Expression of Human Immunodeficiency Virus type 1 (HIV-1) coat protein genes in plants using cotton leaf curl Multan betasatellite-based vector.

It has already been demonstrated that a betasatellite associated with cotton leaf curl Multan virus (CLCuMB) can be used as a plant and animal gene delivery vector to plants. To examine the ability of CLCuMB as a tool to transfer coat protein genes of HIV-1 to plants, two recombinant CLCuMB constructs in which the CLCuMB βC1 ORF was replaced with two HIV-1 genes fractions including a 696 bp DNA fragment related to the HIV-1 p24 gene and a 1501 bp DNA fragment related to the HIV-1 gag gene were constructed. Gag is the HIV-1 coat protein gene and p24 is a component of the particle capsid. Gag and p24 are used for vaccine production. Recombinant constructs were inoculated to Nicotiana glutinosa and N. benthamiana plants in the presence of an Iranian isolate of Tomato yellow leaf curl virus (TYLCV-[Ab]) as a helper virus. PCR analysis of inoculated plants indicated that p24 gene was successfully replicated in inoculated plants, but the gag gene was not. Real-time PCR and ELISA analysis of N. glutinosa and N. benthamiana plants containing the replicative forms of recombinant construct of CLCuMB/p24 indicated that p24 was expressed in these plants. This CLCuMB-based expression system offers the possibility of mass production of recombinant HIV-1 p24 protein in plants

The schematic map of (A) pBinβΔC1/p24 and (B) pBinβΔC1/gag constructs.

<p>In (A) the CLCuMBΔC1 DNA with a direct repeat of 282 bp flanked the 696 bp DNA fragment of HIV-1 p24 and in (B) flanked the 1501 bp DNA fragment of HIV-1 gag. The direct repeat of the CLCuMB DNA is represented by the dotted rods. The restriction enzymes used in the construction of the cassettes and their nucleotide positions in CLCuMB DNA (Acc. No. AJ298903) are shown by the vertical bars. The positions of the polyadenylation signal and the TATA box in βC1 promoter, upstream of the initiation codon of the βC1 ORF are shown by the A and TATA signals on the diagram, respectively. The one sided arrows are used to show the position of the primers used in the construction of the constructs and further analysis of the resulted DNAs in inoculated plants. The two sided arrows are used to show the expected size of the amplified fragments. These constructs cloned separately as <i>Kpn</i>I/<i>Sna</i>BI fragments into the pBin20 binary vector were used in agroinoculation experiments.</p

Amplification of the 142 bp p24 DNA fragments (A) and 112 bp tobacco elongation factor (Ef) 1-alpha DNA fragments (B).

<p>The DNA fragments were obtained by RT-PCR using the total RNA extracted from <i>N</i>. <i>glutinosa</i> plants inoculated with the constructs as shown at the top of the lanes using a pair of either p24 (A) or tobacco EF1 (B) specific primer pairs as shown at the bottom of each panel. The p24 142 bp DNA fragment and 112 bp tobacco EF1 DNA fragment were absent (lane 7 of each panel) in PCR control reactions without RT from the extracted RNA of <i>N</i>. <i>glutinosa</i> plants co-inculated with βΔC1/p24 construct in the presence of TYLCV-[Ab].</p

Optical density values at 405 nm of the ELISA plate readings of protein samples from inoculated plants.

<p>Plants, as indicated at the top of the columns, inoculated with the constructs as indicated at the bottom of the columns. The value represented for each treatment is the mean of three replicates. Bars with different letter are significantly (<i>p</i> ≤ 0.05) different. Quantitation of HIV-1 p24 protein in plant extracts was estimated by ELISA using the positive antigen (Ag) control of the ELISA kit with a definite (100 pgml-1) concentration. The p24 protein concentration was estimated to be 407.1 ngml^-1 in a <i>N</i>. <i>bethamiana</i> plant extract (corresponding to a yield of 814.2 ng per g fresh plant tissue) whereas the concentration of the same protein in four <i>N</i>. <i>glutinosa</i> plant extracts ranged from 39.4 to 231.3 ngml^-1 (corresponding to a yield of 78.8 to 462.6 ng per g fresh plant tissue). This showed that <i>N</i>. <i>bethamiana</i> was a more suitable host for production of HIV-1 <i>p24</i> protein using the CLCuMB-based expression vector.</p

Transreplicon of recombinant βΔC1/p24 DNA (1735 bp) in <i>N</i>. <i>glutinosa</i> and <i>N</i>. <i>benthamiana</i> plants.

<p>The DNA fragments were obtained from plants, as indicated at the bottom of the lanes, co-agroinoculated with the pBinβΔC1/p24 construct and the helper virus TYLCV-[Ab] (lanes 1–8) by PCR using specific adjacent beta1285^V/1290^C primer pair (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190403#pone.0190403.t001" target="_blank">Table 1</a>). No DNA was amplified from the extracts of plants inoculated with the helper virus alone (lanes 9–10) and plants co-inoculated with the pBinβΔC1/gag construct and the helper virus (lanes 11–15) using the same primer pair. M, marker DNA ladder (Fermentas).</p

List, sequences and other details of oligonucleotide primers used in this study.

<p>List, sequences and other details of oligonucleotide primers used in this study.</p

The effects of vitamin D supplementation on indices of glycemic control in Iranian diabetics: A systematic review and meta-analysis

Background and purpose: This systematic review and meta-analysis aimed to assess the effects of vitamin D supplements on indices of glycemic control [homeostatic model assessment-insulin resistance (HOMA-IR), hemoglobin A1C (HbA1C), fasting blood glucose (FBG), and quantitative insulin-sensitivity check index (QUICKI) and lipid profile in diabetic patients. Methods: Eight databases were searched, for randomized controlled trials (RCTs) or cross-sectional and cohort studies that have been published up to December 2017. We used the comprehensive meta-analysis (CMA) software for all statistical analysis and used the I 2 index for assessing heterogeneity. A p value of <0.05 was considered as statistically significant. Results: We found 621 articles, and after the exclusion of ineligible publications, 82 studies remained to be assessed of which 37 were used for meta-analysis. Vitamin D supplementation was associated with a significant improvement in FBG (p = 0.001 and 95% CI: −0.526 to −0.136) and HbA1C (p = 0.003 and 95% CI: 1.719 to −0.361) in individuals with type 2 diabetes mellitus (T2DM); while in women with gestational diabetes mellitus (GDM) the reduction in FBG (p = 0.071 and 95% CI: −0.873 to −0.035) and HbA1C (p = 0.199 and 95% CI: 3.270 to 0.681) failed to reach statistical significance. Treatment with vitamin D supplements was associated with an improvement in HOMA-IR in pregnant diabetic women (p = 0.028 and 95% CI: 0.924 to −0.053) and for individuals with diabetes mellitus (p = 0.005 and 95% CI: 1.772 to −0.319). The pooled result of the cross-sectional meta-analysis indicated that serum vitamin D concentrations were significantly lower in diabetic patients than in healthy controls (p = 0.018 and 95% CI: 0.587 to −0.054). Conclusion: This meta-analysis suggests that vitamin D supplementation improves indices of glycemic control (FBG, HOMA-IR, and HbA1C) in patients with diabetes mellitus. Hence, vitamin D supplements may be of potential therapeutic value in diabetic patients, as an adjuvant therapy along with other treatment

COVID-19 and Acute Kidney Injury: A Systematic Review

Introduction:& nbsp;Acute kidney injury (AKI) has been associated with an increased mortality rate among hospitalized patients with Coronavirus disease 2019 (COVID-19). The current review aimed to evaluate the symptoms, complications, and treatments performed to manage AKI in patients with COVID-19.& nbsp;Methods:& nbsp;We searched PubMed/Medline, Web of Science, and Embase for the relevant scientific literature published up to February 1, 2022. The following keywords were used: "COVID-19 ", "SARS-CoV-2 ", and "Acute kidney injury ".& nbsp;Results:& nbsp;Forty-four studies with a total number of 114 COVID-19 patients with AKI (Mean age: 53.6 years) were included in our systematic review. The most common comorbidities in patients with COVID-19 suffering from AKI were the history of diabetes, hypertension, and hyperlipidemia. Twelve out of the 44 included studies reported a history of chronic kidney disease (CKD) in this group of patients. Focal segmental glomerulosclerosis (FSGS) and acute tubular necrosis (ATN) were the most common pathological evidence. The average length of hospital stay was 19 days, and the average duration of need for mechanical ventilation was 3 days.& nbsp;Conclusions:& nbsp;The current systematic review shows that AKI frequently complicates the course of COVID-19 hospitalizations and is associated with increased severity of illness, prolonged duration of hospitalization, and poor prognosis. Given the extent of the adverse impact of AKI, early detection of comorbidities and renal complications is essential to improve the outcomes of COVID-19 patients