Biocomposite thermoplastic polyurethanes containing evolved bacterial spores as living fillers to facilitate polymer disintegration.

The field of hybrid engineered living materials seeks to pair living organisms with synthetic materials to generate biocomposite materials with augmented function since living systems can provide highly-programmable and complex behavior. Engineered living materials have typically been fabricated using techniques in benign aqueous environments, limiting their application. In this work, biocomposite fabrication is demonstrated in which spores from polymer-degrading bacteria are incorporated into a thermoplastic polyurethane using high-temperature melt extrusion. Bacteria are engineered using adaptive laboratory evolution to improve their heat tolerance to ensure nearly complete cell survivability during manufacturing at 135 °C. Furthermore, the overall tensile properties of spore-filled thermoplastic polyurethanes are substantially improved, resulting in a significant improvement in toughness. The biocomposites facilitate disintegration in compost in the absence of a microbe-rich environment. Finally, embedded spores demonstrate a rationally programmed function, expressing green fluorescent protein. This research provides a scalable method to fabricate advanced biocomposite materials in industrially-compatible processes.

Phenotypically complex living materials containing engineered cyanobacteria.

The field of engineered living materials lies at the intersection of materials science and synthetic biology with the aim of developing materials that can sense and respond to the environment. In this study, we use 3D printing to fabricate a cyanobacterial biocomposite material capable of producing multiple functional outputs in response to an external chemical stimulus and demonstrate the advantages of utilizing additive manufacturing techniques in controlling the shape of the fabricated photosynthetic material. As an initial proof-of-concept, a synthetic riboswitch is used to regulate the expression of a yellow fluorescent protein reporter in Synechococcus elongatus PCC 7942 within a hydrogel matrix. Subsequently, a strain of S. elongatus is engineered to produce an oxidative laccase enzyme; when printed within a hydrogel matrix the responsive biomaterial can decolorize a common textile dye pollutant, indigo carmine, potentially serving as a tool in environmental bioremediation. Finally, cells are engineered for inducible cell death to eliminate their presence once their activity is no longer required, which is an important function for biocontainment and minimizing environmental impact. By integrating genetically engineered stimuli-responsive cyanobacteria in volumetric 3D-printed designs, we demonstrate programmable photosynthetic biocomposite materials capable of producing functional outputs including, but not limited to, bioremediation.

The coordinated outcome of STIM1-Orai1 and superoxide signalling is crucial for headkidney macrophage apoptosis and clearance of Mycobacterium fortuitum.

The mechanisms underlying M. fortuitum-induced pathogenesis remains elusive. Using headkidney macrophages (HKM) from Clarias gariepinus, we report that TLR-2-mediated internalization of M. fortuitum is imperative to the induction of pathogenic effects. Inhibiting TLR-2 signalling alleviated HKM apoptosis, thereby favouring bacterial survival. Additionally, TLR-2-mediated cytosolic calcium (Ca2+)c elevation was instrumental for eliciting ER-stress in infected HKM. ER-stress triggered the activation of membrane-proximal calcium entry channels comprising stromal interaction molecule 1 (STIM1) and calcium-release activated calcium channel 1 (Orai1). RNAi studies suggested STIM1-Orai1 signalling initiate calpain-mediated cleavage of nitric oxide synthase interacting protein, prompting the release of pro-apoptotic nitric oxide. Inhibiting STIM1-Orai1 signalling attenuated superoxide production (O2•-) and vice versa. We conclude, TLR-2-induced ER-stress triggers STIM1/Orai1 expression and that the reciprocal association between STIM1-Orai1 signalling and oxidative stress is critical for sustaining (Ca2+)c level, thereby prolonging ER-stress and maintenance of pro-oxidant rich environment to induce HKM apoptosis and bacterial clearance.

Integrating Systems Biology and an Ex Vivo Human Tumor Model Elucidates PD-1 Blockade Response Dynamics.

Ex vivo human tumor models have emerged as promising, yet complex tools to study cancer immunotherapy response dynamics. Here, we present a strategy that integrates empirical data from an ex vivo human system with computational models to interpret the response dynamics of a clinically prescribed PD-1 inhibitor, nivolumab, in head and neck squamous cell carcinoma (HNSCC) biopsies (N = 50). Using biological assays, we show that drug-induced variance stratifies samples by T helper type 1 (Th1)-related pathways. We then built a systems biology network and mathematical framework of local and global sensitivity analyses to simulate and estimate antitumor phenotypes, which implicate a dynamic role for the induction of Th1-related cytokines and T cell proliferation patterns. Together, we describe a multi-disciplinary strategy to analyze and interpret the response dynamics of PD-1 blockade using heterogeneous ex vivo data and in silico simulations, which could provide researchers a powerful toolset to interrogate immune checkpoint inhibitors.

The ß-turn-supporting motif in the polyglutamine binding peptide QBP1 is essential for inhibiting huntingtin aggregation.

Aggregation of polyglutamine proteins is a hallmark of several neurodegenerative diseases. The 11-residue polyglutamine binding peptide Ac-SNWKWWPGIFD-am, known as QBP1, inhibits polyglutamine aggregation. Besides, a minimal 8-residue stretch in the QBP1 peptide (Ac-WKWWPGIF-am) is reported in the literature to retain this activity. Both peptides harbor a Pro-Gly dipeptide motif, a feature characteristic of potential ß-turn regions. Here, we investigated whether the presence of this ß-turn motif is necessary for the inhibition of huntingtin aggregation, a polyglutamine protein implicated in Huntington's disease. Using single amino acid substitutions to generate analogs that could support, introduce, or eliminate the ß-turn, we show that the turn-supporting motif is essential for QBP1-mediated inhibition of huntingtin aggregation.

Water-Alcohol Bigels from Fatty Acylated Dipeptides.

Peptide-based gels are emerging as an interesting class of biocompatible soft materials. 9-Fluorenylmethoxycarbonyl-protected amino acids and short peptides have gained considerable attention as promising gelators. Peptide amphiphiles, wherein an alkyl chain is appended to a polar peptidic moiety, are another important class of peptide-based gelators. Here, we report the alcohol/water bigels formed by the rather simple fatty acylated dipeptides wherein the peptidic moiety is made up of hydrophobic amino acids, viz., Val, Ile, and Leu. Lauroyl, myristoyl, and palmitoyl were investigated as the N-terminal fatty acyl groups. None of the lauroylated peptides caused gelation of methanol/water and ethanol/water mixtures up to 2 wt % peptide concentration. Eight out of the 27 peptides resulted in distinct bigels. The gels are composed of fibrous aggregates as characterized by electron microscopy. Infrared spectroscopy suggests the ß-sheet conformation of the peptidic region in the gels. Using the Ma-IV ethanol/water bigel as the representative gel, entrapment and steady release of the anticancer drug docetaxel are demonstrated. Such bigels from rather simple amphipathic peptides that are easily synthesized and purified through solvent extraction could be attractive gelator candidates with potential application in drug delivery.

Aromatic-rich C-terminal region of LCI is a potent antimicrobial peptide in itself.

LCI is a 47-residue antimicrobial peptide produced by Bacillus subtilis. The peptide displays potent activity against plant pathogens, Xanthomonas and Pseudomonas. The peptide takes a compact 3-dimensional structure characterized by a four-stranded ß-sheet. The peptide is unusually rich in aromatic residues; 10 of the 47 residues are aromatic and 8 of them lie in the C-terminal region, LCI22-47. Here we report the antimicrobial activity of this C-terminal region against Gram-positive and Gram-negative bacteria. The C-terminal-amidated peptide displays potent activity against E. coli, methicillin and gentamicin-resistant S. aureus, and Xanthomonas oryzae pv. oryzae with lethal concentrations ≤4 µM. Membrane-binding assays indicate preferential binding to the negatively-charged lipids. The peptide permeabilizes the outer-membrane of E. coli indicating membrane-permeabilization as one of the mechanisms of killing. Interestingly, however, no inner-membrane permeabilization was observed, indicating that the membrane-permeabilization may not be the sole mechanism of action.

Insights to the Assembly of a Functionally Active Leptospiral ClpP1P2 Protease Complex along with Its ATPase Chaperone ClpX.

Leptospira interrogans genome is predicted to encode multiple isoforms of caseinolytic proteases (ClpP1 and ClpP2). The ClpP proteins with the aid of its ATPase chaperone are known to be involved in establishing cellular proteostasis and have emerged as a target for developing new antibiotics. We report the molecular characterization of recombinant ClpP1 (rClpP1) and rClpP2 of Leptospira along with its ATPase chaperone rClpX. The two isoforms of rClpPs when coupled together in an equivalent concentration exhibit optimum activity on small fluorogenic peptide substrates, whereas the pure rClpP isoforms are enzymatically inactive. Isothermal titration calorimetry analysis suggests that the two rClpP isoforms bind each other moderately in a 1:1 stoichiometry with a dissociation constant of 2.02 ± 0.1 µM at 37 °C and is thermodynamically favored. Size exclusion chromatography fractionates the majority of pure rClpP1 at ≥308 kDa (14-21-mer) and the pure rClpP2 at 308 kDa (tetradecamer), whereas the functionally active rClpP isoform mixture fractionates as a tetradecamer. The distinct and unprecedented oligomeric form of rClpP1 was also evident through native-gel and dynamic light scattering. Moreover, the rClpP isoform mixture formed after the site-directed mutation of either or both the isoforms at one of the catalytic triad residues (Ser 98/97 to Ala 98/97) resulted in the complete loss of protease activity. The rClpP isoform mixture gets stimulated to degrade the casein substrate in the presence of rClpX and in an energy-dependent manner. On the contrary, pure rClpP1 or the rClpP2 isoform in association with rClpX are incapable of forming operative protease. The reported finding suggests that in Leptospira, the enzymatic activity of the rClpP protease complex in the presence or absence of cochaperone is performed solely by the tetradecamer structure which is hypothesized to be composed of 2-stacked ClpP heptameric rings, wherein each ring is a homo-oligomer of ClpP1 and ClpP2 subunits. Understanding the activities and regulation principle of multi-isoforms of ClpP in pathogenic bacteria may aid in intervening disease outcomes particularly to the co-evolving antibiotic resistance strains.

TLR-2 mediated cytosolic-Ca2+ surge activates ER-stress-superoxide-NO signalosome augmenting TNF-α production leading to apoptosis of Mycobacterium smegmatis-infected fish macrophages.

The implications of TLR-2 mediated alterations in cytosolic-Ca2+((Ca2+)c) levels in M. smegmatis infections is not well known. Using headkidney macrophages (HKM) from Clarias gariepinus, we observed TLR-2 signalling is required in the phagocytosis of M. smegmatis. M. smegmatis induced caspase-dependent HKM apoptosis in MOI, time and growth-phase dependent manner. RNAi and inhibitor studies demonstrated critical role of TLR-2 in eliciting (Ca2+)c-surge and c-Src-PI3K-PLC axis playing an intermediary role in the process. The (Ca2+)c-surge triggered downstream ER-stress and superoxide (O2-) generation. The cross-talk between ER-stress and O2- amplified TNF-α production, which led to HKM apoptosis and bacterial clearance. Release of nitric oxide (NO) was also observed and silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase activity. Pre-treatment with diphenyleneidonium chloride inhibited NO production implicating O2--NO axis imperative in M. smegmatis-induced HKM apoptosis. NO positively impacted CHOP expression and TNF-α production in infected HKM. We conclude that, TLR-2 induced (Ca2+)c-surge and ensuing cross-talk between ER-stress and O2- potentiates HKM pathology by amplifying pro-inflammatory TNF-α production. Moreover, the pro-oxidant environment triggers NO release which prolonged ER-stress and TNF-α production, culminating in HKM apoptosis and bacterial clearance. Together, our study suggests HKM an alternate model to study macrophage-mycobacteria interactions.

Limpid hydrogels from ß-turn motif-connected tandem repeats of Aß16-22.

Self-assembling peptides constitute an important class of functional biomaterials. A number of short amyloidogenic stretches have been identified from amyloid proteins. Such peptides, as such or through subtle modifications, can turn out to be promising candidates for functional biomaterials. End-capped Aß16-22, the well-studied amyloidogenic stretch from ß-amyloid, is reported to be non-hydrogelating up to 20 mM concentration. Here we investigated the hydrogelation propensity of Aß16-22 repeats connected through ß-turn-supporting motifs. The peptide repeats connected through Asn-Gly, Aib-DPro, and DPro-Gly formed transparent hydrogels at concentrations ≥2 mM. The repeats of the aromatic analog Aß16-22(F20Y) also resulted in similar hydrogels. Like other peptide-based gels reported earlier, these gels could trap the anticancer drug doxorubicin and displayed steady release in water. In addition, the gels supported the growth of mammalian cell lines, HEK-293 and RIN-5F. These data show that turn-inducing motifs can have marked effects on the hydrogelating propensity of self-assembling peptides.

Mycobacterium fortuitum-induced ER-Mitochondrial calcium dynamics promotes calpain/caspase-12/caspase-9 mediated apoptosis in fish macrophages.

Mycobacterium fortuitum is a natural fish pathogen. It induces apoptosis in headkidney macrophages (HKM) of catfish, Clarias sp though the mechanism remains largely unknown. We observed M. fortuitum triggers calcium (Ca2+) insult in the sub-cellular compartments which elicits pro-apototic ER-stress factor CHOP. Alleviating ER-stress inhibited CHOP and attenuated HKM apoptosis implicating ER-stress in the pathogenesis of M. fortuitum. ER-stress promoted calpain activation and silencing the protease inhibited caspase-12 activation. The study documents the primal role of calpain/caspase-12 axis on caspase-9 activation in M. fortuitum-pathogenesis. Mobilization of Ca2+ from ER to mitochondria led to increased mitochondrial Ca2+ (Ca2+)m load,, mitochondrial permeability transition (MPT) pore opening, altered mitochondrial membrane potential (ΔΨm) and cytochrome c release eventually activating the caspase-9/-3 cascade. Ultra-structural studies revealed close apposition of ER and mitochondria and pre-treatment with (Ca2+)m-uniporter (MUP) blocker ruthenium red, reduced Ca2+ overload suggesting (Ca2+)m fluxes are MUP-driven and the ER-mitochondria tethering orchestrates the process. This is the first report implicating role of sub-cellular Ca2+ in the pathogenesis of M. fortuitum. We summarize, the dynamics of Ca2+ in sub-cellular compartments incites ER-stress and mitochondrial dysfunction, leading to activation of pro-apoptotic calpain/caspase-12/caspase-9 axis in M. fortuitum-infected HKM.

Calcium and Superoxide-Mediated Pathways Converge to Induce Nitric Oxide-Dependent Apoptosis in Mycobacterium fortuitum-Infected Fish Macrophages.

Mycobacterium fortuitum causes 'mycobacteriosis' in wide range of hosts although the mechanisms remain largely unknown. Here we demonstrate the role of calcium (Ca+2)-signalling cascade on M. fortuitum-induced apoptosis in headkidney macrophages (HKM) of Clarias sp. M. fortuitum could trigger intracellular-Ca+2 influx leading to the activation of calmodulin (CaM), protein kinase C alpha (PKCα) and Calmodulin kinase II gamma (CaMKIIg). Gene silencing and inhibitor studies established the role of CaM in M. fortuitum pathogenesis. We noted that CaMKIIg activation is regulated by CaM as well as PKCα-dependent superoxide anions. This is altogether first report of oxidised CaMKIIg in mycobacterial infections. Our studies with targeted-siRNA and pharmacological inhibitors implicate CaMKIIg to be pro-apoptotic and critical for the activation of extra-cellular signal regulated kinase 1/2 (ERK1/2). Inhibiting the ERK1/2 pathway attenuated nitric oxide synthase 2 (NOS2)-induced nitric oxide (NO) production. Conversely, inhibiting the NOS2-NO axis by specific-siRNA and inhibitors down-regulated ERK1/2 activation suggesting the crosstalk between ERK1/2 and NO is essential for pathogenesis induced by the bacterium. Silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase-8 mediated activation of caspase-3 in the infected HKM. Our findings unveil hitherto unknown mechanism of M. fortuitum pathogenesis. We propose that M. fortuitum triggers intracellular Ca+2 elevations resulting in CaM activation and PKCα-mediated superoxide generation. The cascade converges in common pathway mediated by CaMKIIg resulting in the activation of ERK1/2-NOS2 axis. The crosstalk between ERK1/2 and NO shifts the balance in favour of caspase dependent apoptosis of M. fortuitum-infected HKM.