Bridging the Gap: Can COVID-19 Research Help Combat African Swine Fever?

African swine fever (ASF) is a highly contagious and economically devastating disease affecting domestic pigs and wild boar, caused by African swine fever virus (ASFV). Despite being harmless to humans, ASF poses significant challenges to the swine industry, due to sudden losses and trade restrictions. The ongoing COVID-19 pandemic has spurred an unparalleled global research effort, yielding remarkable advancements across scientific disciplines. In this review, we explore the potential technological spillover from COVID-19 research into ASF. Specifically, we assess the applicability of the diagnostic tools, vaccine development strategies, and biosecurity measures developed for COVID-19 for combating ASF. Additionally, we discuss the lessons learned from the pandemic in terms of surveillance systems and their implications for managing ASF. By bridging the gap between COVID-19 and ASF research, we highlight the potential for interdisciplinary collaboration and technological spillovers in the battle against ASF.

A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance.

Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection-such as multiplexed detection for viral variant surveillance-may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)-including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)-all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool-CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured-may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.

Discovery and Genetic Code Expansion of a Polyethylene Terephthalate (PET) Hydrolase from the Human Saliva Metagenome for the Degradation and Bio-Functionalization of PET.

We report a bioinformatic workflow and subsequent discovery of a new polyethylene terephthalate (PET) hydrolase, which we named MG8, from the human saliva metagenome. MG8 has robust PET plastic degradation activities under different temperature and salinity conditions, outperforming several naturally occurring and engineered hydrolases in degrading PET. Moreover, we genetically encoded 2,3-diaminopropionic acid (DAP) in place of the catalytic serine residue of MG8, thereby converting a PET hydrolase into a covalent binder for bio-functionalization of PET. We show that MG8(DAP), in conjunction with a split green fluorescent protein system, can be used to attach protein cargos to PET as well as other polyester plastics. The discovery of a highly active PET hydrolase from the human metagenome-currently an underexplored resource for industrial enzyme discovery-as well as the repurposing of such an enzyme into a plastic functionalization tool, should facilitate ongoing efforts to degrade and maximize reusability of PET.

Mechanism of transcription regulation by Acinetobacter baumannii HpaR in the catabolism of p-hydroxyphenylacetate.

HpaR is a transcription regulator in the MarR family that controls the expression of the gene cluster responsible for conversion of p-hydroxyphenylacetate to pyruvate and succinate for cellular metabolism. Here, we report the biochemical and structural characterization of Acinetobacter baumannii HpaR (AbHpaR) and its complex with cognate DNA. Our study revealed that AbHpaR binds upstream of the divergently transcribed hpaA gene and the meta-cleavage operon, as well as the hpaR gene, thereby repressing their transcription by blocking access of RNA polymerase. Structural analysis of AbHpaR-DNA complex revealed that the DNA binding specificity can be achieved via a combination of both direct and indirect DNA sequence readouts. DNA binding of AbHpaR is weakened by 3,4-dihydroxyphenylacetate (DHPA), which is the substrate of the meta-cleavage reactions; this likely leads to expression of the target genes. Based on our findings, we propose a model for how A. baumannii controls transcription of HPA-metabolizing genes, which highlights the independence of global catabolite repression and could be beneficial for metabolic engineering toward bioremediation applications. DATABASES: The structural data that support these findings are openly available in the wwPDB at https://doi.org/10.2210/pdb7EL2/pdb and https://doi.org/10.2210/pdb7EL3/pdb for AbHpaR and AbHpaR-DNA complex, respectively.

MANORAA: A machine learning platform to guide protein-ligand design by anchors and influential distances.

The MANORAA platform uses structure-based approaches to provide information on drug design originally derived from mapping tens of thousands of amino acids on a grid. In-depth analyses of the pockets, frequently occurring atoms, influential distances, and active-site boundaries are used for the analysis of active sites. The algorithms derived provide model equations that can predict whether changes in distances, such as contraction or expansion, will result in improved binding affinity. The algorithm is confirmed using kinetic studies of dihydrofolate reductase (DHFR), together with two DHFR-TS crystal structures. Empirical analyses of 881 crystal structures involving 180 ligands are used to interpret protein-ligand binding affinities. MANORAA links to major biological databases for web-based analysis of drug design. The frequency of atoms inside the main protease structures, including those from SARS-CoV-2, shows how the rigid part of the ligand can be used as a probe for molecular design (http://manoraa.org).

Combined effects of double mutations on catalytic activity and structural stability contribute to clinical manifestations of glucose-6-phosphate dehydrogenase deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans, affecting ~ 500 million worldwide. A detailed study of the structural stability and catalytic activity of G6PD variants is required to understand how different mutations cause varying degrees of enzyme deficiency, reflecting the response of G6PD variants to oxidative stress. Furthermore, for G6PD double variants, investigating how two mutations jointly cause severe enzyme deficiency is important. Here, we characterized the functional and structural properties of nine G6PD variants: G6PD Gaohe, G6PD Mahidol, G6PD Shoklo, G6PD Canton, G6PD Kaiping, G6PD Gaohe + Kaiping, G6PD Mahidol + Canton, G6PD Mahidol + Kaiping and G6PD Canton + Kaiping. All variants were less catalytically active and structurally stable than the wild type enzyme, with G6PD double mutations having a greater impact than single mutations. G6PD Shoklo and G6PD Canton + Kaiping were the least catalytically active single and double variants, respectively. The combined effects of two mutations were observed, with the Canton mutation reducing structural stability and the Kaiping mutation increasing it in the double mutations. Severe enzyme deficiency in the double mutants was mainly determined by the trade-off between protein stability and catalytic activity. Additionally, it was demonstrated that AG1, a G6PD activator, only marginally increased G6PD enzymatic activity and stability.

A Peek Inside the Machines of Bacterial Nucleotide Excision Repair.

Double stranded DNA (dsDNA), the repository of genetic information in bacteria, archaea and eukaryotes, exhibits a surprising instability in the intracellular environment; this fragility is exacerbated by exogenous agents, such as ultraviolet radiation. To protect themselves against the severe consequences of DNA damage, cells have evolved at least six distinct DNA repair pathways. Here, we review recent key findings of studies aimed at understanding one of these pathways: bacterial nucleotide excision repair (NER). This pathway operates in two modes: a global genome repair (GGR) pathway and a pathway that closely interfaces with transcription by RNA polymerase called transcription-coupled repair (TCR). Below, we discuss the architecture of key proteins in bacterial NER and recent biochemical, structural and single-molecule studies that shed light on the lesion recognition steps of both the GGR and the TCR sub-pathways. Although a great deal has been learned about both of these sub-pathways, several important questions, including damage discrimination, roles of ATP and the orchestration of protein binding and conformation switching, remain to be addressed.

Emerging roles of Wss1 in the survival of Candida albicans under genotoxic stresses.

This perspective aims to discuss the potential physiological roles and regulation mechanisms of the recently identified Candida albicans Wss1 protease important in DNA-protein crosslink (DPC) tolerance and repair. DPC is a bulky DNA lesion that blocks essential DNA transactions; thus, it poses a significant threat to genome integrity if left unrepaired. Discoveries of Wss1 in Saccharomyces cerevisiae and SPRTN in human as DPC proteases have demonstrated the importance of protease function in DPC repair. Our recent study revealed that Wss1 in C. albicans, an opportunistic pathogen that can cause life-threatening infection in immunocompromised individuals, also promotes DPC tolerance similarly to both S. cerevisiae Wss1 and human SPRTN. However, its molecular mechanism and regulation are still poorly understood. Here, we briefly discuss the recent insights into C. albicans Wss1 based on the information from S. cerevisiae, as well as outline the aspect of this protein that could make it a potential target for antifungal drug development.

Real-time investigation of the roles of ATP hydrolysis by UvrA and UvrB during DNA damage recognition in nucleotide excision repair.

Nucleotide excision repair (NER) stands out among other DNA repair systems for its ability to process a diverse set of unrelated DNA lesions. In bacteria, NER damage detection is orchestrated by the UvrA and UvrB proteins, which form the UvrA2-UvrB2 (UvrAB) damage sensing complex. The highly versatile damage recognition is accomplished in two ATP-dependent steps. In the first step, the UvrAB complex samples the DNA in search of lesion. Subsequently, the presence of DNA damage is verified within the UvrB-DNA complex after UvrA has dissociated. Although the mechanism of bacterial NER damage detection has been extensively investigated, the role of ATP binding and hydrolysis by UvrA and UvrB during this process remains incompletely understood. Here, we report a pre-steady state kinetics Förster resonance energy transfer (FRET) study of the real-time interaction between UvrA, UvrB, and damaged DNA during lesion detection. By using UvrA and UvrB mutants harboring site-specific mutations in the ATP binding sites, we show for the first time that the dissociation of UvrA from the UvrAB-DNA complex does not require ATP hydrolysis by UvrB. We find that ATP hydrolysis by UvrA is not essential, but somehow facilitates the formation of UvrB-DNA complex, with ATP hydrolysis at the proximal site of UvrA playing a more critical role. Consistent with previous reports, our results indicated that the ATPase activity of UvrB is essential for the formation of UvrB-DNA complex but is not required for the binding of the UvrAB complex to DNA.

Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA.

Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes coronavirus disease 2019 (COVID-19)-in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100% specific and 100% sensitive with a fluorescence readout, and 100% specific and 97% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100% specific and 96% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.

Functional analysis of BPSS2242 reveals its detoxification role in Burkholderia pseudomallei under salt stress.

A bpss2242 gene, encoding a putative short-chain dehydrogenase/oxidoreductase (SDR) in Burkholderia pseudomallei, was identified and its expression was up-regulated by ten-fold when B. pseudomallei was cultured under high salt concentration. Previous study suggested that BPSS2242 plays important roles in adaptation to salt stress and pathogenesis; however, its biological functions are still unknown. Herein, we report the biochemical properties and functional characterization of BPSS2242 from B. pseudomallei. BPSS2242 exhibited NADPH-dependent reductase activity toward diacetyl and methylglyoxal, toxic electrophilic dicarbonyls. The conserved catalytic triad was identified and found to play critical roles in catalysis and cofactor binding. Tyr162 and Lys166 are involved in NADPH binding and mutation of Lys166 causes a conformational change, altering protein structure. Overexpression of BPSS2242 in Escherichia coli increased bacterial survival upon exposure to diacetyl and methylglyoxal. Importantly, the viability of B. pseudomallei encountered dicarbonyl toxicity was enhanced when cultured under high salt concentration as a result of BPSS2242 overexpression. This is the first study demonstrating that BPSS2242 is responsible for detoxification of toxic metabolites, constituting a protective system against reactive carbonyl compounds in B. pseudomallei..

Wss1 homolog from Candida albicans and its role in DNA-protein crosslink tolerance.

Candida albicans is an opportunistic yeast that can cause life-threatening systemic infection in immunocompromised individuals. During infections, C. albicans has to cope with genotoxic stresses generated by the host immune system. DNA-protein crosslink (DPC), the covalent linkage of proteins with DNA, is one type of DNA damages that can be caused by the host immune response. DPCs are bulky lesions that interfere with the progression of replication and transcription machineries, and hence threaten genomic integrity. Accordingly, either a DPC tolerance mechanism or a DPC repair pathway is essential for C. albicans to maintain genomic stability and survive in the host. Here, we identified Wss1 (weak suppressor of Smt3) in C. albicans (CaWss1) using bioinformatics, genetic complementation, and biochemical studies. We showed that CaWss1 promotes cell survival under genotoxic stress conditions that generate DPCs and that the catalytic metalloprotease domain of CaWss1 is essential for its cellular function. Interactions of CaWss1 with Cdc48 and small ubiquitin-like modifier, although not strictly required, contribute to the function of CaWss1 in the suppression of the growth defects under DPC-inducing conditions. This report is the first investigation of the role of CaWss1 in DPC tolerance in C. albicans.

Application of WST-8 based colorimetric NAD(P)H detection for quantitative dehydrogenase assays.

BACKGROUND: The reduction of tetrazolium salts by NAD(P)H to formazan product has been widely used to determine the metabolic activity of cells, and as an indicator of cell viability. However, the application of a WST-8 based assay for the quantitative measurement of dehydrogenase enzyme activity has not been described before. In this study, we reported the application of an assay based on the tetrazolium salt WST-8 for the quantitative measurement of dehydrogenase activity. The assay is performed in a microplate format, where a single endpoint is measured at 450 nm. RESULTS: The optimized dehydrogenase-WST-8 assay conditions, the limit of detection (LOD), accuracy, and precision for measuring NAD(P)H, were demonstrated. The sensitivity of the WST-8 assay for detecting NAD(P)H was 5-fold greater than the spectrophotometric measurement of NAD(P)H absorption at 340 nm (LOD of 0.3 nmole vs 1.7 nmole, respectively). In the dehydrogenase assay, the colorimetric WST-8 method exhibits excellent assay reproducibility with a Z' factor of 0.9. The WST-8 assay was also used to determine dehydrogenase activity in biological samples, and for screening the substrate of uncharacterized short-chain dehydrogenase/oxidoreductase from Burkholderia pseudomallei. CONCLUSION: The results suggest that the WST-8 assay is a sensitive and rapid method for determining NAD(P)H concentration and dehydrogenase enzyme activity, which can be further applied for the high-throughput screening of dehydrogenases.

Mechanisms of opening and closing of the bacterial replicative helicase.

Assembly of bacterial ring-shaped hexameric replicative helicases on single-stranded (ss) DNA requires specialized loading factors. However, mechanisms implemented by these factors during opening and closing of the helicase, which enable and restrict access to an internal chamber, are not known. Here, we investigate these mechanisms in the Escherichia coli DnaB helicase•bacteriophage λ helicase loader (λP) complex. We show that five copies of λP bind at DnaB subunit interfaces and reconfigure the helicase into an open spiral conformation that is intermediate to previously observed closed ring and closed spiral forms; reconfiguration also produces openings large enough to admit ssDNA into the inner chamber. The helicase is also observed in a restrained inactive configuration that poises it to close on activating signal, and transition to the translocation state. Our findings provide insights into helicase opening, delivery to the origin and ssDNA entry, and closing in preparation for translocation.

Crystal structure of the flavin reductase of Acinetobacter baumannii p-hydroxyphenylacetate 3-hydroxylase (HPAH) and identification of amino acid residues underlying its regulation by aromatic ligands.

The first step in the degradation of p-hydroxyphenylacetic acid (HPA) is catalyzed by the two-component enzyme p-hydroxyphenylacetate 3-hydroxylase (HPAH). The two components of Acinetobacter baumannii HPAH are known as C1 and C2, respectively. C1 is a flavin reductase that uses NADH to generate reduced flavin mononucleotide (FMNH-), which is used by C2 in the hydroxylation of HPA. Interestingly, although HPA is not directly involved in the reaction catalyzed by C1, the presence of HPA dramatically increases the FMN reduction rate. Amino acid sequence analysis revealed that C1 contains two domains: an N-terminal flavin reductase domain, and a C-terminal MarR domain. Although MarR proteins typically function as transcription regulators, the MarR domain of C1 was found to play an auto-inhibitory role. Here, we report a crystal structure of C1 and small-angle X-ray scattering (SAXS) studies that revealed that C1 undergoes a substantial conformational change in the presence of HPA, concomitant with the increase in the rate of flavin reduction. Amino acid residues that are important for HPA binding and regulation of C1 activity were identified by site-directed mutagenesis. Amino acid sequence similarity analysis revealed several as yet uncharacterized flavin reductases with N- or C-terminal fusions.

Synthesis, structure, and biological evaluation of a platinum-carbazole conjugate.

Cisplatin resistance is caused, in part, by the efficient removal of the helix-distorting cisplatin 1,2-intrastrand cross-links by nucleotide excision repair (NER) machinery. To make a platinum-DNA adduct that causes less helical distortion than the cisplatin 1,2-intrastrand adduct, we designed and synthesized a monofunctional platinum-carbazole conjugate (carbazoplatin). The 2.5 Å crystal structure of carbazoplatin-DNA adduct revealed both the monoplatination of the N7 of a guanine (G) base and the intercalation into two G:C base pairs, while causing a minor distortion of the DNA helix. A 50-mer dsDNA containing a single carbazoplatin lesion was poorly processed by UvrABC endonuclease, the prokaryotic NER machinery that detects helical distortion and performs dual incision around the lesion. Our cell viability assay indicated that the cytotoxic pathways of carbazoplatin might be different from those of cisplatin; carbazoplatin was 5-8 times more cytotoxic than cisplatin against PANC-1 and MDA-MB-231 cancer cell lines.

Movement of the ß-hairpin in the third zinc-binding module of UvrA is required for DNA damage recognition.

Nucleotide excision repair (NER) is distinguished from other DNA repair pathways by its ability to process various DNA lesions. In bacterial NER, UvrA is the key protein that detects damage and initiates the downstream NER cascade. Although it is known that UvrA preferentially binds to damaged DNA, the mechanism for damage recognition is unclear. A ß-hairpin in the third Zn-binding module (Zn3hp) of UvrA has been suggested to undergo a conformational change upon DNA binding, and proposed to be important for damage sensing. Here, we investigate the contribution of the dynamics in the Zn3hp structural element to various activities of UvrA during the early steps of NER. By restricting the movement of the Zn3hp using disulfide crosslinking, we showed that the movement of the Zn3hp is required for damage-specific binding, UvrB loading and ATPase activities of UvrA. We individually inactivated each of the nucleotide binding sites in UvrA to investigate its role in the movement of the Zn3hp. Our results suggest that the conformational change of the Zn3hp is controlled by ATP hydrolysis at the distal nucleotide binding site. We propose a bi-phasic damage inspection model of UvrA in which movement of the Zn3hp plays a key role in damage recognition.

Tyr217 and His213 are important for substrate binding and hydroxylation of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1.

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that contains FAD as a redox-active cofactor. The enzyme catalyzes para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on the enzyme crystal structure, residue His213 is located close to the hydroxyl moiety, whereas Tyr217 is close to the carboxylate group of 3HB. Y217A and Y217S did not show any perturbation of flavin absorption upon addition of 3HB, whereas Y217F has a Kd value for 3HB binding of 7.5 mm, which is ~ 50-fold larger than that found for wild-type enzyme. The results clearly indicate that Tyr217 is necessary for substrate binding. All His213 variants can bind to 3HB with similar affinity as the wild-type enzyme and form C4a-hydroperoxy intermediate. H213S, H213D and H213E produce 2,5-DHB with yields of 28 ± 5%, 52 ± 7% and 92 ± 6%, respectively, whereas H213A cannot catalyze hydroxylation. The results indicate that the interaction between the hydroxyl group of 3HB and residue 213 is important for substrate hydroxylation. Interestingly, the hydroxylation rate constant of H213E (35 s(-1) ) is similar to that of wild-type enzyme (36 s(-1) ) and this variant has an efficiency of hydroxylation (92 ± 6%) similar to the wild-type enzyme (86 ± 2%). Difference spectra of enzyme-bound substrate suggest that 3HB binds to H213E in the phenolic form. The results indicate that His213 and Glu213 in H213E may act as a catalytic base to initiate the substrate deprotonation and facilitate the electrophilic aromatic substitution of 3HB.

The reaction kinetics of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1 provide an understanding of the para-hydroxylation enzyme catalytic cycle.

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that catalyzes the para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on results from stopped-flow spectrophotometry, the reduced enzyme-3HB complex reacts with oxygen to form a C4a-peroxy flavin with a rate constant of 1.13 ± 0.01 × 10(6) m(-1) s(-1) (pH 8.0, 4 °C). This intermediate is subsequently protonated to form a C4a-hydroperoxyflavin with a rate constant of 96 ± 3 s(-1). This step shows a solvent kinetic isotope effect of 1.7. Based on rapid-quench measurements, the hydroxylation occurs with a rate constant of 36 ± 2 s(-1). 3HB6H does not exhibit substrate inhibition on the flavin oxidation step, a common characteristic found in most ortho-hydroxylation enzymes. The apparent kcat at saturating concentrations of 3HB, NADH, and oxygen is 6.49 ± 0.02 s(-1). Pre-steady state and steady-state kinetic data were used to construct the catalytic cycle of the reaction. The data indicate that the steps of product release (11.7 s(-1)) and hydroxylation (36 ± 2 s(-1)) partially control the overall turnover.

Small-angle X-ray scattering reveals architecture and A2B2 stoichiometry of the UvrA-UvrB DNA damage sensor.

The UvrA-UvrB (AB) protein complex operates in the bacterial nucleotide excision repair pathway as the main sensor of DNA damage. Crystallographic analysis of the AB complex revealed a linear UvrB-UvrA-UvrA-UvrB arrangement of subunits with an internal two-fold axis that became incorporated into the crystal. Here, we have used small-angle X-ray scattering (SAXS) to show close correspondence between the crystal structure and the entity in solution. This result confirms the number and disposition of subunits in the crystallographic model and rules out other possible arrangements suggested by packing in the crystal. The current SAXS analysis failed to detect significant changes to the structure as a function of nucleotide.