Agro-industrial waste management employing benefits of artificial intelligence.

By 2050, the world's population is predicted to reach over 9 billion, which requires 70% increased production in agriculture and food industries to meet demand. This presents a significant challenge for the agri-food sector in all aspects. Agro-industrial wastes are rich in bioactive substances and other medicinal properties. They can be used as a different source for manufacturing products like biogas, biofuels, mushrooms, and tempeh, the primary ingredients in various studies and businesses. Increased importance is placed on resource recovery, recycling, and reusing (RRR) any waste using advanced technology like IoT and artificial intelligence. AI algorithms offer alternate, creative methods for managing agro-industrial waste management (AIWM). There are contradictions and a need to understand how AI technologies work regarding their application to AIWM. This research studies the application of AI-based technology for the various areas of AIWM. The current work aims to discover AI-based models for forecasting the generation and recycling of AIWM waste. Research shows that agro-industrial waste generation has increased worldwide. Infrastructure needs to be upgraded and improved by adapting AI technology to maintain a balance between socioeconomic structures. The study focused on AI's social and economic impacts and the benefits, challenges, and future work in AIWM. The present research will increase recycling and reproduction with a balance of cost, efficiency, and human resources consumption in agro-industrial waste management.

MRTF-A gain-of-function in mice impairs homeostatic renewal of the intestinal epithelium.

The actin-regulated transcription factor MRTF-A represents a central relay in mechanotransduction and controls a subset of SRF-dependent target genes. However, gain-of-function studies in vivo are lacking. Here we characterize a conditional MRTF-A transgenic mouse model. While MRTF-A gain-of-function impaired embryonic development, induced expression of constitutively active MRTF-A provoked rapid hepatocyte ballooning and liver failure in adult mice. Specific expression in the intestinal epithelium caused an erosive architectural distortion, villus blunting, cryptal hyperplasia and colonic inflammation, resulting in transient weight loss. Organoids from transgenic mice repeatedly induced in vitro showed impaired self-renewal and defective cryptal compartments. Mechanistically, MRTF-A gain-of-function decreased proliferation and increased apoptosis, but did not induce fibrosis. MRTF-A targets including Acta2 and Pai-1 were induced, whereas markers of stem cells and differentiated cells were reduced. Our results suggest that activated MRTF-A in the intestinal epithelium shifts the balance between proliferation, differentiation and apoptosis.

Targeting oncogenic KRasG13C with nucleotide-based covalent inhibitors.

Mutations within Ras proteins represent major drivers in human cancer. In this study, we report the structure-based design, synthesis, as well as biochemical and cellular evaluation of nucleotide-based covalent inhibitors for KRasG13C, an important oncogenic mutant of Ras that has not been successfully addressed in the past. Mass spectrometry experiments and kinetic studies reveal promising molecular properties of these covalent inhibitors, and X-ray crystallographic analysis has yielded the first reported crystal structures of KRasG13C covalently locked with these GDP analogues. Importantly, KRasG13C covalently modified with these inhibitors can no longer undergo SOS-catalysed nucleotide exchange. As a final proof-of-concept, we show that in contrast to KRasG13C, the covalently locked protein is unable to induce oncogenic signalling in cells, further highlighting the possibility of using nucleotide-based inhibitors with covalent warheads in KRasG13C-driven cancer.

miRNA mediated downregulation of cyclase-associated protein 1 (CAP1) is required for myoblast fusion.

Myoblast fusion is essential for the formation, growth, and regeneration of skeletal muscle, but the molecular mechanisms that govern fusion and myofiber formation remain poorly understood. Past studies have shown an important role of the actin cytoskeleton and actin regulators in myoblast fusion. The Cyclase-Associated Proteins (CAP) 1 and 2 recently emerged as critical regulators of actin treadmilling in higher eukaryotes including mammals. Whilst the role of CAP2 in skeletal muscle development and function is well characterized, involvement of CAP1 in this process remains elusive. Here we report that CAP1, plays a critical role in cytoskeletal remodeling during myoblast fusion and formation of myotubes. Cap1 mRNA and protein are expressed in both murine C2C12 and human LHCN-M2 myoblasts, but their abundance decreases during myogenic differentiation. Perturbing the temporally controlled expression of CAP1 by overexpression or CRISPR-Cas9 mediated knockout impaired actin rearrangement, myoblast alignment, expression of profusion molecules, differentiation into multinucleated myotubes, and myosin heavy chain expression. Endogenous Cap1 expression is post-transcriptionally downregulated during differentiation by canonical myomiRs miR-1, miR-133, and miR-206, which have conserved binding sites at the 3' UTR of the Cap1 mRNA. Deletion of the endogenous 3' UTR by CRISPR-Cas9 in C2C12 cells phenocopies overexpression of CAP1 by inhibiting myotube formation. Our findings implicates Cap1 and its myomiR-mediated downregulation in the myoblast fusion process and the generation of skeletal muscle.

Rep15 interacts with several Rab GTPases and has a distinct fold for a Rab effector.

In their GTP-bound (active) form, Rab proteins interact with effector proteins that control downstream signaling. One such Rab15 effector is Rep15, which is known to have a role in receptor recycling from the endocytic recycling compartment but otherwise remains poorly characterized. Here, we report the characterization of the Rep15:Rab15 interaction and identification of Rab3 paralogs and Rab34 as Rep15 interacting partners from a yeast two-hybrid assay. Biochemical validation of the interactions is presented and crystal structures of the Rep15:Rab3B and Rep15:Rab3C complexes provide additional mechanistic insight. We find that Rep15 adopts a globular structure that is distinct from other reported Rab15, Rab3 and Rab34 effectors. Structure-based mutagenesis experiments explain the Rep15:Rab interaction specificity. Rep15 depletion in U138MG glioblastoma cells impairs cell proliferation, cell migration and receptor recycling, underscoring the need for further clarification of the role of Rep15 in cancer.

Role of advanced respiratory support in acute respiratory failure in clinically frail patients with COVID-19.

Background: The main aim of this study was to assess the efficacy of advanced respiratory support (ARS) for acute respiratory failure in do-not-attempt cardiopulmonary resuscitation order (DNACPR) COVID-19 patients. Methods: In this single-center study, the impact of different types of ARS modality, PaO2/FiO2 (PF) ratio, clinical frailty score (CFS) and 4C score on mortality was evaluated. Results: There was no significant difference in age, type of ARS modality, PF ratio and 4C scores between those who died and those who survived. Overall survival rates/hospital discharge of patients still requiring ARS at 5 and 7 days post admission were 20 and 17%, respectively. Conclusion: Our study showed that ARS can be a useful tool in frail, elderly and high-risk COVID-19 patients irrespective of high 4C mortality score.

New and efficient purification process for recombinant human insulin produced in Escherichia coli.

A new and efficient purification process for recombinant human insulin production was developed by exploring new resins and optimizing purification steps from E. coli inclusion body washing to insulin polishing. A combined additives inclusion body wash protocol drastically improved efficiency in clarifying ZZ-proinsulin samples. ZZ-proinsulin recovery increased three-fold under optimized solubilization and sulfitolysis incubation temperature and duration. Desalting with Bio-Gel P4 and P6 resulted in higher sample loading and product recovery compared to conventional resins. A higher recovery (96%) and purity (81%) of ZZ-proinsulin were achievable with the Nuvia S cation exchanger for proinsulin purification compared to a reported process using expensive affinity chromatography resin. As the first step for insulin purification, process scale-up is more economical and practical when Nuvia HR-S cation exchanger was used instead of commonly used reversed-phase chromatography. Nuvia HR-S was highly effective in removing ZZ fusion protein (90% removal) after enzymatic cleavage, although ZZ fusion protein has a very close theoretical pI to human insulin, which was supposedly challenging to be removed by cation exchange chromatography. Also, insulin can be eluted at a lower ethanol % using Nuvia HR-S compared to other reported processes and is thus more environmentally sustainable. Recombinant human insulin was obtained with over 98% purity in just a single reversed-phase polishing step, which is comparable to the reference standard. The process workflow presented here can be potentially applied for the development of purification workflow for insulin analogs or other peptide products derived from E. coli inclusion body.Key points• Drastic efficiency improvement for inclusion body wash with combined additives.• High recovery of proinsulin purification with high capacity cation exchange resin.• Effective removal of fusion tag at lower ethanol % with high-resolution resin.

Structural and Biochemical Characterization of a Dye-Decolorizing Peroxidase from Dictyostelium discoideum.

A novel cytoplasmic dye-decolorizing peroxidase from Dictyostelium discoideum was investigated that oxidizes anthraquinone dyes, lignin model compounds, and general peroxidase substrates such as ABTS efficiently. Unlike related enzymes, an aspartate residue replaces the first glycine of the conserved GXXDG motif in Dictyostelium DyPA. In solution, Dictyostelium DyPA exists as a stable dimer with the side chain of Asp146 contributing to the stabilization of the dimer interface by extending the hydrogen bond network connecting two monomers. To gain mechanistic insights, we solved the Dictyostelium DyPA structures in the absence of substrate as well as in the presence of potassium cyanide and veratryl alcohol to 1.7, 1.85, and 1.6 Å resolution, respectively. The active site of Dictyostelium DyPA has a hexa-coordinated heme iron with a histidine residue at the proximal axial position and either an activated oxygen or CN- molecule at the distal axial position. Asp149 is in an optimal conformation to accept a proton from H2O2 during the formation of compound I. Two potential distal solvent channels and a conserved shallow pocket leading to the heme molecule were found in Dictyostelium DyPA. Further, we identified two substrate-binding pockets per monomer in Dictyostelium DyPA at the dimer interface. Long-range electron transfer pathways associated with a hydrogen-bonding network that connects the substrate-binding sites with the heme moiety are described.

The mechanism of activation of the actin binding protein EHBP1 by Rab8 family members.

EHBP1 is an adaptor protein that regulates vesicular trafficking by recruiting Rab8 family members and Eps15-homology domain-containing proteins 1/2 (EHD1/2). It also links endosomes to the actin cytoskeleton. However, the underlying molecular mechanism of activation of EHBP1 actin-binding activity is unclear. Here, we show that both termini of EHBP1 have membrane targeting potential. EHBP1 associates with PI(3)P, PI(5)P, and phosphatidylserine via its N-terminal C2 domain. We show that in the absence of Rab8 family members, the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domain forms an intramolecular complex with its central calponin homology (CH) domain and auto-inhibits actin binding. Rab8 binding to the bMERB domain relieves this inhibition. We have analyzed the CH:bMERB auto-inhibited complex and the active bMERB:Rab8 complex biochemically and structurally. Together with structure-based mutational studies, this explains how binding of Rab8 frees the CH domain and allows it to interact with the actin cytoskeleton, leading to membrane tubulation.

The developing and evaluation of an electronic tool to assess the effect of undergraduate training in palliative care: the electronic international medical education in palliative care (IMEP-e) assessment tool.

BACKGROUND: With increased demand for palliative care (PC), the World Health Organisation (WHO) have called for PC teaching to be made routine. However, medical students report feeling unprepared in dealing with end-of-life care. Necessary benchmarking of the preparedness of clinicians to provide PC is required to identify where current training is sub-optimal and ensure future doctors are equipped to meet the needs of their patients. The aim of this study is to assess the utility of an electronic International Medical Education in Palliative Care (IMEP-e) assessment tool that examines the preparedness of clinicians to provide PC. METHODS: A multi-phase pilot study. Phase 1: To transpose the Self-Efficacy Palliative Care Scale (SEPCs) and the Thanatophobia Scale (TS) to an electronic format and evaluate its utility. Phase 2: To assess the effects of PC teaching by comparing data from year three (Y3) and year five (Y5 - who have participated in PC placement) medical students. Scales: The 23 item SEPC and 7 item TS assess attitudes towards caring for dying patients. RESULTS: Total questionnaires sent =360 (280 Y3, 80 Y5). Total response rate = 46.39%, n = 167 (127 Y3, 40 Y5). Completed data: n = 125 (95 Y3, 30 Y5). Analysis identified statistically significant differences (p < 0.001) between year groups across all subscales of the SEPC; communication skills (t = - 13.52), Pain and Treatment management (t = - 14.25) and multidisciplinary management (t = - 7.89). The TS shows a statistically significant increased positive attitudes (z = - 2.85 p < 0.005). From the focus group, three themes were identified from the qualitative feedback including university based teaching, hospice based teaching and utility of IMEP-e tool. CONCLUSION: The IMEP-e tool is a viable and comparable method for collecting data on the preparedness to practice PC. A larger scale study is needed to determine and evaluate if, and how, preparing clinicians to work in PC has been adapted in to routine training.

Multivalency in Rab effector interactions.

Rab proteins regulate vesicular transport in eukaryotic cells and establish connections to various cellular structures and processes by interacting with so-called effector molecules. Several of these effectors are known to not only bind a single Rab protein, but to be able to bind multiple different Rabs simultaneously. In this review we will give a short overview of effectors in general and (putative) functions of the aforementioned multivalent Rab:effector interactions.

Human MICAL1: Activation by the small GTPase Rab8 and small-angle X-ray scattering studies on the oligomerization state of MICAL1 and its complex with Rab8.

Human MICAL1 is a member of a recently discovered family of multidomain proteins that couple a FAD-containing monooxygenase-like domain to typical protein interaction domains. Growing evidence implicates the NADPH oxidase reaction catalyzed by the flavoprotein domain in generation of hydrogen peroxide as a second messenger in an increasing number of cell types and as a specific modulator of actin filaments stability. Several proteins of the Rab families of small GTPases are emerging as regulators of MICAL activity by binding to its C-terminal helical domain presumably shifting the equilibrium from the free - auto-inhibited - conformation to the active one. We here extend the characterization of the MICAL1-Rab8 interaction and show that indeed Rab8, in the active GTP-bound state, stabilizes the active MICAL1 conformation causing a specific four-fold increase of kcat of the NADPH oxidase reaction. Kinetic data and small-angle X-ray scattering (SAXS) measurements support the formation of a 1:1 complex between full-length MICAL1 and Rab8 with an apparent dissociation constant of approximately 8 µM. This finding supports the hypothesis that Rab8 is a physiological regulator of MICAL1 activity and shows how the protein region preceding the C-terminal Rab-binding domain may mask one of the Rab-binding sites detected with the isolated C-terminal fragment. SAXS-based modeling allowed us to propose the first model of the free full-length MICAL1, which is consistent with an auto-inhibited conformation in which the C-terminal region prevents catalysis by interfering with the conformational changes that are predicted to occur during the catalytic cycle.

bMERB domains are bivalent Rab8 family effectors evolved by gene duplication.

In their active GTP-bound form, Rab proteins interact with proteins termed effector molecules. In this study, we have thoroughly characterized a Rab effector domain that is present in proteins of the Mical and EHBP families, both known to act in endosomal trafficking. Within our study, we show that these effectors display a preference for Rab8 family proteins (Rab8, 10, 13 and 15) and that some of the effector domains can bind two Rab proteins via separate binding sites. Structural analysis allowed us to explain the specificity towards Rab8 family members and the presence of two similar Rab binding sites that must have evolved via gene duplication. This study is the first to thoroughly characterize a Rab effector protein that contains two separate Rab binding sites within a single domain, allowing Micals and EHBPs to bind two Rabs simultaneously, thus suggesting previously unknown functions of these effector molecules in endosomal trafficking.

A pull-down procedure for the identification of unknown GEFs for small GTPases.

Members of the family of small GTPases regulate a variety of important cellular functions. In order to accomplish this, tight temporal and spatial regulation is absolutely necessary. The two most important factors for this regulation are GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), the latter being responsible for the activation of the GTPase downstream pathways at the correct location and time. Although a large number of exchange factors have been identified, it is likely that a similarly large number remains unidentified. We have therefore developed a procedure to specifically enrich GEF proteins from biological samples making use of the high affinity binding of GEFs to nucleotide-free GTPases. In order to verify the results of these pull-down experiments, we have additionally developed two simple validation procedures: An in vitro transcription/translation system coupled with a GEF activity assay and a yeast two-hybrid screen for detection of GEFs. Although the procedures were established and tested using the Rab protein Sec4, the similar basic principle of action of all nucleotide exchange factors will allow the method to be used for identification of unknown GEFs of small GTPases in general.

Expression, purification and crystallization of a dye-decolourizing peroxidase from Dictyostelium discoideum.

Dye-decolourizing peroxidases are haem-containing peroxidases with broad substrate specificity. Using H2O2 as an electron acceptor, they efficiently decolourize various dyes that are of industrial and environmental relevance, such as anthraquninone- and azo-based dyes. In this study, the dye-decolourizing peroxidase DdDyP from Dictyostelium discoideum was overexpressed in Escherichia coli strain Rosetta(DE3)pLysS, purified and crystallized using the vapour-diffusion method. A native crystal diffracted to 1.65 Å resolution and belonged to space group P4(1)2(1)2, with unit-cell parameters a = b = 141.03, c = 95.56 Å, α = ß = γ = 90°. The asymmetric unit contains two molecules.

Functional dissection of the dictyostelium discoideum dynamin B mitochondrial targeting sequence.

Most mitochondrial proteins are nuclear encoded and synthesized in the cytosol with an N-terminal mitochondrial targeting sequence or presequence for subsequent import into mitochondria. Here, we describe the proteolytic processing and inner membrane potential-dependent translocation of a dynamin family member by the Dictyostelium discoideum mitochondrial import system. Our results show that the unusual D. discoideum dynamin B presequence is removed through a processing mechanism that is common for mitochondrial matrix proteins. We identified a minimal segment of the dynamin B presequence containing seven lysine residues. This 47-residue region is, in combination with consensus matrix protease cleavage sites, necessary and sufficient for mitochondrial targeting. The correct positioning of these lysine residues plays a critical role for the proper processing and mitochondrial import of dynamin B in D. discoideum. Fluorescent proteins tagged with the dynamin B presequence or presequence regions supporting mitochondrial import in D. discoideum are imported with similar efficiency into the mitochondrial matrix of mammalian cells, indicating that the basic mechanisms underlying mitochondrial protein import are highly conserved from amoebozoa to mammalia.

An in vitro evaluation of the anthelmintic activity of Hedychium spichatum rhizomes and Zingiber zerumbet rhizomes on the Pheritima Posthuma model: A comparative study.

Ethanolic extracts of Hedychium spichatum rhizomes and Zingiber zerumbet rhizomes were taken for in vitro comparative studies on the anthelmintic activity against Pheritima posthuma. Different concentrations (25, 50, 100 mg/ml) of both the extracts were used for the activity. Varying albendazole concentrations (25, 50, 100 mg/ml) were used as a reference standard and normal saline (0.9% NaCl) was used for the control treatment. The results were expressed in terms of time in minutes to report the paralysis and time of death of the earthworms. The results obtained from the study indicate toward the anthelmintic activity, supporting folk use of both the plants when compared with the standard. The results also established that Z. zerumbet was a more potent candidature of as compared with H. spichatum.

Dictyostelium dynamin B modulates cytoskeletal structures and membranous organelles.

Dictyostelium discoideum cells produce five dynamin family proteins. Here, we show that dynamin B is the only member of this group of proteins that is initially produced as a preprotein and requires processing by mitochondrial proteases for formation of the mature protein. Our results show that dynamin B-depletion affects many aspects of cell motility, cell-cell and cell-surface adhesion, resistance to osmotic shock, and fatty acid metabolism. The mature form of dynamin B mediates a wide range and unique combination of functions. Dynamin B affects events at the plasma membrane, peroxisomes, the contractile vacuole system, components of the actin-based cytoskeleton, and cell adhesion sites. The modulating effect of dynamin B on the activity of the contractile vacuole system is unique for the Dictyostelium system. Other functions displayed by dynamin B are commonly associated with either classical dynamins or dynamin-related proteins.