CRISPR/Cas-mediated knockdown of vacuolar invertase gene expression lowers the cold-induced sweetening in potatoes.

MAIN CONCLUSION: VInv gene editing in potato using CRISPR/Cas9 resulted in knockdown of expression and a lower VInv enzymatic activity resulting in a decrease in post-harvest cold-storage sugars formation and sweetening in potatoes. CRISPR-Cas9-mediated knockdown of vacuolar invertase (VInv) gene was carried out using two sgRNAs in local cultivar of potato plants. The transformation efficiency of potatoes was found to be 11.7%. The primary transformants were screened through PCR, Sanger sequencing, digital PCR, and ELISA. The overall editing efficacy was determined to be 25.6% as per TIDE analysis. The amplicon sequencing data showed maximum indel frequency for potato plant T12 (14.3%) resulting in 6.2% gene knockout and 6% frame shift. While for plant B4, the maximum indel frequency of 2.0% was found which resulted in 4.4% knockout and 4% frameshift as analyzed by Geneious. The qRT-PCR data revealed that mRNA expression of VInv gene was reduced 90-99-fold in edited potato plants when compared to the non-edited control potato plant. Following cold storage, chips analysis of potatoes proved B4 and T12 as best lines. Reducing sugars' analysis by titration method determined fivefold reduction in percentage of reducing sugars in tubers of B4 transgenic lines as compared to the control. Physiologically genome-edited potatoes behaved like their conventional counterpart. This is first successful report of knockdown of potato VInv gene in Pakistan that addressed cold-induced sweetening resulting in minimum accumulation of reducing sugars in genome edited tubers.

Allele frequency data of 15 autosomal STRs in Arain population of Pakistan.

Fifteen autosomal STRs were evaluated using Identifiler plus kit in 121 Arain samples of Pakistan. The highly discriminatory locus was D2S1338 with value of 0.968. Allele 8 at TPOX was the most frequent with value of 0.467. No significant deviations from Hardy-Weinberg equilibrium were seen except D3S1358 and D18S51. Combined power of discrimination, combined power of exclusion, and combined matching probability were obtained as 0.9999999999999999925, 0.99999815, and 7.4897 × -18, respectively. Population differentiation test demonstrated significant differences between Arain and geographically distinct populations.

Enhancing the resilience of transgenic cotton for insect resistance.

BACKGROUND: The efficacy of Bt crystal proteins has been compromised due to their extensive utilization in the field. The second-generation Bt vegetative insecticidal proteins could be the best-suited alternative to combat resistance build-up due to their broad range affinity with midgut receptors of insects. MATERIAL AND RESULTS: The codon-optimized synthetic vegetative insecticidal proteins (Vip3Aa) gene under the control of CaMV35S promoter was transformed into a locally developed transgenic cotton variety (CKC-01) expressing cry1Ac and cry2A genes. Transformation efficiency of 1.63% was recorded. The highest Vip3Aa expression (51.98-fold) was found in MS3 transgenic cotton plant. Maximum Vip3Aa protein concentration (4.23 µg/mL) was calculated in transgenic cotton plant MS3 through ELISA. The transgenic cotton plant (MS3) showed one copy number on both chromatids in the homozygous form at chromosome 8 at the telophase stage. Almost 99% mortality of H. armigera was recorded in transgenic cotton plants expressing double crystal proteins pyramided with Vip3Aa gene as contrasted to transgenic cotton plant expressing only double crystal protein with 70% mortality. CONCLUSIONS: The results obtained during this study suggest that the combination of Bt cry1Ac, cry2A, and Vip3Aa toxins is the best possible alternative approach to combat chewing insects.

Enhanced expression of plasma membrane intrinsic protein 2 improves cotton fiber length in Gossypium arboreum.

BACKGROUND: Gossypium arboreum is a cotton crop native to tropical and subtropical regions that are naturally resistant to cotton leaf curl virus (CLCuV). However, its cultivation is unfavorable due to the lower quality and shorter fiber length of cotton when compared to the market leading G. hirsutum. Plasma membrane intrinsic protein 2 (PIP2) is an aquaporin responsible for the transport of water and small molecules across cellular membranes. This fluid transport influences cell elongation and cotton fibre development. Hence, increased PIP2 expression may yield plants with enhanced fiber qualities including length. METHODS AND RESULTS: To test this hypothesis, G. arboreum was transformed with a PIP2 gene construct (35SCpPIP2) using the Agrobacterium-mediated shoot apex cutting method. Relative expression of the CpPIP2 gene in transgenic plants increased up to 35-fold when compared with non-transgenic controls. Transgenic plants displayed a corresponding increase of staple length (up to 150%) when compared with non-transgenic controls. Transgene integration was examined using FISH and karyotyping and revealed the presence of a single transgene located on chromosome 6. CONCLUSION: Since G. arboreum is naturally whitefly and CLCuV resistant, this improvement of fiber length evidenced for CpPIP2 transgenic plants renders their crop production more economically viable.

Novel Coronavirus: A Review from Origin to Current Status of Therapeutic Strategies.

Coronaviruses (CoVs) are continuously emerging, highly transmissible, and pathogenic agents that primarily target the human respiratory system. Previous outbreaks of severe acute respiratory syndrome-CoV and Middle East respiratory syndrome-CoV remain life-threatening and global public health concerns. A novel CoV outbreak that occurred in December 2019 in Wuhan, China was declared a pandemic outbreak that has since killed millions of individuals worldwide. Rapid transmission, genetic variations, and unavailability of specific therapeutic drugs are major factors that led to this alarming and deadly situation. Currently, > 200 clinical vaccine trials are underway to combat infection. This review summarizes reports related to CoV origin, genetic variations, drug options, status of nine vaccines that were in phase III trials, and novel therapies including convalescent plasma and stem cell treatment.

Dengue Vaccines: Ongoing Challenges and Current Status in the Advancement of Different Candidates.

Dengue is a vector-borne highly systemic infectious disease of the tropical and subtropical countries and is devastating millions of lives worldwide. It may be self-eliminated like a mild fever or may cause life-threatening fatal complications as dengue hemorrhagic fever and dengue shock syndrome. The lack of specific and effective antiviral drugs and vaccines amplify its transmission rate across the world. The development of the dengue vaccine has been an ambitious task due to the presence of four different dengue serotypes capable of carrying antibody enhancement complex mechanisms. In this review, we have summarized the ongoing challenges in the construction of a dengue vaccine and the current status of the vaccine development. Limited knowledge of immune responses against dengue infection, lack of human or animal model of disease, and suboptimal assay strategies to detect immune responses after infection or vaccination, are some barriers to vaccine and drug development. A tetravalent vaccine with low cost, high efficiency, and capable of eliciting immune responses against all four serotypes is needed to minimize the epidemics. Currently, only one live attenuated chimeric dengue vaccine, the CYD Dengue Vaccine, has completed its third phase and has been licensed. DENVax and TetraVax-DV-TV003 (TV003) are in the third phase while others are still in the first trial phase.

Method for efficient soluble expression and purification of recombinant human interleukin-15.

Periplasmic expression of recombinant proteins ensures the production of biologically active proteins in a correctly folded state with several key advantages. This research focused on the in-frame cloning of rhIL-15 in pET-20 (+) vector with pelB-leader sequence to direct the protein to the bacterial periplasm. The target construct periplasmic expression was evaluated in four strains, BL21 (DE3), BL21 (DE3) pLysS, Rosetta 2 (DE3) and Rosetta-gami 2 (DE3). Soluble periplasmic expression of IL-15 was highest in Rosetta-gami 2 (DE3) followed by Rossetta 2 (DE3) whereas negligible expression was observed with rest of two expression host. Best expression clone was selected for purification by dye ligand affinity chromatography. Purified rhIL-15 was characterized by SDS-PAGE, Western blotting and SEC-HPLC. This is the first report of functional recombinant human interleukin-15 being expressed and purified with yield of 120 mg/L in the periplasmic space of E. coli.

Development of broad-spectrum and sustainable resistance in cotton against major insects through the combination of Bt and plant lectin genes.

KEY MESSAGE: Second generation Bt insecticidal toxin in comibination with Allium sativum leaf agglutinin gene has been successfully expressed in cotton to develop sustainable resistance against major chewing and sucking insects. The first evidence of using the Second-generation Bt gene in combination with Allium sativum plant lectin to develop sustainable resistance against chewing and sucking insects has been successfully addressed in the current study. Excessive use of Bt δ-endotoxins in the field is delimiting its insecticidal potential. Second-generation Bt Vip3Aa could be the possible alternative because it does not share midgut receptor sites with any known cry proteins. Insecticidal potential of plant lectins against whitefly remains to be evaluated. In this study, codon-optimized synthetic Bt Vip3Aa gene under CaMV35S promoter and Allium sativum leaf agglutinin gene under phloem-specific promoter were transformed in a local cotton variety. Initial screening of putative transgenic cotton plants was done through amplification, histochemical staining and immunostrip assay. The mRNA expression of Vip3Aa gene was increased to be ninefold in transgenic cotton line L6P3 than non-transgenic control while ASAL expression was found to be fivefold higher in transgenic line L34P2 as compared to non-transgenic control. The maximum Vip3Aa concentration was observed in transgenic line L6P3. Two copy numbers in homozygous form at chromosome number 9 and one copy number in hemizygous form at chromosome number 10 was observed in transgenic line L6P3 through fluorescent in situ hybridization. Significant variation was observed in transgenic cotton lines for morphological characteristics, whereas physiological parameters of plants and fiber characteristics (as assessed by scanning electron microscopic) remained comparable in transgenic and non-transgenic cotton lines. Leaf-detach bioassay showed that all the transgenic lines were significantly resistant to Helicoverpa armigera showing mortality rates between 78% and 100%. Similarly, up to 95% mortality of whiteflies was observed in transgenic cotton lines when compared with non-transgenic control lines.

Evaluation the effect of subchronic feeding of transgenic cotton line (CKC1) on the faecal microbiota of albino rabbits.

Recent studies have demonstrated a strong relationship between the intestinal microbiota and the host health. As such, consumers are increasingly becoming more concerned about the potential effect of certain foods/feeds, particularly of transgenic origin on the gut microbiota. Although the European Food Safety Authority has recommended in their guidelines, to study the effect of transgenic food/feed on host-microbiota, yet, few studies have focused on the evaluation of such effects mainly due to culturing difficulties. Therefore, this study was intended to evaluate the potential adverse effects of transgenic diet consumption on some specific gut microflora (Lactobacillus group, Bifidobacterium genus, Escherichia coli subgroup and Enterococcus genus) of rabbits. A total of forty-eight rabbits were randomly assigned into four groups and fed a diet containing a variable proportion of transgenic cottonseeds at 0, 20, 30 and 40% inclusion level, respectively. Changes in the specific or total faecal bacterial population were monitored at five different experimental stages (i.e. 0, 45, 90, 135 and 180 days) using both the traditional plate count method (TM) and quantitative real-time PCR (qPCR). No significant differences (p > .05) were observed concerning numbers of specific bacteria or total bacteria between the control and experimental groups, though qPCR showed numerically higher values in terms of 16S rRNA gene copies as compared to the values obtained from TM. However, such numerical differences were biologically insignificant (p > .05). Similarly, no significant variations were noticed in the calculated B/E (log10 copies of Bifidobacterium per g faces/log10 copies of E. coli genome per g faeces) ratios in all the groups. All the ratios were in the range of 1.24 to 1.30 throughout the experiment, indicating a good balance of intestinal microflora and greater resistance to intestinal disorders. It is therefore concluded that feeding transgenic cottonseeds could not adversely affect the gut microflora of rabbits during a long-term study.

Kashmiris phylogenetic depictions through uniparental and biparental genetic markers.

In this study, the Kashmiri population was genetically screened for fifteen autosomal STRs, seventeen Y-chromosomal STRs and mitochondrial DNA control region SNPs. Autosomal STRs exhibited that D2S1338 was the most discriminatory locus with value of 0.963 whereas combine power of discrimination was 0.999999999999999. On the other hand, analysis of Y-chromosomal STRs generated 59 distinct haplotypes of which 52 were unique with haplotype diversity value of 0.99752. Mitochondrial D-loop analysis resulted that haplogroups M and U were found most prevalent with 25% each. Haplotypic data of Y-STRs was submitted to YHRD under accession number YA004153 whereas mitochondrial DNA control region (CR) sequences were submitted to EMPOP with accession number EMP00683.

Phylogenetic analysis and haplotype diversity in Christian residents of Lahore, Pakistan, using 17 Y-chromosomal STR loci.

This population data pertains to 250 unrelated male residents of the Christian population of Lahore, Pakistan. AmpF/STR®Yfiler PCR amplification kit was utilized for evaluation of 17 Y-chromosomal STRs loci. Ancestral lineages and parameters of forensic importance were examined leading to recognition of 135 unique out of 175 total haplotypes with diversity value of 0.991. All Y-STRs portrayed high discrimination power where DYS385 depicted the maximum value of 0.917. Multidimensional Scaling (MDS) and Analysis of Molecular Variance (AMOVA) were generated using YHRD (Y-Chromosome STR Haplotype Reference Database) tools. A pair-wise genetic distance comparison using Rst, Fst, and associated p values lead to the conclusion that studied Christian community was significantly demarcated from the rest of the global populations.

Assessing the fate of recombinant plant DNA in rabbit's tissues fed genetically modified cotton.

Various feeding studies have been conducted with the different species of animals to evaluate the possible transfer of transgenic DNA (tDNA) from genetically modified (GM) feed into the animal tissues. However, the conclusions drawn from most of such studies are sometimes controversial. Thus, in the present study, an attempt has been made to evaluate the fate of tDNA in rabbits raised on GM cotton-based diet through PCR analysis of the DNA extracted specifically from blood, liver, kidney, heart and intestine (jejunum). A total of 48 rabbits were fed a mixed diet consisting variable proportions of transgenic cottonseeds meal (i.e. 0% w/w, 20% w/w, 30% w/w and 40% w/w) for 180 days. The presence of transgenic DNA fragments (Cry1Ac, Cry2A and CP4 EPSPS) or plant endogenous gene (Sad1) was traced in those specific tissues and organs. The presence of ß-actin (ACTB) was also monitored as an internal control. Neither the transgenic fragments (459 bp of Cry1Ac gene, 167 bp of Cry2A gene and111 bp of CP4 EPSPS gene) nor cotton endogenous reference gene (155 bp of Sad1) could be detected in any of the DNA samples extracted from the rabbit's tissues in both control and transgenic groups. However, 155 bp fragment of the rabbit's reference gene (ACTB) was recovered in all the DNA samples extracted from rabbit tissues. The results obtained from this study revealed that both plant endogenous and transgenic DNA fragments have same fate in rabbit's tissues and were efficiently degraded in the gastrointestinal tract (GIT).

Effect of dietary supplementation of recombinant Cry and Cp4 epsps proteins on haematological indices of growing rabbits.

Genetically modified (GM) crops expressing insect resistance and herbicide tolerance provide a novel approach for improved crop production but their advent at the same time presents serious challenges in terms of food safety. Although prevailing scientific proof has suggested that transgenic crops are analogous to their conventional counterparts, their use in human and animal diet gave rise to emotional public discussion. A number of studies had been conducted to evaluate the potential unintended effects of transgenic crops expressing single transgene, but very few studies for those with multiple transgenes. As the crops with single and multiple transgenes could impart different effects on non-target organisms, thus, risk evaluation of transgenic crops expressing more than one transgene is required to declare their biosafety. The present study was therefore designed to assess the effects of different levels of dietary transgenic cottonseed expressing recombinants proteins produced by Cry1Ac, Cry2A and Cp4epsps genes on haematological indices of growing rabbits. A total of 48 rabbits were assigned to four dietary treatments containing different levels of transgenic cottonseeds (i.e., 0% w/w, 20% w/w, 30% w/w and 40% w/w) with 0% w/w serving as control. Haematological parameters were measured at periodic intervals (0, 45, 90, 135 and 180) days. No significant (p > 0.05) dose-dependent effects were observed in most of the haematological parameters evaluated. Though, significant differences (p < 0.05) were recorded in the level of MCHC, MCH and HCT in some of experimental male and female rabbits, yet, they were not biologically significant, as all the differences were within the normal reference values. Our study suggested that feeding transgenic cottonseed of up to 40% could not adversely affect rabbit's haematological profile. However, further study needs to be conducted with different cotton genotypes expressing both single and polygenic traits before recommending the utilization of transgenic cottonseed in routine livestock feeding.

Male individualization using 12 rapidly mutating Y-STRs in Araein ethnic group and shared paternal lineage of Pakistani population.

A multiplex assay has been developed with newly designed primer sets comprising high mutation rate 12 RM Y-STR markers (DYS570, DYF399S1, DYS547, DYS612, DYF387S1, DYS449, DYS576, DYS5626, DYF403S1 (a + b), DYS627, DYS526, and DYF404S1). Rapidly mutating Y-STRs were evaluated in 167 male individuals among 97 were unrelated from Araein ethnic group and 70 belonged to shared paternal lineage including 20 pairs of father-son and 15 pairs of brother-brother relationship collected from Punjabi population of Pakistan. Forensic competency parameters were implemented for each marker and exceptionally significant results found wherein polymorphism information content (PIC) was in range of 0.7494 (DYS576) to 0.8994 (DYS627). Samples were also analyzed with Y-filer kit for comparison and marked differentiations observed. Haplotype discrimination capacity was 100% as no haplotype shared among all the unrelated individuals of same ethnic group as compared to 17 Y-filer loci (78%). While in closely related males, discrimination capacity was 96.4% with haplotype diversity value of 0.98. Resulted high mutation rate 1 × 10-2 to 7.14 × 10-2 as compared to Y-filer (1 × 10-4 to 1 × 10-3) manifested the power of RM Y-STRs for considering absolute individualization of interrelated and unrelated male individuals. However, multiplex assay would be useful for male discrimination in mixed DNA specimen, azoospermic males, and multiple male DNA contributors in sexual assault cases and mass disasters victim's identification as well as anthropological studies.

Evidence of genetically diverse virulent mating types of Phytophthora capsici from Capsicum annum L.

Chili pepper (Capsicum annum L.) is an important economic crop that is severely destroyed by the filamentous oomycete Phytophthora capsici. Little is known about this pathogen in key chili pepper farms in Punjab province, Pakistan. We investigated the genetic diversity of P. capsici strains using standard taxonomic and molecular tools, and characterized their colony growth patterns as well as their disease severity on chili pepper plants under the greenhouse conditions. Phylogenetic analysis based on ribosomal DNA (rDNA), ß-tubulin and translation elongation factor 1α loci revealed divergent evolution in the population structure of P. capsici isolates. The mean oospore diameter of mating type A1 isolates was greater than that of mating type A2 isolates. We provide first evidence of an uneven distribution of highly virulent mating type A1 and A2 of P. capsici that are insensitive to mefenoxam, pyrimorph, dimethomorph, and azoxystrobin fungicides, and represent a risk factor that could ease outpacing the current P. capsici management strategies.

Genetic variations of 15 autosomal and 17 Y-STR markers in Sindhi population of Pakistan.

This study comprises genetic characterization of 15 autosomal and 17 Y-chromosomal STR loci in 103 unrelated male inhabitants of the Sindhi population to establish its lineage and parameters of forensic interest. The examined autosomal STRs revealed high combined power of discrimination, combined power of exclusion, and the combined matching probability as 0.99999999999999999042580, 0.9999977141, and 9.5742 × 10-18, respectively. A total of 89 unique haplotypes were obtained, of which 84 were observed once with a haplotype diversity value of 0.999677. The resulted Y-STR haplotypes exhibited a high degree of geographical demarcation by comparing with other populations at the local and global levels.

Development of new PCR multiplex system by the simultaneous detection of 10 miniSTRs, SE33, Penta E, Penta D, and four Y-STRs.

The 18 loci multiplex system has been instigated for co-amplification and fluorescent detection of Amelogenin and 17 STRs, including 10 MiniSTRs (CSF1PO, D18S51, D7S820, D2S1338, TPOX, D13S317, FGA, D5S818, D21S11, D16S539), SE33, Penta E, Penta D, and four Y-STRs (DYS385a/b, DYS438, DYS392). This multiplex system was developed for the simultaneous analysis of compromised DNA samples, Y-amelogenin marker mutation, motherless paternity issues where single allele sharing occurs at autosomal STRs in unrelated individuals, and other complex forensic cases. Selection of loci, primers, and allelic ladders were designed and created in-house with a design strategy to work in this multiplex. The multiplex system was evaluated by sensitivity, specificity, stability, precision and accuracy, case-type samples, mixture studies, PCR-based and population distribution studies to establish the robustness and reliability of the system as the current requirements of the forensic case work. Among all the markers evaluated for this study, 209 alleles including 44 variants were observed with combined power of discrimination, combined power of exclusion, and the combined probability of matching calculated as 0.999999999999999999893916339344, 0.999993816173890, and 5.90019 × 10-19, respectively. Due to highly polymorphic characteristics of these loci particularly SE33 and Penta E which are most discriminatory (PD = 0.991 and 0.983, respectively) in the Pakistani population, this multiplex would be highly valuable for individual identification in complex forensic cases and paternity issues as well as population database.

Risk assessment of Bt crops on the non-target plant-associated insects and soil organisms.

Transgenic plants containing Bacillus thuringiensis (Bt) genes are being cultivated worldwide to express toxic insecticidal proteins. However, the commercial utilisation of Bt crops greatly highlights biosafety issues worldwide. Therefore, assessing the risks caused by genetically modified crops prior to their commercial cultivation is a critical issue to be addressed. In agricultural biotechnology, the goal of safety assessment is not just to identify the safety of a genetically modified (GM) plant, rather to demonstrate its impact on the ecosystem. Various experimental studies have been made worldwide during the last 20 years to investigate the risks and fears associated with non-target organisms (NTOs). The NTOs include beneficial insects, natural pest controllers, rhizobacteria, growth promoting microbes, pollinators, soil dwellers, aquatic and terrestrial vertebrates, mammals and humans. To highlight all the possible risks associated with different GM events, information has been gathered from a total of 76 articles, regarding non-target plant and soil inhabiting organisms, and summarised in the form of the current review article. No significant harmful impact has been reported in any case study related to approved GM events, although critical risk assessments are still needed before commercialisation of these crops. © 2016 Society of Chemical Industry.

Allele frequency distribution of 10 MiniSTRs in the Pashtun population of Pakistan.

Two hundred individual samples of Pashtun population from Khyber Pakhtunkhwa province of Pakistan were randomly evaluated through 10 MiniSTR loci (CSF1PO, D7S820, TPOX, D18S51, D2S1338, D13S317, FGA, D5S818, D21S11, and D16S539). The PCR product size was reduced in the range of 65 to 280 bp. A total of 112 alleles were observed containing allelic frequency ranging from 0.0025 to 0.4325. Statistical values for forensic and parentage analysis were calculated including combined power of discrimination (PD), combined power of exclusion (PE), and cumulative probability of matching (PM) and equaled to 0.99999999999768, 0.99984944, and 2.33 × 10(-12), respectively. These MiniSTRs show a high degree of polymorphism information content and discriminatory power which would be helpful to resolve forensic cases and establish DNA database for major population groups of Pakistan. In contrast to different populations, significant differences were also observed on these loci.

Chloroplast localization of Cry1Ac and Cry2A protein--an alternative way of insect control in cotton.

BACKGROUND: Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants. RESULTS: Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 µg/g tissue of Cry1Ac and 0.568 µg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein. CONCLUSION: Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.