Detection of Sulfoquinovosidase Activity in Cell Lysates Using Activity-Based Probes.

The sulfolipid sulfoquinovosyl diacylglycerol (SQDG), produced by plants, algae, and cyanobacteria, constitutes a major sulfur reserve in the biosphere. Microbial breakdown of SQDG is critical for the biological utilization of its sulfur. This commences through release of the parent sugar, sulfoquinovose (SQ), catalyzed by sulfoquinovosidases (SQases). These vanguard enzymes are encoded in gene clusters that code for diverse SQ catabolic pathways. To identify, visualize and isolate glycoside hydrolase CAZY-family 31 (GH31) SQases in complex biological environments, we introduce SQ cyclophellitol-aziridine activity-based probes (ABPs). These ABPs label the active site nucleophile of this enzyme family, consistent with specific recognition of the SQ cyclophellitol-aziridine in the active site, as evidenced in the 3D structure of Bacillus megaterium SQase. A fluorescent Cy5-probe enables visualization of SQases in crude cell lysates from bacteria harbouring different SQ breakdown pathways, whilst a biotin-probe enables SQase capture and identification by proteomics. The Cy5-probe facilitates monitoring of active SQase levels during different stages of bacterial growth which show great contrast to more traditional mRNA analysis obtained by RT-qPCR. Given the importance of SQases in global sulfur cycling and in human microbiota, these SQase ABPs provide a new tool with which to study SQase occurrence, activity and stability.

Capture-and-release of a sulfoquinovose-binding protein on sulfoquinovose-modified agarose.

The solute-binding protein (SBP) components of periplasmic binding protein-dependent ATP-binding cassette (ABC)-type transporters often possess exquisite selectivity for their cognate ligands. Maltose binding protein (MBP), the best studied of these SBPs, has been extensively used as a fusion partner to enable the affinity purification of recombinant proteins. However, other SBPs and SBP-ligand based affinity systems remain underexplored. The sulfoquinovose-binding protein SmoF, is a substrate-binding protein component of the ABC transporter cassette in Agrobacterium tumefaciens involved in importing sulfoquinovose (SQ) and its derivatives for SQ catabolism. Here, we show that SmoF binds with high affinity to the octyl glycoside of SQ (octyl-SQ), demonstrating remarkable tolerance to extension of the anomeric substituent. The 3D X-ray structure of the SmoF·octyl-SQ complex reveals accommodation of the octyl chain, which projects to the protein surface, providing impetus for the synthesis of a linker-equipped SQ-amine using a thiol-ene reaction as a key step, and its conjugation to cyanogen bromide modified agarose. We demonstrate the successful capture and release of SmoF from SQ-agarose resin using SQ as competitive eluant, and selectivity for release versus other organosulfonates. We show that SmoF can be captured and purified from a cell lysate, demonstrating the utility of SQ-agarose in capturing SQ binding proteins from complex mixtures. The present work provides a pathway for development of 'capture-and-release' affinity resins for the discovery and study of SBPs.

Approach to fiber-based optical polarimetry for tissue assessment: system calibration and application.

The potential of optical polarimetry is increasingly explored to unravel the tissue structure through several optical instrument configurations. Fiber-based solutions offer portability and are accommodative in an endoscopic examination environment. Here, we address the challenges in realizing a fiber-based optical polarimetry system through an approach involving an all-fiber polarization controller. The methods of device calibration and application in bulk tissues are discussed, and results are presented.

Characterizing colon cancer stages through optical polarimetry-assisted digital staining.

Tissue polarimetry has been gaining importance in extracting useful diagnostic information from the structural attributes of tissues, which vary in response to the tissue health status and hence find great potential in cancer diagnosis. However, the complexities associated with cancer make it challenging to isolate the characteristic changes as the tumor progresses using polarimetry. This study attempts to experimentally characterize the polarimetric behavior in colon cancer associated with various stages of development. Bulk and unstained sections of normal and tumor colon tissue were imaged in the reflection and transmission polarimetry configurations at low and high imaging resolutions using an in-house developed Mueller polarimeter. Through this study, we observed that the information about the major contributors of scattering in colon tissue, manifesting in depolarization and retardance, can be obtained from the bulk tissue and unstained sections. These parameters aid in characterizing the polarimetric changes as the colon tumor progresses. While the unstained colon section best indicated the depolarization contrast between normal and tumor, the contrast through the retardance parameter was more pronounced in the bulk colon tissue. The results suggest that the polarimetric "digitally stained" images obtained by Mueller polarimetry are comparable with the bulk tissue counterparts, making it useful for characterizing colon cancer tissues across different stages of development.

Investigation of The Effect of Metal-based Medicine Arsenicum album on Humoral Immune Response in SRBC-immunized Mice.

BACKGROUND: In complementary and alternative medicinal systems, the Arsenicum album in ultra-high dilution was used in various therapeutic conditions, considering its effects on the body's immune system, including the COVID-19 pandemic. However, scientific evidence regarding its immunomodulatory effects is insufficient. OBJECTIVE: The current study aimed to investigate the immunomodulatory effects of Arsenicum album in an experimental mouse model. MATERIALS AND METHODS: Immunomodulatory activity of potentized dilutions of Arsenicum album i.e., 6C, 30C, 200C in BALB/c mice was evaluated by humoral antibody titer and delayed- type hypersensitivity assays wherein a fixed concentration (0.5 ml of 1× 109 cells/ml) of freshly prepared sheep RBC was administered as a foreign antigen to generate primary and secondary antibodies. RESULTS: Arsenicum album showed significant immunomodulatory activity by increasing primary antibody titer evaluated on day 21 of the treatment in all the dilutions as compared to SRBC and vehicle control group in humoral immune response assay without showing any effect on delayed-type hypersensitivity. CONCLUSION: The results of this preliminary study indicate that oral administration of Arsenicum album has the potential to augment primary humoral response at all dilutions. Hence, the possibility of using the Arsenicum album could be explored to treat immunological conditions, infections, etc., as an alternative therapy alongwith modern medicines.

Correction: Molecular basis of sulfolactate synthesis by sulfolactaldehyde dehydrogenase from Rhizobium leguminosarum.

[This corrects the article DOI: 10.1039/D3SC01594G.].

Acute and Sub-acute Oral Toxicity Assessment of Ferrum phosphoricum in Rats.

BACKGROUND: Ferrum phosphoricum (FP) has been used by traditional medicine practitioners for various ailments since ancient times. However, scientific evidence on the safety of FP is still unavailable. Thus, the current study aimed to investigate the acute and sub-acute oral toxicity of homeopathic FP in experimental rats. METHODS: In an acute toxicity investigation, a single dose of 2,000 µL/kg of FP 6c, 30c and 200c was administered to female Wistar rats, which were monitored for up to 14 days according to the Organization for Economic Cooperation and Development (OECD) guideline 423. For a sub-acute toxicity study, FP 6c, 30c and 200c (200 µL/kg) were administered to male and female rats for 28 days as per the OECD guideline 407. All the animals were observed for mortality, clinical signs and body weight during the study. At the end of the experiment, hematological, biochemical and histopathological assessments were performed. RESULTS: During the acute toxicity study, no mortality was observed in rats administered with FP, and thus the median lethal dose (LD50) was identified as >2,000 µL/kg. In the sub-acute study, no mortality or adverse clinical signs were noticed with FP treatment. Moreover, weekly body weight gain was normal. Hematological and biochemical investigations revealed no abnormalities. Furthermore, histological analysis of FP-treated rats' vital organs revealed no pathological changes. CONCLUSION: Overall, our findings imply that FP 6c, 30c and 200c potencies are safe and do not cause toxicity when given orally to Wistar albino rats for an extended period at a dose of 200 µL/kg.

Widespread Family of NAD+-Dependent Sulfoquinovosidases at the Gateway to Sulfoquinovose Catabolism.

The sulfosugar sulfoquinovose (SQ) is produced by photosynthetic plants, algae, and cyanobacteria on a scale of 10 billion tons per annum. Its degradation, which is essential to allow cycling of its constituent carbon and sulfur, involves specialized glycosidases termed sulfoquinovosidases (SQases), which release SQ from sulfolipid glycoconjugates, so SQ can enter catabolism pathways. However, many SQ catabolic gene clusters lack a gene encoding a classical SQase. Here, we report the discovery of a new family of SQases that use an atypical oxidoreductive mechanism involving NAD+ as a catalytic cofactor. Three-dimensional X-ray structures of complexes with SQ and NAD+ provide insight into the catalytic mechanism, which involves transient oxidation at C3. Bioinformatic survey reveals this new family of NAD+-dependent SQases occurs within sulfoglycolytic and sulfolytic gene clusters that lack classical SQases and is distributed widely including within Roseobacter clade bacteria, suggesting an important contribution to marine sulfur cycling.

Molecular basis of sulfolactate synthesis by sulfolactaldehyde dehydrogenase from Rhizobium leguminosarum.

Sulfolactate (SL) is a short-chain organosulfonate that is an important reservoir of sulfur in the biosphere. SL is produced by oxidation of sulfolactaldehyde (SLA), which in turn derives from sulfoglycolysis of the sulfosugar sulfoquinovose, or through oxidation of 2,3-dihydroxypropanesulfonate. Oxidation of SLA is catalyzed by SLA dehydrogenases belonging to the aldehyde dehydrogenase superfamily. We report that SLA dehydrogenase RlGabD from the sulfoglycolytic bacterium Rhizobium leguminsarum SRDI565 can use both NAD+ and NADP+ as cofactor to oxidize SLA, and indicatively operates through a rapid equilibrium ordered mechanism. We report the cryo-EM structure of RlGabD bound to NADH, revealing a tetrameric quaternary structure and supporting proposal of organosulfonate binding residues in the active site, and a catalytic mechanism. Sequence based homology searches identified SLA dehydrogenase homologs in a range of putative sulfoglycolytic gene clusters in bacteria predominantly from the phyla Actinobacteria, Firmicutes, and Proteobacteria. This work provides a structural and biochemical view of SLA dehydrogenases to complement our knowledge of SLA reductases, and provide detailed insights into a critical step in the organosulfur cycle.

Diversity, Antimicrobial, Antioxidant, and Anticancer Activity of Culturable Fungal Endophyte Communities in Cordia dichotoma.

Endophytic fungi are a significant source of secondary metabolites, which are chemical compounds with biological activities. The present study emphasizes the first-time isolation and identification of such fungi and their pharmacological activities from the medicinal plant Cordia dichotoma, which is native to Jammu, India. The Shannon Wiener diversity index revealed a wide range of fungal endophytes in root (1.992), stem (1.645), and leaf (1.46) tissues. A total of 19 endophytic fungi belonging to nine different genera were isolated from this plant and the majority belonged to the Ascomycota phylum. ITS rRNA gene sequencing was used to identify the fungal strains and they were submitted in NCBI GenBank. The most potent fungal isolate Cladosporium cladosporioides OP870014 had strong antimicrobial, antioxidant, and anticancer activity against MCF-7, HCT-116, and PC-3 cancer cell lines. The LC-MS and GC-MS analyses of the ethyl acetate extract of C. cladosporioides were examined to identify the bioactive metabolites. The major compounds of the crude extract derived from C. cladosporioides OP870014, according to GC-MS, are spiculisporic acid; dibutyl phthalate; phenylethyl alcohol; cyclohexanone, 2,3,3-trimethyl-2-3-methylbutyl; pyrrolo[1,2-a]pyrazine-1,4-dione,hexahydro-3-(phenylmethyl);2,5-piperazinedione,3,6-bis(2-methylpropyl); and heneicosane which possessed antimicrobial, anticancerous, and antioxidant activities. The findings revealed that C. dichotoma has the capacity to host a wide variety of fungal endophytes and that secondary metabolites from the endophytic fungus may be a source of alternative naturally occurring antimicrobial, antioxidant, and cytotoxic compounds.

Defining the molecular architecture, metal dependence, and distribution of metal-dependent class II sulfofructose-1-phosphate aldolases.

Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) is a sulfosugar that is the anionic head group of plant, algal, and cyanobacterial sulfolipids: sulfoquinovosyl diacylglycerols. SQ is produced within photosynthetic tissues, forms a major terrestrial reservoir of biosulfur, and is an important species within the biogeochemical sulfur cycle. A major pathway for SQ breakdown is the sulfoglycolytic Embden-Meyerhof-Parnas pathway, which involves cleavage of the 6-carbon chain of the intermediate sulfofructose-1-phosphate (SFP) into dihydroxyacetone and sulfolactaldehyde, catalyzed by class I or II SFP aldolases. While the molecular basis of catalysis is understood for class I SFP aldolases, comparatively little is known about class II SFP aldolases. Here, we report the molecular architecture and biochemical basis of catalysis of two metal-dependent class II SFP aldolases from Hafnia paralvei and Yersinia aldovae. 3D X-ray structures of complexes with substrate SFP and product dihydroxyacetone phosphate reveal a dimer-of-dimers (tetrameric) assembly, the sulfonate-binding pocket, two metal-binding sites, and flexible loops that are implicated in catalysis. Both enzymes were metal-dependent and exhibited high KM values for SFP, consistent with their role in a unidirectional nutrient acquisition pathway. Bioinformatic analysis identified a range of sulfoglycolytic Embden-Meyerhof-Parnas gene clusters containing class I/II SFP aldolases. The class I and II SFP aldolases have mututally exclusive occurrence within Actinobacteria and Firmicutes phyla, respectively, while both classes of enzyme occur within Proteobacteria. This work emphasizes the importance of SQ as a nutrient for diverse bacterial phyla and the different chemical strategies they use to harvest carbon from this sulfosugar.

Structure of the imine reductase from Ajellomyces dermatitidis in three crystal forms.

The NADPH-dependent imine reductase from Ajellomyces dermatitidis (AdRedAm) catalyzes the reductive amination of certain ketones with amine donors supplied in an equimolar ratio. The structure of AdRedAm has been determined in three forms. The first form, which belongs to space group P3121 and was refined to 2.01 Šresolution, features two molecules (one dimer) in the asymmetric unit in complex with the redox-inactive cofactor NADPH4. The second form, which belongs to space group C21 and was refined to 1.73 Šresolution, has nine molecules (four and a half dimers) in the asymmetric unit, each complexed with NADP+. The third form, which belongs to space group P3121 and was refined to 1.52 Šresolution, has one molecule (one half-dimer) in the asymmetric unit. This structure was again complexed with NADP+ and also with the substrate 2,2-difluoroacetophenone. The different data sets permit the analysis of AdRedAm in different conformational states and also reveal the molecular basis of stereoselectivity in the transformation of fluorinated acetophenone substrates by the enzyme.

Novel Evoked Synaptic Activity Potentials (ESAPs) Elicited by Spinal Cord Stimulation.

Spinal cord stimulation (SCS) evokes fast epidural evoked compound action potential (ECAP) that represent activity of dorsal column axons, but not necessarily a spinal circuit response. Using a multimodal approach, we identified and characterized a delayed and slower potential evoked by SCS that reflects synaptic activity within the spinal cord. Anesthetized female Sprague Dawley rats were implanted with an epidural SCS lead, epidural motor cortex stimulation electrodes, an epidural spinal cord recording lead, an intraspinal penetrating recording electrode array, and intramuscular electromyography (EMG) electrodes in the hindlimb and trunk. We stimulated the motor cortex or the epidural spinal cord and recorded epidural, intraspinal, and EMG responses. SCS pulses produced characteristic propagating ECAPs (composed of P1, N1, and P2 waves with latencies <2 ms) and an additional wave ("S1") starting after the N2. We verified the S1-wave was not a stimulation artifact and was not a reflection of hindlimb/trunk EMG. The S1-wave has a distinct stimulation-intensity dose response and spatial profile compared with ECAPs. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; a selective competitive antagonist of AMPA receptors (AMPARs)] significantly diminished the S1-wave, but not ECAPs. Furthermore, cortical stimulation, which did not evoke ECAPs, produced epidurally detectable and CNQX-sensitive responses at the same spinal sites, confirming epidural recording of an evoked synaptic response. Finally, applying 50-Hz SCS resulted in dampening of S1-wave but not ECAPs. Therefore, we hypothesize that the S1-wave is synaptic in origin, and we term the S1-wave type responses: evoked synaptic activity potentials (ESAPs). The identification and characterization of epidurally recorded ESAPs from the dorsal horn may elucidate SCS mechanisms.

Structure and mechanism of sulfofructose transaldolase, a key enzyme in sulfoquinovose metabolism.

Sulfoquinovose (SQ) is a key component of plant sulfolipids (sulfoquinovosyl diacylglycerols) and a major environmental reservoir of biological sulfur. Breakdown of SQ is achieved by bacteria through the pathways of sulfoglycolysis. The sulfoglycolytic sulfofructose transaldolase (sulfo-SFT) pathway is used by gut-resident firmicutes and soil saprophytes. After isomerization of SQ to sulfofructose (SF), the namesake enzyme catalyzes the transaldol reaction of SF transferring dihydroxyacetone to 3C/4C acceptors to give sulfolactaldehyde and fructose-6-phosphate or sedoheptulose-7-phosphate. We report the 3D cryo-EM structure of SF transaldolase from Bacillus megaterium in apo and ligand bound forms, revealing a decameric structure formed from two pentameric rings of the protomer. We demonstrate a covalent "Schiff base" intermediate formed by reaction of SF with Lys89 within a conserved Asp-Lys-Glu catalytic triad and defined by an Arg-Trp-Arg sulfonate recognition triad. The structural characterization of the signature enzyme of the sulfo-SFT pathway provides key insights into molecular recognition of the sulfonate group of sulfosugars.

Quantitative assessment of antioxidant potential of selected homeopathic preparations in clinical practice.

OBJECTIVES: Antioxidant property like radical scavenging is a primary target to elucidate the efficacy mechanism of a drug against diseases linked to oxidative stress such as cancer, metabolic disorders, rheumatoid arthritis, etc. In alternative therapies, homeopathy is one of the preferred choices by patients and clinicians due to its potential to cure chronic and complex illnesses. However, the efficacy of homeopathic preparations at high diluted potencies attracts rational criticism due to insufficient scientific knowledge supporting the mechanism of action. Therefore, an attempt was made to estimate the total phenolic content (TPC) and radical scavenging activity of clinically prescribed homeopathic drugs. METHODS: With gallic acid as a reference control, mother tinctures (MTs) and different potencies of Eucalyptus globulus (EG), Syzygium jambolanum (SJ), Ruta graveolens (RG), and Thuja occidentalis (TO) were used to perform Folin-Ciocalteu test, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. RESULTS: The results showed TPC of MTs equivalent to µg/mL of gallic acid viz; EG (4,872.5 ± 133.2), SJ (8,840.5 ± 14.8), RG (985.6 ± 39.1), and TO (341.5 ± 19.5) with significant ABTS and DPPH radical scavenging potential. Whereas 30C and 200C potencies of each homeopathic drug showed undetectable phenolic content and insignificant radical scavenging potential compared to vehicle control, i.e., alcohol 90% (2.0 ± 1.5). CONCLUSIONS: The reported efficacy of 30C and 200C potencies of homeopathic medicines against oxidative stress-related illnesses might be due to mechanisms other than radical scavenging. Furthermore, the assays studied can be helpful in drug standardization and quality control of MTs that are used as starting material in homeopathic preparations.

Mosquito-larvicidal Binary (BinA/B) proteins for mosquito control programs -advancements, challenges, and possibilities.

The increasing global burden of mosquito-borne diseases require targeted, environmentally friendly, and sustainable approaches for effective vector control without endangering the non-target beneficial insect population. Biological interventions such as biopesticides, Wolbachia-mediated biological controls, or sterile insect techniques are used worldwide. Here we review Binary or BinAB toxin-the mosquito-larvicidal component of WHO-recognized Lysinibacillus sphaericus bacterium employed in mosquito control programs. Binary (BinAB) toxin is primarily responsible for the larvicidal effect of the bacterium. BinAB is a single-receptor-specific toxin and is effective against larvae of Culex and Anopheles, but not against Aedes aegypti. The receptor in Culex, the Cqm1 protein, has been extensively studied. It is a GPI-anchored amylomaltase and is located apically in the lipid rafts of the larval-midgut epithelium. The interaction of the toxin components with the receptor is crucial for the mosquito larvicidal activity of the BinAB toxin. Here we extend support for the pore formation model of BinAB toxin internalization and the role of toxin-glycan interactions in the endoplasmic reticulum in mediating larval death. BinAB is phylogenetically safe for humans, as Cqm1-like protein is not expected in the human proteome. This review aims to initiate targeted R&D efforts, such as applying fusion technologies (chimera of BinA, chemical modification of BinA), for efficient mosquito control interventions. In addition, the review also examines other areas such as bioremediation and cancer therapeutics, in which L. sphaericus is proving useful and showing potential for further development.

Environmental friendly Polyacrylonitrile nanofiber mats encapsulated and coated with green algae mediated Titanium oxide nanoparticles for efficient oil spill adsorption.

Oil spill causes extreme environmental damage, from aquatic life to seabirds, disrupting the entire ecosystem. Herein, we have synthesized high scale, economical and bio-compatible, green algae mediated Titanium oxide (TiO2) nanoparticles and Polyacrylonitrile (PAN) nanofiber mats. We have studied the effect of encapsulation and coating of TiO2 nanoparticles over nanofiber mats for highly efficient oil spill adsorption. TiO2 encapsulated and coated PAN (TECP) nanofibers showed a maximum of 62.34 g g-1 adsorption capacity of petroleum oil from the water surface. Moreover, the composite mats show maximum adsorption within 45 s for up to 5 repeated cycles. Further, it has been observed that the adsorption capacity has increased by increasing the weight of the composite nanofiber mats, which confirms its commercial applicability. Thus, this work provides rapid, large-scale, economical, bio-compatible, and highly effective adsorbents for oil spill cleaning and extraction over natural waterbodies.

Diversity, Antimicrobial Activity, and Antibiotic Susceptibility Pattern of Endophytic Bacteria Sourced From Cordia dichotoma L.

Endophytic bacteria isolated from medicinal plants are crucial for the production of antimicrobial agents since they are capable of possessing bioactive compounds with diverse structures and activities. Cordia dichotoma, a plant of medicinal importance native to the Jammu region of India, was selected for the isolation and characterization of culturable endophytic bacteria and evaluation of their antimicrobial activities. Standardized surface sterilization methods were employed to isolate thirty-three phenotypically distinguishable endophytic bacteria from the root, stem, and leaf parts of the plant. Shannon Wiener diversity index clearly divulged diverse endophytes in roots (0.85), stem (0.61), and leaf (0.54) tissues. Physio-biochemical features of the isolates differentiated the distinct variations in their carbohydrate utilization profile and NaCl tolerance. The endophytes produced an array of enzymes, namely, catalase, oxidase, amylase, cellulase, nitrate reductase, and lipase. The bacterial isolates belong to the genera Bacillus, Pseudomonas, Paenibacillus, Acidomonas, Streptococcus, Ralstonia, Micrococcus, Staphylococcus, and Alcalignes predominantly. However, the antibiotic susceptibility pattern indicated that the isolates were mostly sensitive to erythromycin and streptomycin, while they were resistant to rifampicin, amoxicillin, and bacitracin. Interestingly, majority of the bacterial endophytes of C. dichotoma showed antimicrobial activity against Bacillus subtilis followed by Klebsiella pneumoniae. The 16S rRNA sequence of Bacillus thuringiensis has been deposited in the NCBI GenBank database under accession number OM320575. The major compounds of the crude extract derived from endophytic B. thuringiensis OM320575, according to the metabolic profile examination by GC-MS, are dibutyl phthalate, eicosane, tetrapentacontane, heneicosane, and hexadecane, which possessed antibacterial activities. In conclusion, results indicated the potential of C. dichotoma to host a plethora of bacterial endophytes that produce therapeutic bioactive metabolites.