The Bacteroidetes Aequorivita sp. and Kaistella jeonii Produce Promiscuous Esterases With PET-Hydrolyzing Activity

Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation.

Electrospinning of Liquefied Banana Stem Residue in Conjugation with Hydroxyapatite Nanocrystals: Towards New Scaffolds for Bone Tissue Engineering

Electrospun fibers have high structural similarity to the extracellular matrix (ECM) of natural bone. Some researchers have tried to fabricate cellulose nanofibers using electrospinning method, although the fabricated fibers usually exhibited a non-uniform texture. Moreover, the fabricated mats always suffer from low biological, mechanical and structural properties. Thus, the objective of this study was first to produce a naturally occurring cellulose from banana pseudo-stem through the combination of liquefaction and bleaching processes. The native cellulose was then tried to electrospun in order to determine how a systematic approach based on a Taguchi L9 orthogonal array can be used to fabricate a defect-free fibrous mat. Finally, the electrospun cellulose mats incorporated with hydroxyapatite (HA) nanoparticles were fabricated to generate a new fibrous nanocomposite with enhanced biological and mechanical characteristics. The results revealed that among the electrospinning parameters, cellulose concentration of solution and applied voltage had the greatest effect on the morphology of the fibers. The morphological characterization of the fibrous nanocomposites showed that fibrous cellulose/HA mats had a uniform fiber texture without any significant bead, splashing or particle agglomeration. According to the mechanical tests, the samples containing the higher concentration of HA had a significantly higher elastic modulus and tensile strength. The results obtained from bioactivity analysis indicated an interesting morphological transformation into a flake-like structure which confirmed the high bioactivity of the scaffolds. Accordingly, encapsulation of HA nanoparticles inside the cellulose in the fibrous form can be promising for bone regeneration applications.

Microwave Imaging of Breast Skin Utilizing Elliptical UWB Antenna and Reverse Problems Algorithm

Skin cancer is one of the most widespread and fast growing of all kinds of cancer since it affects the human body easily due to exposure to the Sun’s rays. Microwave imaging has shown better outcomes with higher resolution, faster processing time, mobility, and less cutter and artifact effects. A miniaturized elliptical ultra-wideband (UWB) antenna and its semi-spherical array arrangement were used for signal transmission and reception from the defected locations in the breast skin. Several conditions such as various arrays of three, six, and nine antenna elements, smaller tumor, multi-tumors, and skin on a larger breast sample of 30 cm were considered. To assess the ability of the system, a breast shape container with a diameter of 130 mm and height of 60 mm was 3D printed and then filled with fabricated skin and breast fat to perform the experimental investigation. An improved modified time-reversal algorithm (IMTR) was used to recreate 2D images of tumors with the smallest radius of 1.75 mm in any location within the breast skin. The reconstructed images using both simulated and experimental data verified that the system can be a reliable imaging system for skin cancer diagnosis having a high structural similarity index and resolution.

The structure of Prp2 bound to RNA and ADP-BeF3 − reveals structural features important for RNA unwinding by DEAH-box ATPases

Noncoding intron sequences present in precursor mRNAs need to be removed prior to translation, and they are excised via the spliceosome, a multimegadalton molecular machine composed of numerous protein and RNA components. The DEAH-box ATPase Prp2 plays a crucial role during pre-mRNA splicing as it ensures the catalytic activation of the spliceosome. Despite high structural similarity to other spliceosomal DEAH-box helicases, Prp2 does not seem to function as an RNA helicase, but rather as an RNA-dependent ribonucleoprotein particle-modifying ATPase. Recent crystal structures of the spliceosomal DEAH-box ATPases Prp43 and Prp22, as well as of the related RNA helicase MLE, in complex with RNA have contributed to a better understanding of how RNA binding and processivity might be achieved in this helicase family. In order to shed light onto the divergent manner of function of Prp2, an N-terminally truncated construct of Chaetomium thermophilum Prp2 was crystallized in the presence of ADP-BeF3 − and a poly-U12 RNA. The refined structure revealed a virtually identical conformation of the helicase core compared with the ADP-BeF3 −- and RNA-bound structure of Prp43, and only a minor shift of the C-terminal domains. However, Prp2 and Prp43 differ in the hook-loop and a loop of the helix-bundle domain, which interacts with the hook-loop and evokes a different RNA conformation immediately after the 3′ stack. On replacing these loop residues in Prp43 by the Prp2 sequence, the unwinding activity of Prp43 was abolished. Furthermore, a putative exit tunnel for the γ-phosphate after ATP hydrolysis could be identified in one of the Prp2 structures.

Considerations on activity determinants of fungal polyphenol oxidases based on mutational and structural studies

Polyphenol oxidases (PPOs) are an industrially relevant family of enzymes, being involved in the post-harvest browning of fruits and vegetables, as well as in human melanogenesis. Their involvement lies in their ability to oxidize phenolic or polyphenolic compounds, that subsequently form pigments. PPO family includes tyrosinases and catechol oxidases, which in spite of their high structural similarity, exhibit different catalytic activities. Long-standing research efforts have not yet managed to decipher the structural determinants responsible for this differentiation, as every new theory is disproved by a more recent study. In the present work, we combined biochemical along with structural data, in order to rationalize the function of a previously characterized PPO from Thermothelomyces thermophila (TtPPO). The crystal structure of a TtPPO variant, determined at 1.55 Å resolution, represents the second known structure of an ascomycete PPO. Kinetic data of structure-guided mutants prove the implication of “gate” residue L306, residue HB1+1 (G292) and HB2+1 (Y296) in TtPPO function against various substrates. Our findings demonstrate the role of L306 in the accommodation of bulky substrates and that residue HB1+1 is unlikely to determine monophenolase activity as suggested from previous studies. IMPORTANCE PPOs are enzymes of biotechnological interest. They have been extensively studied both biochemically and structurally, with a special focus on the plant-derived counterparts. Even so, explicit description of the molecular determinants of their substrate specificity is still pending. Especially for ascomycete PPOs, only one crystal structure has been determined so far, thus limiting our knowledge on this tree branch of the family. In the present study, we report the second crystal structure of an ascomycete PPO. Combined with site-directed mutagenesis and biochemical studies, we depict the amino acids in the vicinity of the active site that affect enzyme activity, and perform a detailed analysis on a variety of substrates. Our findings improve current understanding of structure-function relations of microbial PPOs, which is a prerequisite for the engineering of biocatalysts of desired properties.

Considerations on activity determinants of fungal polyphenol oxidases based on mutational and structural studies

ABSTRACTPolyphenol oxidases (PPOs) are an industrially relevant family of enzymes, being involved in the post-harvest browning of fruits and vegetables, as well as in human melanogenesis. Their involvement lies in their ability to oxidize phenolic or polyphenolic compounds, that subsequently form pigments. PPO family includes tyrosinases and catechol oxidases, which in spite of their high structural similarity, exhibit different catalytic activities. Long-standing research efforts have not yet managed to decipher the structural determinants responsible for this differentiation, as every new theory is disproved by a more recent study. In the present work, we combined biochemical along with structural data, in order to rationalize the function of a previously characterized PPO from Thermothelomyces thermophila (TtPPO). The crystal structure of a TtPPO variant, determined at 1.55 Å resolution, represents the second known structure of an ascomycete PPO. Kinetic data of structure-guided mutants prove the implication of “gate” residue L306, residue HB1+1 (G292) and HB2+1 (Y296) in TtPPO function against various substrates. Our findings demonstrate the role of L306 in the accommodation of bulky substrates and that residue HB1+1 is unlikely to determine monophenolase activity as suggested from previous studies.IMPORTANCEPPOs are enzymes of biotechnological interest. They have been extensively studied both biochemically and structurally, with a special focus on the plant-derived counterparts. Even so, explicit description of the molecular determinants of their substrate specificity is still pending. Especially for ascomycete PPOs, only one crystal structure has been determined so far, thus limiting our knowledge on this tree branch of the family. In the present study, we report the second crystal structure of an ascomycete PPO. Combined with site-directed mutagenesis and biochemical studies, we depict the amino acids in the vicinity of the active site that affect enzyme activity, and perform a detailed analysis on a variety of substrates. Our findings improve current understanding of structure-function relations of microbial PPOs, which is a prerequisite for the engineering of biocatalysts of desired properties.

An in Silico Scientific Basis for LL-37 as a Therapeutic and Vitamin D as Preventive for Covid-19.

<p></p><p>A multi-pronged approach with help in all forms possible is essential to <i>completely</i> overcome the Covid-19 pandemic. There is a requirement to research as many new and different types of approaches as possible to cater to the entire world population, complementing the vaccines with promising results. The need is also because SARS-CoV-2 has several unknown or variable facets which get revealed from time to time. In this report, <i>in silico</i> scientific findings are presented, which are indicative of the potential for the use of the LL-37 human anti-microbial peptide as a therapeutic against SARS-CoV-2. This indication is based on the high structural similarity of LL-37 to the N-terminal helix, with which the virus interacts, of the receptor for SARS-CoV-2, Angiotensin Converting Enzyme 2. Moreover, there is positive prediction of binding of LL-37 to the receptor-binding domain of SARS-CoV-2. As Vitamin D is known to upregulate the expression of LL-37, the vitamin is a candidate preventive molecule. This report provides the possible basis for why there is an inverse correlation between Vitamin D levels in the body and the severity of or susceptibility to Covid-19. The path for development of LL-37 as a therapeutic could be of lesser duration, as LL-37 is native to the human body. With the scientific link put forth herein, Vitamin D could be used at an effective, medically prescribed, safe dose as a preventive. The information in this report would be valuable in bolstering the worldwide efforts to control the pandemic as early as possible.</p><br><p></p>

An in Silico Scientific Basis for LL-37 as a Therapeutic and Vitamin D as Preventive for Covid-19.

<p></p><p>A multi-pronged approach with help in all forms possible is essential to <i>completely</i> overcome the Covid-19 pandemic. There is a requirement to research as many new and different types of approaches as possible to cater to the entire world population, complementing the vaccines with promising results. The need is also because SARS-CoV-2 has several unknown or variable facets which get revealed from time to time. In this report, <i>in silico</i> scientific findings are presented, which are indicative of the potential for the use of the LL-37 human anti-microbial peptide as a therapeutic against SARS-CoV-2. This indication is based on the high structural similarity of LL-37 to the N-terminal helix, with which the virus interacts, of the receptor for SARS-CoV-2, Angiotensin Converting Enzyme 2. Moreover, there is positive prediction of binding of LL-37 to the receptor-binding domain of SARS-CoV-2. As Vitamin D is known to upregulate the expression of LL-37, the vitamin is a candidate preventive molecule. This report provides the possible basis for why there is an inverse correlation between Vitamin D levels in the body and the severity of or susceptibility to Covid-19. The path for development of LL-37 as a therapeutic could be of lesser duration, as LL-37 is native to the human body. With the scientific link put forth herein, Vitamin D could be used at an effective, medically prescribed, safe dose as a preventive. The information in this report would be valuable in bolstering the worldwide efforts to control the pandemic as early as possible.</p><br><p></p>

The Crystal Structures of Bacillithiol Disulfide Reductase YpdA Reveal Structural and Functional Insight into a New Type of FAD-Containing NADPH-Dependent Oxidoreductases

Low G+C Gram-positive Firmicutes, such as the clinically important pathogens Staphylococcus aureus and Bacillus cereus, use the low-molecular weight (LMW) thiol bacillithiol (BSH) as a defense mechanism to buffer the intracellular redox environment and counteract oxidative stress encountered by human neutrophils during infections. The protein YpdA has recently been shown to function as an essential NADPH-dependent reductase of oxidized bacillithiol disulfide (BSSB) resulting from stress responses and is crucial in maintaining the reduced pool of BSH and cellular redox balance. In this work, we present the first crystallographic structures of YpdAs, namely from S. aureus and B. cereus. Our analyses reveal a uniquely organized biological tetramer; however, the monomeric subunit has high structural similarity to other flavin disulfide reductases. The absence of a redox active cysteine in the vicinity of the FAD isoalloxazine ring implies a new direct disulfide reduction mechanism, which is backed by the presence of a potentially gated channel, serving as a putative binding site for BSSB in proximity to the FAD cofactor. We also report enzymatic activity for both YpdAs, which along with the structures presented in this work provide important structural and functional insight into a new class of FAD-containing NADPH-dependent oxidoreductases, related to the emerging fight against pathogenic bacteria.

A Self-Healing Hierarchical Fiber Hydrogel That Mimics ECM Structure

Although there have been many studies on using hydrogels as substitutes for natural extracellular matrices (ECMs), hydrogels that mimic the structure and properties of ECM remain a contentious topic in current research. Herein, a hierarchical biomimetic fiber hydrogel was prepared using a simple strategy, with a structure highly similar to that of the ECM. Cell viability experiments showed that the hydrogel not only has good biocompatibility but also promotes cell proliferation and growth. It was also observed that cells adhere to the fibers in the hydrogel, mimicking the state of cells in the ECM. Lastly, through a rat skin wound repair experiment, we demonstrated that this hydrogel has a good effect on promoting rat skin healing. Its high structural similarity to the ECM and good biocompatibility make this hydrogel a good candidate for prospective applications in the field of tissue engineering.