DNA-proximity-linkage technologies to investigate intraspecific diversity of the microbiomes, including host-resolved viruses, in individuals of a model marine sponge. We find strong impacts of the sponge host and the cellular hosts of viruses on strain-level organization of the holobiont, whereas substantial overlap in nucleotide diversity between holobionts suggests frequent exchanges of microbial cells and viruses at intrastrain level in the local sponge population. Immune-evasive arms races likely restricted virus-host co-evolution at the intrastrain level, generated holobiont-specific genome variations, and linked virus-host genetics through recombination. Our work shows that a decoupling of strain- and intrastrain-level interactions is a key factor in the genetic diversification of holobionts.

Exploring the cyanobacterial diversity in Portugal: Description of four new genera from LEGE-CC using thepolyphasic approach.

Culture collections such as the Blue Biotechnology and Ecotoxicology Culture Collection (LEGE-CC) hold approximately 1200 cyanobacterial strains and are critical community resources. However, many isolates in this and other collections have not been described with a polyphasic approach, and this limits further study. Here, we employed a polyphasic methodology that integrates 16S rRNA gene phylogenetic analyses, similarity (p-distance), 16S-23S ITS rRNA region secondary structures, morphological analyses, and habitat assessments to describe four novel cyanobacterial genera from the LEGE-CC, Portugal. Pseudolimnococcus planktonicus gen. et sp. nov. (Chroococcales) is phylogenetically and morphologically related to Limnococcus. The 16S rRNA gene similarity between the types of both genera is only 93.1%. Morphologically, Pseudolimnococcus cells do not reach the original spherical shape before the next division or have aerotopes and firm mucilage, while Limnococcus cells reach the original shape, lack aerotopes, and have diffluent mucilage. Eucapsopsis lusitanus gen. et sp. nov. (Chroococcales) is morphologically similar to Eucapsis but differs from it by having aerotopes and diffluent envelope. Eucapsis lacks aerotopes and has firm mucilaginous envelopes, rarely diffluent. Both genera are phylogenetically very distant from each other and have only 90.68% 16S rRNA gene similarity. Pseudoacaryochloris arrabidensis gen. et sp. nov. (Acaryochloridales) differs from Acaryochloris by the lack of mucilaginous envelope, which is present in Acaryochloris. Both genera are phylogenetically distant and have only 94.1% 16S rRNA gene similarity. Moreover, Acaryochloris is marine (sponge symbiont), while Pseudoacaryochloris is from freshwater. Vasconcelosia minhoensis gen. et sp. nov. (Nodosilineales) is phylogenetically related to Cymatolege but has only 94.3% similarity with this genus. Morphologically both genera are distinct. Vasconcelosia has a Romeria-like structure, while Cymatolege has a Phormidium-like structure. In all cases the 16S-23S ITS rRNA region secondary structures are in agreement with the other analyses. These novel genera expand the diversity of cyanobacteria in culture collections.

Structural insights and antimicrobial synergy of a proto-galectin from the marine sponge Aiolochroia crassa

In this study, we isolated a novel lectin from the marine sponge Aiolochroia crassa, named AcrL. The lectin showed a preference for glycans containing sialic acid terminal residues, as indicated by the strongest inhibition with fetuin and bovine submaxillary mucin. Primary structure determination by mass spectrometry revealed that AcrL is a galectin with conserved amino acid residues typically involved in carbohydrate binding. Structural modeling indicated that AcrL adopts a typical galectin β-sandwich motif, featuring two anti-parallel β-sheets with five strands each. Docking calculations revealed a carbohydrate-binding site composed of a main site, capable of hosting galactopyranosides, and an extended site, facilitating the binding of complex carbohydrates. AcrL inhibited significant biofilm formation against Staphylococcus aureus, S. epidermidis, and Escherichia coli with concentrations ranging from 500 to 15.6 μg.mL−1 for S. aureus, 7.8 μg.mL−1 for S. epidermidis, and 500 μg.mL−1 for E. coli. Furthermore, when combined with different antibiotics, AcrL potentiated their effect against pathogenic bacteria. The antimicrobial mechanism of AcrL was investigated using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The analysis indicates that AcrL induces damage to the bacterial membrane. These findings underscore the discovery of a novel galectin in a basal organism and the comprehensive biochemical characterization conducted in this research, highlighting the potential of AcrL as a novel antibacterial agent and emphasizing its importance in combating bacterial infections.

Biochemical and structural insights of a recombinant AA16 LPMO from the marine and sponge-symbiont Peniophora sp

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidize polysaccharides, leading to their cleavage. LPMOs are classified into eight CAZy families (AA9-11, AA13-17), with the functionality of AA16 being poorly characterized. This study presents biochemical and structural data for an AA16 LPMO (PnAA16) from the marine sponge symbiont Peniophora sp. Phylogenetic analysis revealed that PnAA16 clusters separately from previously characterized AA16s. However, the structural modelling of PnAA16 showed the characteristic immunoglobulin-like fold of LPMOs, with a conserved his-brace motif coordinating a copper ion. The copper-bound PnAA16 showed greater thermal stability than its apo-form, highlighting copper's role in enzyme stability. Functionally, PnAA16 demonstrated oxidase activity, producing 5 μM H₂O₂ after 30 min, but showed 20 times lower peroxidase activity (0.27 U/g) compared to a fungal AA9. Specific activity assays indicated that PnAA16 acts only on cellohexaose, generating native celloligosaccharides (C3 to C5) and oxidized products with regioselective oxidation at C1 and C4 positions. Finally, PnAA16 boosted the activity of a cellulolytic cocktail for cellulose saccharification in the presence of ascorbic acid, hydrogen peroxide, or both. In conclusion, the present work provides insights into the AA16 family, expanding the understanding of their structural and functional relationships and biotechnological potential.

Bio-Informed Porous Mineral-Based Composites.

Certain biominerals, such as sea sponges and echinoderm skeletons, display a fascinating combination of mechanical properties and adaptability due to the well-defined structures spanning various length scales. These materials often possess high density normalized mechanical properties because they contain well-defined pores. The density-normalized mechanical properties of synthetic minerals are often inferior because the pores are stochastically distributed, resulting in an inhomogeneous stress distribution. The mechanical properties of synthetic materials are limited by the degree of structural and compositional control currently available fabrication methods offer. In the first part of this review, examples of structural elements nature uses to impart exceptional density normalized Young's moduli to its porous biominerals are showcased. The second part highlights recent advancements in the fabrication of bio-informed mineral-based composites possessing pores with diameters that span a wide range of length scales. The influence of the processing of mineral-based composites on their structures and mechanical properties is summarized. Thereby, it is aimed at encouraging further research directed to the sustainable, energy-efficient fabrication of synthetic lightweight yet stiff mineral-based composites.

A new sponge from the Marjum Formation of Utah documents the Cambrian origin of the hexactinellid body plan.

Modern poriferans are classified into four classes-Calcarea, Demospongiae, Hexactinellida and Homoscleromorpha-the recognition of which in fossil specimens almost exclusively relies on spicule morphology and arrangement. Early fossil representatives of the phylum Porifera are morphologically diverse, and many of them problematically display characteristics that are incompatible with the classification scheme developed for modern taxa. Critically, hexactine spicules-a diagnostic feature of hexactinellids among modern taxa-are found in various Cambrian and Ordovician taxa that cannot be accommodated within the hexactinellid body plan. Here we describe a new poriferan from the Drumian Marjum Formation of Utah, Polygoniella turrelli gen. et sp. nov., which exhibits a unique combination of complex anatomical features for a Cambrian form, including a syconoid-like organization, a thick body wall, and a multi-layered hexactin-based skeleton. The hexactinellid-like body wall architecture of this new species supports a Cambrian origin of the hexactinellid body plan and provides valuable insights into character evolution in early glass sponges.

Microbulbifer spongiae sp. nov., isolated from marine sponge Diacarnus erythraeanus.

A novel bacterium, designated as MI-GT, was isolated from marine sponge Diacarnus erythraeanus. Cells of strain MI-GT are Gram-stain-negative, aerobic, and rod or coccoid-ovoid in shape. MI-GT is able to grow at 10-40 °C (optimum, 28 °C), with 1.0-8.0% (w/v) NaCl (optimum, 4.0%), and at pH 5.5-9.0 (optimum, pH 8.0). The 16S rRNA gene sequence of strain MI-GT shows 98.35, 97.32 and 97.25% similarity to those of Microbulbifer variabilis Ni-2088T, Microbulbifer maritimus TF-17T and Microbulbifer echini AM134T, respectively. Phylogenetic analysis also exhibits that strain MI-GT falls within a clade comprising members of the genus Microbulbifer (class Gammaproteobacteria). The genome size of strain MI-GT is 4478124 bp with a G+C content of 54.51 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain MI-GT and other type strains are 71.61-76.44% (ANIb), 83.27-84.36% (ANIm) and 13.4-18.7% (dDDH), respectively. These values are significantly lower than the recommended threshold values for bacterial species delineation. Percentage of conserved proteins and average amino acid identity values among the genomes of strain MI-GT and other closely related species are 52.04-59.13% and 67.47-77.21%, respectively. The major cellular fatty acids of MI-GT are composed of summed feature 8 (C18 : 1 ω7c or C18 : 1 ω6c), iso-C11 : 0 3-OH, iso-C15 : 0, C16 : 0, and summed feature 9 (C17 : 1 iso ω9c or C16 : 0 10-methyl). The polar lipids of MI-GT mainly consist of phosphatidylethanolamine, phosphatidylglycerol, aminolipid, and two glycolipids. The major respiratory quinone is Q-8. Based on differential phenotypic and phylogenetic data, strain MI-GT is considered to represent a novel species of genus Microbulbifer, for which the name Microbulbifer spongiae sp. nov. is proposed. The type strain is MI-GT (=MCCC 1K07826T=KCTC 8081T).

Novel Galectins Purified from the Sponge Chondrilla australiensis: Unique Structural Features and Cytotoxic Effects on Colorectal Cancer Cells Mediated by TF-Antigen Binding.

We here report the purification of a novel member of the galectin family, the β-galactoside-binding lectin hRTL, from the marine sponge Chondrilla australiensis. The hRTL lectin is a tetrameric proto-type galectin with a subunit molecular weight of 15.5 kDa, consisting of 141 amino acids and sharing 92% primary sequence identity with the galectin CCL from the congeneric species C. caribensis. Transcriptome analysis allowed for the identification of additional sequences belonging to the same family, bringing the total number of hRTLs to six. Unlike most other galectins, hRTLs display a 23 amino acid-long signal peptide that, according to Erdman degradation, is post-translationally cleaved, leaving an N-terminal end devoid of acetylated modifications, unlike most other galectins. Moreover, two hRTLs display an internal insertion, which determines the presence of an unusual loop region that may have important functional implications. The characterization of the glycan-binding properties of hRTL revealed that it had high affinity towards TF-antigen, sialyl TF, and type-1 N-acetyl lactosamine with a Galβ1-3 structure. When administered to DLD-1 cells, a colorectal carcinoma cell line expressing mucin-associated TF-antigen, hRTL could induce glycan-dependent cytotoxicity.

Stonikacidin A, an Antimicrobial 4-Bromopyrrole Alkaloid Containing L-Idonic Acid Core from the Northwestern Pacific Marine Sponge Lissodendoryx papillosa.

Stonikacidin A (1), the first representative of a new class of 4-bromopyrrole alkaloids containing an aldonic acid core, was isolated from the marine sponge Lissodendoryx papillosa. The compound is named in honor of Prof. Valentin A. Stonik, who is one of the outstanding investigators in the field of marine natural chemistry. The structure of 1 was determined using NMR, MS analysis, and chemical correlations. The L-idonic acid core was established by the comparison of GC, NMR, MS, and optical rotation data of methyl-pentaacetyl-aldonates obtained from the hydrolysis products of 1 and standard hexoses. The L-form of the idonic acid residue in 1 was confirmed by GC analysis of pentaacetate of (S)-2-butyl ester of the hydrolysis product from 1 and compared with corresponding derivatives of L- and D-idonic acids. The biosynthetic pathway for stonikacidin A (1) was proposed. The alkaloid 1 inhibited the growth of Staphylococcus aureus and Escherichia coli test strains, as well as affected the formation of S. aureus and E. coli biofilms. Compound 1 inhibited the activity of sortase A. Molecular docking data showed that stonikacidin A (1) can bind with sortase A due to the interactions between its bromine atoms and some amino acid residues of the enzyme.

Natural pain killers from marine sources: a new frontier in neurosurgical pain management.

The development of new analgesics for the management of neurosurgical pain is a great opportunity in marine environments. Particular secondary metabolites with strong biological activity that have developed for defense are produced by marine bioresources. The analgesic potential of marine-derived peptides, alkaloids, terpenes and polyketides-such as discodermolide from marine sponges and ziconotide from cone snail venom-is highlighted in this letter. These substances work through novel methods that minimize the negative effects associated with conventional analgesics while focusing on particular pain pathways. Despite obstacles in sustainable sourcing and regulatory validation, the development of these marine-derived analgesics requires interdisciplinary collaboration to fulfill their potential in enhancing pain management.

Metagenomic mining of two Egyptian Red Sea sponges associated microbial community

The Red Sea is a promising habitat for the discovery of new bioactive marine natural products. Sponges associated microorganisms represent a wealthy source of compounds with unique chemical structures and diverse biological activities. Metagenomics is an important omics-based culture-independent technique that is used as an effective tool to get genomic and functional information on sponge symbionts. In this study, we used metagenomic analysis of two Egyptian Red Sea sponges Hyrtios erectus and Phorbas topsenti microbiomes to study the biodiversity and the biosynthetic potential of the Red Sea sponges to produce bioactive compounds. Our data revealed high biodiversity of the two sponges’ microbiota with phylum Proteobacteria as the most dominant phylum in the associated microbial community with an average of 31% and 70% respectively. The analysis also revealed high biosynthetic potential of sponge Hyrtios erectus microbiome through detecting diverse types of biosynthetic gene clusters (BGCs) with predicted cytotoxic, antibacterial and inhibitory action. Most of these BGCs were predicted to be novel as they did not show any similarity with any MIBiG database known cluster. This study highlights the importance of the microbiome of the collected Red Sea sponge Hyrtios erectus as a valuable source of new bioactive natural products.

Discovery and Folding Dynamics of a Fused Bicyclic Cysteine Knot Undecapeptide from the Marine Sponge Halichondria bowerbanki.

We describe the discovery and structure of an undecapeptide natural product from a marine sponge, termed halichondamide A, that is morphed into a fused bicyclic ring topology via two disulfide bonds. Molecular dynamics simulations allow us to posit that the installation of one disulfide bond biases the intermediate peptide conformation and predisposes the formation of the second disulfide bond. The natural product was found to be mildly cytotoxic against liver and breast cancer cell lines.

Marine sponge-derived natural products: trends and opportunities for the decade of 2011-2020

The discovery of natural products derived from marine sources has demonstrated a consistent upward trajectory for the decade of 2011-2020, holding significant promise for the development of novel drugs and many other marine bioproducts. In recent years, the spotlight has shifted away from marine sponges (Porifera) towards marine microorganisms as the primary source of discovery. Despite reports of marine sponges spanning 20 different orders and being the subject of 769 papers between 2011 and 2020, they only contributed to 19.29% of all new compounds discovered, in contrast to 51.94% by marine microorganisms and phytoplankton. 563 new compounds were reported from marine sponge-associated microbes, more than doubling the number for the previous decade (2001-2010). It heralds a positive outlook for a sustainable resource strategy as the extraction of bioactive compounds produced by pure cultures of sponge-associated microbes could overcome supply challenges that arise with isolation from host sponges for the same compound. However, the application of novel marine natural products (MNPs) remains challenging due to the limited yield of compounds from large amounts of sponges. This review covers the literature published between 2011 and 2020, focusing on MNPs isolated from marine sponges. A total of 2603 new compounds are documented, detailing their chemical classification, biological activities, source country or geographic locations, and the taxonomic information of the source organisms, including order, family, genus, and species.

Characterization of Antibiotic Actinomycin D Isolated from Streptomyces parvulus Collected from Marine Sponge of Barrang Lompo Island, Makassar, Indonesia.

Microorganisms associated with sea sponges have proven to be good natural product resources that are biologically active and pharmaceutically important. This research aimed to identify actinomycetes related to a sponge from Bar-ranglompo Island Makassar and the antibacterial compounds. Identification of actinomycetes was based on molecular characterization of sequence gen16S rRNA. The antibacterial compound was separated using vacuum liquid chromatog-raphy and preparative Thin Layer Chromatography (TLC). The structure determination was done based on spectroscopy 1H-NMR, 13C-NMR, 2D NMR, and mass spectra. Molecular characterization showed that actinomycetes strain BLP 20 had the closest relationship with Streptomyces parvulus and Uncultured Streptomyces sp. with a similarity value of 83%. The results obtained from the characterization of antibacterial compounds based on spectroscopic data indicate that these compounds lead to Actinomycin D. Characterization and identification of Strain 20 / BLP by molecular phylogenetic analysis of 16S rRNA sequences revealed the closest relationship with Uncultured Streptomy-ces sp and S. parvulus with a similarity value of 83 %, which indicated a new species. The structure of the active compound isolated from actinomycetes strain 20 / BLP leads to Actino-mycin D.

Discovery of cycloheptapeptides phakefusins A−E from the marine sponge Phakellia fusca based on molecular networking

Guided by a probe-based molecular networking strategy, five undescribed cycloheptapeptides, phakefusins A−E (1−5), were isolated from the marine sponge Phakellia fusca. Compounds 1 and 2 contain the nonproteinogenic amino acid residues of dioxindolyalanine (Dioia) and β-3-oxindolylalanine (Oia), respectively. Compound 3 possesses a unique methionine sulfoxide, whereas compound 5 includes a glutamic acid ethyl ester unit. Their structures were elucidated through NMR spectroscopy, HR-MS/MS analysis, and the advanced Marfey's method. By synthesizing the (S, S/R)-Oia standard through tryptophan oxidation, we determined the configuration of this amino acid in compound 2 using the advanced Marfey's method. These cycloheptapeptides were evaluated for their antitumor, antibacterial, and antioxidant activities. Compound 1 showed moderate cytotoxicity against MCF-7 and PC9 cells, with IC50 values of 6.8 and 9.6 μM, respectively, while compounds 2−5 demonstrated potential antioxidant effects by upregulating HO-1, NQO1, and SOD2 levels, as well as inducing Nrf2 activation.

Application of marine sponges for biomonitoring active pharmaceutical ingredients (APIs) in coral reefs. Optimization of an SPME and ESI-LC-MS/MS method

Chemical pollution is a threat to coral reefs. To preserve them, it is crucial to monitor novel contaminants and assess the related risks. The occurrence of active pharmaceutical ingredients (APIs) in coral reefs has been poorly investigated until now. Under this light, we tested the use of the marine sponge Cf. Hyrtios as bio-monitors and conducted a pilot study in the Faafu Atoll (Maldives). Analyses were carried out by in vivo solid-phase microextraction (SPME) and liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS). Twelve APIs were selected for method optimization. Limits of quantitation (LOQs) were in the 0.6 and 2.5 ng/g range, accuracy between 86.5 % and 104.7 %, and precision between 3.0 % and 14.9 %. All the sponges located in the inner reefs resulted contaminated with at least one API. Gabapentin and Carbamazepine displayed the highest detection rates, while Ketoprofen had the highest concentration (up to 15.7 ng/g).

North Atlantic deep-sea benthic biodiversity unveiled through sponge natural sampler DNA

The deep-sea remains the biggest challenge to biodiversity exploration, and anthropogenic disturbances extend well into this realm, calling for urgent management strategies. One of the most diverse, productive, and vulnerable ecosystems in the deep sea are sponge grounds. Currently, environmental DNA (eDNA) metabarcoding is revolutionising the field of biodiversity monitoring, yet complex deep-sea benthic ecosystems remain challenging to assess even with these novel technologies. Here, we evaluate the effectiveness of whole-community metabarcoding to characterise metazoan diversity in sponge grounds across the North Atlantic by leveraging the natural eDNA sampling properties of deep-sea sponges themselves. We sampled 97 sponge tissues from four species across four North-Atlantic biogeographic regions in the deep sea and screened them using the universal COI barcode region. We recovered unprecedented levels of taxonomic diversity per unit effort, especially across the phyla Chordata, Cnidaria, Echinodermata and Porifera, with at least 406 metazoan species found in our study area. These assemblages identify strong spatial patterns in relation to both latitude and depth, and detect emblematic species currently employed as indicators for these vulnerable habitats. The remarkable performance of this approach in different species of sponges, in different biogeographic regions and across the whole animal kingdom, illustrates the vast potential of natural samplers as high-resolution biomonitoring solutions for highly diverse and vulnerable deep-sea ecosystems.

Pyrrololactam alkaloids with IL-6 inhibitory activities from the sponge Phakellia fusca collected in the South China Sea

Sixteen undescribed pyrrololactam alkaloids, including five 2-bromopyrrole-ε-lactam (1a, 1b, 4a, 4b and 5), two 3-bromopyrrole-ε-lactam (9 and 10), eight pyrrole-ε-lactam (2a-3 and 6a-8), and one pyrrole-δ-lactam alkaloids (11), along with three previously reported compounds (12–14) were isolated from the marine sponge Phakellia fusca collected in the South China Sea. The planar structures were determined by NMR and MS analyses, while the absolute configurations were clearly elucidated by comparing the experimental and calculated ECD spectra. Compounds 2a, 2b, 4a-7b, 10, 12 and 13 exhibited anti-inflammatory activity in inhibiting the production of inflammatory cytokines IL-6 in LPS-induced RAW264.7 macrophages.

In Vivo Effects of Biosilica and Spongin-Like Collagen Scaffolds on the Healing Process in Osteoporotic Rats.

Due to bioactive properties, introducing spongin-like collagen (SPG) into the biosilica (BS) extracted from marine sponges would present an enhanced biological material for improving osteoporotic fracture healing by increasing bone formation rate. Our aim was to characterize the morphology of the BS/SPG scaffolds by scanning electron microscopy (SEM), the chemical bonds of the material by Fourier transform infrared spectroscopy (FTIR), and evaluating the orthotopic in vivo response of BS/SPG scaffolds in tibial defects of osteoporotic fractures in rats (histology, histomorphometry, and immunohistochemistry) in two experimental periods (15 and 30days). SEM showed that scaffolds were porous, showing the spicules of BS and fibrous aspect of SPG. FTIR showed characteristic peaks of BS and SPG. For the in vivo studies, after 30days, BS and BS/SPG showed a higher amount of newly formed bone compared to the first experimental period, observed both in the periphery and in the central region of the bone defect. For histomorphometry, BS/SPG presented higher %BV/TV compared to the other experimental groups. After 15days, BS presented higher volumes of collagen type I. After 30days, all groups demonstrated higher volumes of collagen type III compared to volumes at 15days. After 30days, BS/SPG presented higher immunostaining of osteoprotegerin compared to the other experimental groups at the same experimental period. The results showed that BS and BS/SPG scaffolds were able to improve bone healing. Future research should focus on the effects of BS/SPG on longer periods in vivo studies.

A regularized variational mechanics theory for modeling the evolution of brittle crack networks in composite materials with sharp interfaces

In the design of structural materials, there is traditionally a tradeoff between achieving high strength and achieving high toughness. Nature offers creative solutions to this problem in the form of structural biomaterials (SBs), intelligent arrangements of mineral and organic phases which possess greater strength and toughness than the constituents. The micro-architecture of SBs like nacre and sea sponge spicules are characterized by weak organic interfaces between brittle mineral phases. To better understand the toughening mechanisms in SBs requires simulation techniques which can resolve arbitrary interface and bulk fracture patterns.In this work, we present a modified regularization of Variational Fracture Theory (VFT) that allows for simulation of fracture in materials and structures with weak interfaces. The core of our approach is to widen the weak interfaces on a length scale proportional to that of the diffuse damage field, and assign a reduced fracture toughness therein. We show that in 2D the modified regularized functionals Γ-converge to that for sharp cracks. The resulting thin weak interfaces have fracture toughness which depends on the bulk material fracture toughness, the widened interface fracture toughness, and the ratio of the widened interface length scale to the crack regularization length scale. We next apply our modified regularization within a computer implementation of regularized VFT, which we term RVFTI. We assess the performance of RVFTI in 2D by reproducing the effective interface fracture toughness predicted by the Γ-convergence theory and simulating crack trapping at a bi-material interface. We then use RVFTI to study toughening in SB-inspired microarchitectures, namely layered materials and materials with wavy interfaces.