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The isolation of good quality metagenomic DNA from diverse soil, in appreciable amount, is a prerequisite for metagenomics. The availability of commercial kits for isolation of genomic DNAs from soil has drastically expedited the application of metagenomics approach for identifying novel sources of industrially important enzymes. The quantitative and qualitative assessment of metagenomic DNA isolated using either the manual method or the kit-based method should be performed prior to its use in downstream applications. The metagenomic DNA isolated from six different soil samples, using three methods, were analyzed in terms of yield, quality and downstream application as template for PCR amplification. The yield of DNA was approximately 3.52, 7.35, and 232.42 μg of DNA per gram of soil sample for the kit-based method, kit-modified method, and manual method, respectively. The manual method seems to be promising based on better yield and lesser humic acid content than the other two methods. The maximum yield was obtained in the soil collected from teak forest with all the three methods, indicating maximum microbial content and diversity. Furthermore, in terms of its suitability as template DNA for PCR amplification using 16S RNA primer, all methods are equally well. Thus, comparative assessment of three methods revealed suitability of manual method based on DNA yield and humic acid content, which could be important for many downstream applications like library preparations during metageomics approach. [ABSTRACT FROM AUTHOR]

The apicoplast of Plasmodium falciparum carries a 35 kb circular genome (plDNA) that replicates at the late trophozoite stage of the parasite intraerythocytic cycle. plDNA replication proceeds predominantly via ad-loop/bi-directional ori mechanism with replication ori localized within inverted repeat region. Although replication of the apicoplast genome is a validated drug target, the proteins involved in the replication process are only partially characterized. We analysed DNA–protein interactions at a plDNA replication ori region and report the identification of a nuclear-encoded DnaJ homologue that binds directly to ori elements of the plDNA molecule. PfDnaJA interacted with the minor groove of the DNA double-helix and recognized a 13 bp sequence within the ori. Inhibition of binding with anti-PfDnaJA antibodies confirmed identity of the protein in DNA-binding experiments with organellar protein fractions. The DNA-binding domain of the ∼69 kDa PfDnaJA lay within the N-terminal 38 kDa region that carries DnaJ signature motifs. In contrast to PfDnaJA in parasite organellar fractions, the recombinant protein interacted with DNA in a sequence non-specific manner. Our results suggest a role for PfDnaJA in replication/repair of the apicoplast genome. [ABSTRACT FROM AUTHOR]

Immunosuppressants, such as methotrexate (MTX), can suppress the COVID-19 vaccine response in patients with autoimmune diseases. Thus, this study aims to evaluate the effects of MTX hold following COVID-19 vaccination on vaccine efficacy response. A systematic review and meta-analysis of relevant studies retrieved from Web of Science, SCOPUS, PubMed, and CENTRAL from inception until Oct 1, 2023, was conducted. Covidence was used to screen the eligible articles, and all relevant outcomes data were synthesized using risk ratios (RRs) or standardized mean differences (SMDs) with 95% confidence intervals (CIs) in meta-analysis models within RevMan 5.4. PROSPERO ID: CRD42024511628. Four studies with a total of 762 patients with autoimmune inflammatory disorders were included. Holding MTX following the COVID-19 vaccination for approximately 2 weeks was associated with significantly higher antibody titer (SMD: 0.70, 95% CI [0.54, 0.87], P < 0.00001). However, the flare rate was significantly higher in the MTX hold group based on CDAI > 10 or DAS28-CRP > 1.2 either after 1st dose (RR: 2.49 with 95% CI [1.39, 4.47], P = 0.002) or 2nd dose (RR: 2.16 with 95% CI [1.37, 3.41], P = 0.0009) and self-reported disease flare (RR: 1.71 with 95% CI [1.35, 2.17], P < 0.00001). Holding MTX for 2 weeks after the COVID-19 vaccination resulted in significantly higher antibody titer but also had a higher disease flare rate, necessitating cautious clinical monitoring during this period. There is still a need to investigate safer MTX hold duration, considering patients' vulnerability to COVID-19, disease status, and demographics while adopting this strategy. [ABSTRACT FROM AUTHOR]

Pectin, also known as pectic compounds, is a fundamental component of plant cell walls that imparts rigidity and structure. Pectin lyases have a unique β-elimination mechanism of directly degrading pectin polymers to 4, 5-unsaturated oligogalacturonides, while other pectinases have preferences for sequential degradation. Members of the pectin lyase family have characteristic Pec_Lyase_C domain and β-helix structure. They have been successfully isolated and characterized from different bacteria, fungi, and yeast. Based on their biochemical properties, pectin lyase is one of the most important enzymes in the commercial sector. In the current biotechnological world, these enzymes play a massive role in juice processing industries, retting of fibres, biosurfactant production, and they possess a biocontrol effect. Pectin lyases are promising for the fruit juice industry as they maintain the typical odor of juice and do not form extremely harmful methanol as a by-product. Several recent innovations related to production, purification, and molecular insights have been reported since the last review published by our group in 2009. This review is an attempt to update the sources, production strategies, biochemical attributes, molecular and expression profiling, bioinformatics, and genomic-driven research exclusively in pectin lyases. • Pectin lyases have a unique β-elimination mechanism of directly degrading pectin. • Microbes are the major sources of Pectin lyases. • Pectin lyases are used in juice industries, retting of fibres, and biosurfactant production. • Productions, purifications, and molecular studies on pectin lyase attempted. [ABSTRACT FROM AUTHOR]

The fungal system holds morphological plasticity and metabolic versatility which makes it unique. Fungal habitat ranges from the Arctic region to the fertile mainland, including tropical rainforests, and temperate deserts. They possess a wide range of lifestyles behaving as saprophytic, parasitic, opportunistic, and obligate symbionts. These eukaryotic microbes can survive any living condition and adapt to behave as extremophiles, mesophiles, thermophiles, or even psychrophile organisms. This behaviour has been exploited to yield microbial enzymes which can survive in extreme environments. The cost-effective production, stable catalytic behaviour and ease of genetic manipulation make them prominent sources of several industrially important enzymes. Pectinases are a class of pectin-degrading enzymes that show different mechanisms and substrate specificities to release end products. The pectinase family of enzymes is produced by microbial sources such as bacteria, fungi, actinomycetes, plants, and animals. Fungal pectinases having high specificity for natural sources and higher stabilities and catalytic activities make them promising green catalysts for industrial applications. Pectinases from different microbial sources have been investigated for their industrial applications. However, their relevance in the food and textile industries is remarkable and has been extensively studied. The focus of this review is to provide comprehensive information on the current findings on fungal pectinases targeting diverse sources of fungal strains, their production by fermentation techniques, and a summary of purification strategies. Studies on pectinases regarding innovations comprising bioreactor-based production, immobilization of pectinases, in silico and expression studies, directed evolution, and omics-driven approaches specifically by fungal microbiota have been summarized. [ABSTRACT FROM AUTHOR]

The two organelles, apicoplast and mitochondrion, of the malaria parasite Plasmodium falciparum have unique morphology in liver and blood stages; they undergo complex branching and looping prior to division and segregation into daughter merozoites. Little is known about the molecular processes and proteins involved in organelle biogenesis in the parasite. We report the identification of an AAA+/FtsH protease homolog (PfFtsH1) that exhibits ATP- and Zn2+-dependent protease activity. PfFtsH1 undergoes processing, forms oligomeric assemblies, and is associated with the membrane fraction of the parasite cell. Generation of a transfectant parasite line with hemagglutinin-tagged PfFtsH1, and immunofluorescence assay with anti-PfFtsH1 Ab demonstrated that the protein localises to P. falciparum mitochondria. Phylogenetic analysis and the single transmembrane region identifiable in PfFtsH1 suggest that it is an i-AAA like inner mitochondrial membrane protein. Expression of PfFtsH1 in Escherichia coli converted a fraction of bacterial cells into division-defective filamentous forms implying a sequestering effect of the Plasmodium factor on the bacterial homolog, indicative of functional conservation with EcFtsH. These results identify a membrane-associated mitochondrial AAA+/FtsH protease as a candidate regulatory protein for organelle biogenesis in P. falciparum. [ABSTRACT FROM AUTHOR]

Pectin lyase (PNL) is an important enzyme of the pectinases group which degrades pectin polymer to 4,5-unsaturated oligogalacturonides by a unique β-elimination mechanism and is used in several industries. The existence of multigene families of pectin lyases has been investigated by mining microbial genomes. In the present study, 52 pectin lyase genes were predicted from sequenced six species of Fusarium, namely F. fujikuroi, F. graminearum, F. proliferatum, F. oxysporum, F. verticillioides and F. virguliforme. These sequences were in silico characterized for several physico-chemical, structural and functional attributes. The translated PNL proteins showed variability with 344–1142 amino acid residues, 35.44–127.41 kDa molecular weight, and pI ranging from 4.63 to 9.28. The aliphatic index ranged from 75.33 to 84.75. Multiple sequence alignment analysis showed several conserved amino acid residues and five distinct groups marked as I, II, III, IV, and V were observed in the phylogenetic tree. The Three-dimensional Structure of five of these PNLs, each representing a distinct group of phylogenetic trees was predicted using I-TASSER Server and validated. The pectin lyase proteins of Fusarium species revealed close similarity with pectin lyase of Aspergillus niger PelA(1IDJ) and PelB(1QCX). Diversity in the structural motifs was observed among Fusarium species with 2 β-sheets, 1 β-hairpin, 7–12 β bulges, 18–25 strands, 6 -11 helices, 1 helix-helix interaction, 32–49 β turns, 2–6 γ turns and 2- 3 disulfide bonds. The unique Pec_lyase domain was uniformly observed among all PNL proteins confirming its identity. The genome-wide mining of Fusarium species was attempted to provide the diversity of PNL genes, which could be explored for diverse applications after performing cloning and expression studies. [ABSTRACT FROM AUTHOR]

The malaria parasite carries a plastid called the apicoplast that has been the subject of intense study in the last 15 years. Having originated from red-algal plastids, the apicoplast has lost its ability to photosynthesize, but carries out other essential functions such as type-II fatty acid synthesis, biosynthesis of haem and isoprenoid synthesis; the DOXP pathway for isoprenoid synthesis has recently been demonstrated to be the only pathway critical for parasite survival in the erythrocytic stage. The apicoplast also has a functional Suf system for assembly of (Fe-S) complexes on target proteins. The organelle has a 35 kb, double-stranded DNA genome that encodes a set of RNAs and proteins, the latter being translated from organellar mRNA by an active translation machinery, a major component of which is encoded by the nucleus. This article reviews current knowledge of housekeeping functions of the Plasmodium apicoplast and its (Fe-S) assembly system and discusses these components as sites for drug intervention against malaria. [ABSTRACT FROM AUTHOR]

Abstract: The plastid of Plasmodium falciparum, the apicoplast, performs metabolic functions essential to the parasite. Various reactions in the plastid require the assembly of [Fe–S] prosthetic groups on participating proteins as well as the reductant activity of ferredoxin that is converted from its apo-form by the assembly of [Fe–S] clusters inside the apicoplast. The [Fe–S] assembly pathway involving sulphur mobilising Suf proteins has been predicted to function in the apicoplast with one component (PfSufB) encoded by the plastid genome itself. We demonstrate the ATPase activity of recombinant P. falciparum nuclear-encoded SufC and its localisation in the apicoplast. Further, an internal region of apicoplast SufB was used to detect PfSufB–PfSufC interaction in vitro; co-elution of SufB from parasite lysate with recombinant PfSufC on an affinity column also indicated an interaction of the two proteins. As a departure from bacterial SufB and similar to reported plant plastid SufB, apicoplast SufB exhibited ATPase activity, suggesting the evolution of specialised functions in the plastid counterparts. Our results provide experimental evidence for an active Suf pathway in the Plasmodium apicoplast. [Copyright &y& Elsevier]