Rational regulation of reaction specificity of nitrilase for efficient biosynthesis of 2-chloronicotinic acid through a single site mutation

Nitrilase-catalyzed hydrolysis of 2-chloronicotinonitrile (2-CN) is a promising approach for efficient synthesis of 2-chloronicotinic acid (2-CA). Development of nitrilase with ideal catalytic properties is crucial for the biosynthetic route with industrial potentail. Herein, a nitrilase from Rhodococcus zopfii ( Rz NIT), which showed much higher hydration activity than hydrolysis activity, was designed for efficient hydrolysis of 2-CN. Two residues (N165 and W167) significantly affecting the reaction specificity were precisely identified. By tuning these two residues, a single mutation of W167G with abolished hydration activity and 20-fold improved hydrolysis activity was obtained. Molecular dynamics simulation and molecular docking revealed that the mutation generated a larger binding pocket, causing the substrate 2-CN bound more deeply in the pocket and the formation of delocalized π bond between the residues W190 and Y196, which reduced the negative influence of steric hindrance and electron effect caused by chlorine substituent. With mutant W167G as biocatalyst, 100 mM 2-CN was exclusively converted into 2-CA within 16 h. The study provides useful guidance in nitrilase engineering for simultaneous improvement of reaction specificity and catalytic activity, which are highly desirable in value-added carboxylic acids production from nitriles hydrolysis. Importance 2-CA is an important building block for agrochemicals and pharmaceuticals with rapid increase in demand in recent years. It is currently manufactured from 3-cyanopyridine by chemical methods. However, during the final step of 2-CN hydrolysis under high temperature and strong alkaline conditions, by-product 2-CM was generated except for the target product, leading to low yield and tedious separation steps. Nitrilase-mediated hydrolysis is regarded as a promising alternative for 2-CA production, which proceeds under mild conditions. Nevertheless, nitrilase capable of efficient hydrolysis of 2-CN was not reported till now, since the enzymes showed either extremely low activity or surprisingly high hydration activity towards 2-CN. Herein, the reaction specificity of Rz NIT was precisely tuned through a single site mutation. The mutant exhibited remarkably enhanced hydrolysis activity without formation of by-products, providing a robust biocatalyst for 2-CA biosynthesis with industrial potential.

Reversing insecticide resistance with allelic-drive in Drosophila melanogaster

A recurring target-site mutation identified in various pests and disease vectors alters the voltage gated sodium channel (vgsc) gene (often referred to as knockdown resistance or kdr) to confer resistance to commonly used insecticides, pyrethroids and DDT. The ubiquity of kdr mutations poses a major global threat to the continued use of insecticides as a means for vector control. In this study, we generate common kdr mutations in isogenic laboratory Drosophila strains using CRISPR/Cas9 editing. We identify differential sensitivities to permethrin and DDT versus deltamethrin among these mutants as well as contrasting physiological consequences of two different kdr mutations. Importantly, we apply a CRISPR-based allelic-drive to replace a resistant kdr mutation with a susceptible wild-type counterpart in population cages. This successful proof-of-principle opens-up numerous possibilities including targeted reversion of insecticide-resistant populations to a native susceptible state or replacement of malaria transmitting mosquitoes with those bearing naturally occurring parasite resistant alleles.

Revertant Mosaicism in Epidermolysis Bullosa

Epidermolysis bullosa (EB) is a group of genetic blistering diseases characterized by mechanically fragile skin and mucocutaneous involvement. Historically, disease management has focused on supportive care. The development of new genetic, cellular, and recombinant protein therapies has shown promise, and this review summarizes a unique gene and cell therapy phenomenon termed revertant mosaicism (RM). RM is the spontaneous correction of a disease-causing mutation. It has been reported in most EB subtypes, some with relatively high frequency, and has been observed in both keratinocytes and fibroblasts. RM manifests as identifiable patches of unaffected, blister-resistant skin and can occur through a variety of molecular mechanisms, including true back mutation, intragenic crossover, mitotic gene conversion, and second-site mutation. RM cells represent a powerful autologous platform for therapy, and leveraging RM cells as a therapeutic substrate may avoid the inherent mutational risks of gene therapy/editing. However, further examination of the genomic integrity and long-term functionality of RM-derived cells, as well in vivo testing of systemic therapies with RM cells, is required to realize the full therapeutic promise of naturally occurring RM in EB.

AHSA1 is a promising therapeutic target for cellular proliferation and proteasome inhibitor resistance in multiple myeloma

Background Currently, multiple myeloma (MM) is still an incurable plasma cell malignancy in urgent need of novel therapeutic targets and drugs. Methods Bufalin was known as a highly toxic but effective anti-cancer compound. We used Bufalin as a probe to screen its potential targets by proteome microarray, in which AHSA1 was the unique target of Bufalin. The effects of AHSA1 on cellular proliferation and drug resistance were determined by MTT, western blot, flow cytometry, immunohistochemistry staining and xenograft model in vivo. The potential mechanisms of Bufalin and KU-177 in AHSA1/HSP90 were verified by co-immunoprecipitation, mass spectrometry, site mutation and microscale thermophoresis assay. Results AHSA1 expression was increased in MM samples compared to normal controls, which was significantly associated with MM relapse and poor outcomes. Furthermore, AHSA1 promoted MM cell proliferation and proteasome inhibitor (PI) resistance in vitro and in vivo. Mechanism exploration indicated that AHSA1 acted as a co-chaperone of HSP90A to activate CDK6 and PSMD2, which were key regulators of MM proliferation and PI resistance respectively. Additionally, we identified AHSA1-K137 as the specific binding site of Bufalin on AHSA1, mutation of which decreased the interaction of AHSA1 with HSP90A and suppressed the function of AHSA1 on mediating CDK6 and PSMD2. Intriguingly, we discovered KU-177, an AHSA1 selective inhibitor, and found KU-177 targeting the same site as Bufalin. Bufalin and KU-177 treatments hampered the proliferation of flow MRD-positive cells in both primary MM and recurrent MM patient samples. Moreover, KU-177 abrogated the cellular proliferation and PI resistance induced by elevated AHSA1, and decreased the expression of CDK6 and PSMD2. Conclusions We demonstrate that AHSA1 may serve as a promising therapeutic target for cellular proliferation and proteasome inhibitor resistance in multiple myeloma.

Novel Insights for PI3KC3 in Mediating Lipid Accumulation in Yellow Catfish Pelteobagrus Fulvidraco

In this study, the transcriptional regulation of PI3KC3 by three transcript factors (PPARγ, PPARα and STAT3) and the potential role of PI3KC3 in mediating lipid accumulation were determined in yellow catfish Pelteobagrus fulvidraco. The 5’-deletion assay, overexpression assay, site-mutation assay and electrophoretic mobility shift assay suggested that PPARα, PPARγ and STAT3 negatively regulated the promoter activity of pi3kc3. Moreover, the transcriptional inactivation of pi3kc3 was directly mediated by PPARα and PPARγ under fatty acid (FA) treatment. Using primary hepatocytes from yellow catfish, FA incubation significantly increased triacylglyceride (TG), NEFA content, the mRNA level of pparα, pparγ, stat3 and dnmt3b, the protein level of PPARα, PPARγ and STAT3, and the methylation level of pi3kc3, but significantly reduced the mRNA and protein level of PI3KC3. Our findings offer new insights into the mechanisms for transcriptional regulation of PI3KC3 and for PI3KC3-mediated lipid accumulation in fish.

Mosquito Species Composition And Insecticide Resistance Status of Anopheles Arabiensis In Itang Special Woreda, Gambella, Southwestern Ethiopia

Introduction: Anopheles arabiensis, member species of the Anopheles gambiae complex, is the primary vector of malaria widely distributed in Ethiopia. Anopheles funestus, An. pharoensis and An. nili are secondary vectors occurring with limited distribution in the country. Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) are pillars for the interventions against malaria control and elimination efforts in Ethiopia. However, the emergence and widespread of insecticide resistance in the major malaria vector, An. arabiensis, might compromise the efforts of the country. The aim of this study was to investigate composition of mosquito species and insecticide resistance status of An. arabiensis in Itang special woreda (district), Gambella, southwestern Ethiopia.Materials and methods: Adult mosquitoes were sampled from September 2020 to Feburary 2021 using Centers for Disease Control and Prevention (CDC) light trap and Pyrethrum Spray Catch (PSC). Moreover, mosquito larvae were also collected from different breeding sites and reared to adults to assess susceptibility status of populations of An. gambiae s.l. in the study area. Susceptibility tests were conducted on two to three days old non blood fed female An. gambiae s.l using insecticide impregnated papers with deltamethrin (0.05%), alpha-cypermethrin (0.05%), propoxur (0.1%), pirimiphos-methyl (0.25%) and bendiocarb (0.1%) following World Health Organization (WHO) standard susceptibility test procedure. Molecular diagnostics were done for the identification of member species of An. gambiae s.l and detection of knockdown resistance (kdr) allele using species specific polymerase chain reaction (PCR) and allele specific PCR. Results: In total, 468 adult mosquitoes were collected from different houses. Culex mosquitoes were the most dominant (80.4%) followed by Anopheles mosquitoes. Three species of Anopheles mosquitoes (An. coustani, An. pharoensis, and An. gambiae (s.l.)) were identified, of which An. coustani was the dominant (8.1%) species. WHO bioassay tests revealed that the populations of An. gambiae s.l in the study area are resistant against alpha-cypermethrin and deltamethrin whereas, susceptible to bendiocarb, pirimiphos-methyl and propoxur. Out of the total 86 An. gambiae s.l specimens assayed, 79 (92%) successfully amplified and identified as An. arabiensis. West African Kdr (L1014F) mutation was detected with high Kdr allele frequency ranging from 67-88%.Conclusion: The detection of target site mutation, kdr L1014F allele, coupled with the phenotypic resistance against alpha-cypermethrin and deltamethrin call for continuous resistance monitoring.

Novel mutations in PDE6A and CDHR1 cause retinitis pigmentosa in Pakistani families

AIM: To investigate the genetic basis of autosomal recessive retinitis pigmentosa (arRP) in two consanguineous/ endogamous Pakistani families. METHODS: Whole exome sequencing (WES) was performed on genomic DNA samples of patients with arRP to identify disease causing mutations. Sanger sequencing was performed to confirm familial segregation of identified mutations, and potential pathogenicity was determined by predictions of the mutations’ functions. RESULTS: A novel homozygous frameshift mutation [NM_000440.2:c.1054delG, p. (Gln352Argfs*4); Chr5:g.149286886del (GRCh37)] in the PDE6A gene in an endogamous family and a novel homozygous splice site mutation [NM_033100.3:c.1168-1G>A, Chr10:g.85968484G>A (GRCh37)] in the CDHR1 gene in a consanguineous family were identified. The PDE6A variant p. (Gln352Argfs*4) was predicted to be deleterious or pathogenic, whilst the CDHR1 variant c.1168-1G>A was predicted to result in potential alteration of splicing. CONCLUSION: This study expands the spectrum of genetic variants for arRP in Pakistani families.