Tackling Counterfeit Drugs: The Challenges and Possibilities.

Drugs that have been manufactured or packaged fraudulently are referred to as counterfeit/fake/spurious/falsified drugs because they either lack active ingredients or have the incorrect dosages. Counterfeiting of drugs has become a global issue with which the whole world is grappling. The World Health Organization states the frightening figure in which almost 10.5% of the medications worldwide are either subpar or fake. Although developing and low-income countries are the targets of the large-scale drug counterfeiting activities, fake/substandard drugs are also making their way into developed nations including the USA, Canada, and European countries. Counterfeiting of drugs is leading to not only economic loss but is also playing its part in the morbidity and mortality of patients. The recent COVID-19 pandemic fuelled the demand for certain categories of medicines such as antipyretics, remdesivir, corticosteroids, vaccines, etc., thus increasing the demand and manufacture of subpar/fake medicines. This review articulates the current trends and global impact of drug counterfeiting, current and potential measures for its prevention and the role of different stakeholders in tackling the menace of drug counterfeiting.

Chloroplasts evolved an additional layer of translational regulation based on non-AUG start codons for proteins with different turnover rates.

Chloroplasts have evolved from photosynthetic cyanobacteria-like progenitors through endosymbiosis. The chloroplasts of present-day land plants have their own transcription and translation systems that show several similarities with prokaryotic organisms. A remarkable feature of the chloroplast translation system is the use of non-AUG start codons in the protein synthesis of certain genes that are evolutionarily conserved from Algae to angiosperms. However, the biological significance of such use of non-AUG codons is not fully understood. The present study was undertaken to unravel the significance of non-AUG start codons in vivo using the chloroplast genetic engineering approach. For this purpose, stable transplastomic tobacco plants expressing a reporter gene i.e. uidA (GUS) under four different start codons (AUG/UUG/GUG/CUG) were generated and ß-glucuronidase (GUS) expression was compared. To investigate further the role of promoter sequences proximal to the start codon, uidA was expressed under two different chloroplast gene promoters psbA and psbC that use AUG and a non-AUG (GUG) start codons, respectively, and also showed significant differences in the DNA sequence surrounding the start codon. Further, to delineate the role of RNA editing that creates AUG start codon by editing non-AUG codons, if any, which is another important feature of the chloroplast transcription and translation system, transcripts were sequenced. In addition, a proteomic approach was used to identify the translation initiation site(s) of GUS and the N-terminal amino acid encoded when expressed under different non-AUG start codons. The results showed that chloroplasts use non-AUG start codons in combination with the translation initiation site as an additional layer of gene regulation to over-express proteins that are required at high levels due to their high rates of turnover.

Functional Characterization of Pseudoidium neolycopersici Photolyase Reveals Mechanisms Behind the Efficacy of Nighttime UV on Powdery Mildew Suppression.

Powdery mildews can be controlled by brief exposure to ultraviolet (UV) radiation with devastating effect on their developmental stages including conidia germination. The treatment effect can be impaired by subsequent exposure to UV-A/blue light. UV-A/blue light-activated photolyase may be responsible for this and therefore we tested the function of three cryptochrome/photolyase family (CPF)-like genes (OINE01015670_T110144, OINE01000912_T103440, and OINE01005061_T102555) identified in the obligate biotrophic fungus Pseudoidium neolycopersici, the cause of tomato powdery mildew. A photolyase-deficient mutant of Escherichia coli transformed with coding sequence of OINE01000912_T103440 and exposed to brief (UV)-C treatment (peak emission at 254 nm) showed photoreactivation and cell survival when exposed to subsequent blue light, indicating complementation of photolyase activity. In contrast, the same photolyase-deficient E. coli transformed with the coding sequences of other two CPF-like genes did not survive this treatment, even though their expression were confirmed at protein level. This confirmed that OINE01000912_T103440 is a gene encoding photolyase, here named PnPHR1, with functionality similar to the native photolyase in E. coli, and classified as a class I cyclobutane pyrimidine dimer (CPD) photolyase. Modeling of the 634-amino acid sequence of PnPHR1 suggested that it is capable of binding flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF). However, spectroscopic data of the protein produced in an E. coli expression system could only reveal the presence of a reduced form of FAD, i.e., FADH- as an intrinsic chromophore. Within the tested wavelength range of 365-525 nm, the survival of photolyase-deficient mutant E. coli transformed with PnPHR1 showed a broad action spectrum from 365 to 454 nm. This was very similar to the previously characterized action spectrum for survival of P. neolycopersici conidia that had been treated with UV-C. Quantitative RT-PCR revealed that the expression of PnPHR1 in P. neolycopersici conidia was induced by UV-C, and peak expression occurred 4 h after brief UV-C treatment. The expression of PnPHR1 was repressed when incubated in red light after the UV-C treatment, but not when incubated in UV-A/blue light. The results may explain why the disease-reducing effect of short wavelength UV is impaired by exposure to UV-A and blue light.

Wavelength dependent recovery of UV-mediated damage: Tying up the loose ends of optical based powdery mildew management.

Controlled environment chamber experiments at Petri dish level were conducted to examine the wavelength and dose dependent efficacy of ultraviolet (UV) radiation, the recovery action potential of optical radiation applied concomitantly/subsequently to effective UV treatment, and the lapse time between UV treatment and subsequent exposure to recovery wavelength on germination efficiency of Oidium neolycopersici conidia. Conidia of eight- to nine-day-old colonies were dusted on water agar surface in Petri dishes and exposed to UV treatments (without lid). Immediately after UV treatments, Petri dishes were sealed and incubated in darkness or differing optical environments generated using seven different radiation sources (range 290nm to 780nm). Twenty-four hours after UV treatment, fifty conidia from each sample were assessed for germination. Compared to non-UV controls, <10% of the conidia germinated after 30s of exposure to 254nm or 283nm UV and subsequent dark incubation. Conidia germination was almost negligible if the exposure duration increased to 4min. Germination was about 60% with broad spectrum UV after 1min of exposure, and about 35% after 2 to 4min of exposure. There was no reduction of conidia germination with the exposure of ≤4min with 310nm. With the tested wavelength and dose ranges, germination recovery was effective in the 350nm to 500nm range. Germination efficiency of conidia treated with effective UV was significantly higher (>73%) if incubated subsequently in the 350nm to 500nm range (germination recovery). Furthermore, germination recovery depends on the characteristics of UV treatment (wavelength, and duration of exposure) and the lapse time between UV treatment and subsequent exposure to optical radiation in the recovery range. The findings of this study provide key criteria for wavelength selection, combination and application time in the optical radiation range, enabling improved design of optical based management strategies against powdery mildews.

Genome-wide transcriptomic analysis of cotton under drought stress reveal significant down-regulation of genes and pathways involved in fibre elongation and up-regulation of defense responsive genes.

Cotton is an important source of natural fibre used in the textile industry and the productivity of the crop is adversely affected by drought stress. High throughput transcriptomic analyses were used to identify genes involved in fibre development. However, not much information is available on cotton genome response in developing fibres under drought stress. In the present study a genome wide transcriptome analysis was carried out to identify differentially expressed genes at various stages of fibre growth under drought stress. Our study identified a number of genes differentially expressed during fibre elongation as compared to other stages. High level up-regulation of genes encoding for enzymes involved in pectin modification and cytoskeleton proteins was observed at fibre initiation stage. While a large number of genes encoding transcription factors (AP2-EREBP, WRKY, NAC and C2H2), osmoprotectants, ion transporters and heat shock proteins and pathways involved in hormone (ABA, ethylene and JA) biosynthesis and signal transduction were up-regulated and genes involved in phenylpropanoid and flavonoid biosynthesis, pentose and glucuronate interconversions and starch and sucrose metabolism pathways were down-regulated during fibre elongation. This study showed that drought has relatively less impact on fibre initiation but has profound effect on fibre elongation by down-regulating important genes involved in cell wall loosening and expansion process. The comprehensive transcriptome analysis under drought stress has provided valuable information on differentially expressed genes and pathways during fibre development that will be useful in developing drought tolerant cotton cultivars without compromising fibre quality.

Genome-wide transcriptome and proteome analyses of tobacco psaA and psbA deletion mutants.

Photosynthesis in higher land plants is a complex process involving several proteins encoded by both nuclear and chloroplast genomes that require a highly coordinated gene expression. Significant changes in plastid differentiation and biochemical processes are associated with the deletion of chloroplast genes. In this study we report the genome-wide responses caused by the deletion of tobacco psaA and psbA genes coding core components of photosystem I (PSI) and photosystem II (PSII), respectively, generated through a chloroplast genetic engineering approach. Transcriptomic and quantitative proteomic analysis showed the down regulation of specific groups of nuclear and chloroplast genes involved in photosynthesis, energy metabolism and chloroplast biogenesis. Moreover, our data show simultaneous activation of several defense and stress responsive genes including those involved in reactive oxygen species (ROS) scavenging mechanisms. A major finding is the differential transcription of the plastome of deletion mutants: genes known to be transcribed by the plastid encoded polymerase (PEP) were generally down regulated while those transcribed by the nuclear encoded polymerase (NEP) were up regulated, indicating simultaneous activation of multiple signaling pathways in response to disruption of PSI and PSII complexes. The genome wide transcriptomic and proteomic analysis of the ∆psaA and ∆psbA deletion mutants revealed a simultaneous up and down regulation of the specific groups of genes located in nucleus and chloroplasts suggesting a complex circuitry involving both retrograde and anterograde signaling mechanisms responsible for the coordinated expression of nuclear and chloroplast genomes.