Reverse Transcriptase Enzyme Research Articles

FNC: An Advanced Anticancer Therapeutic or Just an Underdog?

Azvudine (FNC) is a novel cytidine analogue that has both antiviral and anticancer activities. This minireview focuses on its underlying molecular mechanisms of suppressing viral life cycle and cancer cell growth and discusses applications of this nucleoside drug for advanced therapy of tumors and malignant blood diseases. FNC inhibits positive-stand RNA viruses, like HCV, EV, SARS-COV-2, HBV, and retroviruses, including HIV, by suppressing their RNA-dependent polymerase enzymes. It may also inhibit such enzyme (reverse transcriptase) in the human retrotransposons, including human endogenous retroviruses (HERVs). As the activation of retrotransposons can be the major factor of ongoing cancer genome instability and consequently higher aggressiveness of tumors, FNC has a potential to increase the efficacy of multiple anticancer therapies. Furthermore, FNC also showed other aspects of anticancer activity by inhibiting adhesion, migration, invasion, and proliferation of malignant cells. It was also reported to be involved in cell cycle arrest and apoptosis, thereby inhibiting the progression of cancer through different pathways. To the date, the grounds of FNC effects on cancer cells are not fully understood and hence additional studies are needed for better understanding molecular mechanisms of its anticancer activities to support its medical use in oncology.

CRISPR Genome Editing: Into the Second Decade

CRISPR Genome Editing: Into the Second Decade

Single-Particle Tracking Reveals the Interplay between HIV-1 Reverse Transcription and Uncoating.

Reverse transcription uses the reverse transcriptase enzyme to synthesize deoxyribonucleic acid (DNA) from a ribonucleic acid (RNA) template. This plays an essential role in viral replication. There are still, however, many unknown facts regarding the timing and dynamic processes involved in this life stage. Here, three types of dual-fluorescence human immunodeficiency virus type-1 (HIV-1) particles were constructed with high infectivity, and the sequential process of reverse transcription was observed by real-time imaging of a single HIV-1 particle. Viral uncoating occurred at 60-120 min post infection. Subsequently, at 120-180 min post infection, the viral genome was separated into two parts and reverse-transcribed to generate a DNA product. Nevirapine (NVP), a reverse transcriptase inhibitor, can delay the dynamic process. This study revealed a delicate, sequential, and complex relationship between uncoating and reverse transcription, which may facilitate the development of antiviral drugs.

In Silico Design, Synthesis, and Biological Evaluation of Anticancer Arylsulfonamide Endowed with Anti-Telomerase Activity.

Telomerase, a reverse transcriptase enzyme involved in DNA synthesis, has a tangible role in tumor progression. Several studies have evidenced telomerase as a promising target for developing cancer therapeutics. The main reason is due to the overexpression of telomerase in cancer cells (85–90%) compared with normal cells where it is almost unexpressed. In this paper, we used a structure-based approach to design potential inhibitors of the telomerase active site. The MYSHAPE (Molecular dYnamics SHared PharmacophorE) approach and docking were used to screen an in-house library of 126 arylsulfonamide derivatives. Promising compounds were synthesized using classical and green methods. Compound 2C revealed an interesting IC50 (33 ± 4 µM) against the K-562 cell line compared with the known telomerase inhibitor BIBR1532 IC50 (208 ± 11 µM) with an SI ~10 compared to the BALB/3-T3 cell line. A 100 ns MD simulation of 2C in the telomerase active site evidenced Phe494 as the key residue as well as in BIBR1532. Each moiety of compound 2C was involved in key interactions with some residues of the active site: Arg557, Ile550, and Gly553. Compound 2C, as an arylsulfonamide derivative, is an interesting hit compound that deserves further investigation in terms of optimization of its structure to obtain more active telomerase inhibitors

Homology modeling of clinically-relevant rilpivirine-resistant HIV-RT variants identifies novel rilpivirine analogs with retained binding affinity against NNRTI-resistant HIV mutations

Human immunodeficiency virus (HIV), which affects tens of millions of individuals worldwide, can lead to acquired immunodeficiency syndrome (AIDS). Though there is currently no cure for HIV, the development of small molecule antiretroviral agents has greatly improved the prognosis of infected individuals, especially in developed countries. In particular, compounds such as rilpivirine have been developed as non-nucleoside reverse transcriptase inhibitors (NNRTIs), which allosterically target the reverse transcriptase enzyme in the retrovirus. However, mutations to the reverse transcriptase enzyme threaten to undermine the efficacy of this class of antivirals. Recent advances in computational biophysical modeling enable the structural analyses of such variants without the complications of obtaining crystal structures. Here, we employ homology modeling and molecular docking towards the identification of novel rilpivirine analogs that retain high binding affinity to clinically relevant rilpivirine-resistant mutations of the HIV reverse transcriptase enzyme.

Determination of RNA Structure with In Vitro SHAPE Experiments.

The structure of an RNA molecule is often critical for its correct functioning, post-transcriptional regulation, and/or translation. Predicting RNA secondary structures with in silico tools is relatively straightforward with the large array of software and webservers available. However, for long RNAs and RNA at high temperatures, in silico predictions are less accurate and require experimental validation. To this end, a variety of structural probing reagents are commonly used, both for in vitro and in vivo mapping of RNA structure. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments make use of a nonbase-specific modifying reagent, acylating all conformationally flexible (mainly single-stranded or unpaired) nucleotides and have been employed both for in vitro and in vivo modification of RNA. Here, we describe a protocol for an in vitro SHAPE experiment, starting from in vitro transcribed RNA using a T7 polymerase system. This RNA is folded and subsequently modified in vitro with the SHAPE-reagent N-methyl isatoic anhydride (NMIA). Primer extension employing a radioactive 32P-labeled primer that binds the RNA downstream of the structure of interest will generate cDNA until the reverse transcriptase enzyme is halted by the introduced SHAPE modifications. Denaturing acrylamide gel electrophoresis of the pool of 32P-labeled cDNAs and the corresponding sequencing ladders, followed by autoradiography, will expose these stops in reverse transcription (RT) and will therefore enable to identify single-stranded nucleotides in the RNA of interest. These RT stops and NMIA-modification efficiencies can be quantified with ImageJ software and can be used to validate or increase the accuracy of RNA secondary structure predictions.

A Combined Approach of Pharmacophore Modeling, QSAR Study, Molecular Docking and In silico ADME/Tox Prediction of 4-Arylthio & 4-Aryloxy-3- Iodopyridine-2(1H)-one Analogs to Identify Potential Reverse Transcriptase Inhibitor: Anti-HIV Agents.

Reverse transcriptase is an important therapeutic target to treat AIDS caused by the Human Immunodeficiency Virus (HIV). Despite many effective anti-HIV drugs, reverse transcriptase (RT) inhibitors remain the cornerstone of the drug regimen to treat AIDS. In the present work, we have expedited the use of different computational modules and presented an easy, costeffective, and high throughput screening method to identify potential reverse transcriptase inhibitors. A congeneric series of 4-Arylthio & 4-Aryloxy-3- Iodopyridine-2(1H)-one analogs having anti-HIV activity were subjected to structure-based 2D, 3D QSAR, Pharmacophore Modeling, and Molecular Docking to elucidate the structural properties required for the design of potent HIV-RT inhibitors. Prediction of preliminary Pharmacokinetic and the Drug Likeliness profile was performed for these compounds by in silico ADME study. The 2D and 3D- QSAR models were developed by correlating two and three-dimensional descriptors with activity (pIC50) by sphere exclusion method and k-nearest neighbor molecular field analysis approach, respectively. The significant 2D- QSAR model developed by Partial Least Square is associated with the Sphere Exclusion method (PLS-SE), having r2 and q2 values 0.9509 and 0.8038, respectively. The 3D-QSAR model by Step Wise variable selection method (SW-kNN MFA) is more significant, which has a cross-validated squared correlation coefficient q2= 0.8509 and a non-crossvalidated correlation coefficient pred_r2= 0.8102. The pharmacophore hypothesis was developed, which comprised 5 features includes 3 aliphatic regions (Ala), 1 H-bond donor (HDr) and 1 H-bond acceptor (HAc). Docking studies of the selected inhibitors with the active site of reverse transcriptase enzyme showed hydrogen bond and π - π interaction with LYS-101, LYS-103, TYR- 181, TYR-188 and TRP-229 residues present at the active site. All the candidates with good bioavailability and ADMET drug likeliness properties. The results of the present work provide more useful information and important structural insights for the discovery, design of novel and potent reverse transcriptase inhibitors with high therapeutic windows in the future.

Measurement of paraoxonase and telomerase enzymes and HDL (high density lipoprotein) values and research of their possible relationships with each other in bloodserum of obese cats

Today, obesity is seen as a serious health problem in domestic cats. Paraoxonase1 (PON1) is a negative acute phase protein with anti-inflammatory properties. PON1 is transported by linking to different parts of HDL in the blood and has a protective feature of HDL against oxidation. Telomerase is reverse transcriptase enzyme that synthesizes and protects telomeres. Telomere length becomes shorter due to stress, obesity, and lack of exercise. In this study, paraoxonase1, telomerase enzymes and HDL values in blood serum of obese and non-obese cats were measured and their relationships with each other were evaluated. Body fat ratio in cats with body fat measurement system, those above 30% were evaluated as obese, and those below 30% were evaluated as the control group. Paraoxonase1 (p<0.001), telomerase (p<0.001) and HDL (p<0.008) values showed a statistically significant difference between the both groups, it was found to be lower in the obese group compared to the control group. As a result; paraoxonase1 and telomerase enzymes which are parameters of oxidative stress and are known to decrease in inflammation, were also found to be decreased in obese cats. The fact that HDL values are lower than the control group is an indicator that there are diseases that occur together with obesity.

High-Level Production of MMLV Reverse Transcriptase Enzyme in Escherichia Coli

Reverse transcriptase (RT) of Moloney murine leukemia virus (MMLV) is the most widely used enzyme for cDNA synthesis and RNA amplification. In this study, we aimed to produce MMLV RT enzyme recombinantly due to its importance in molecular studies. In this context, the DNA fragment encoding the MMLV RT enzyme was cloned into pTOLT plasmid and expressed in E. coli BL21 (DE3) pLysE cells. Since the high-level expression of the protein caused the protein molecules to aggregate in the inclusion bodies, co-expression of MMLV RT and chaperone plasmids (pG-KJE8, pGro7, pKJE7, pGTf2, pTf16) was performed to obtain the MMLV RT protein in soluble form. Contrary to our expectations, because it could not be obtained in soluble form, the protein was recovered from the inclusion bodies using refolding process. Finally, the protein was purified by affinity chromatography and the activity of the protein was checked using RT-PCR technique.

An in silico molecular modeling approach of halolactone derivatives as potential inhibitors for human immunodeficiency virus type-1 reverse transcriptase enzyme

Acquired Immune Deficiency Syndrome (AIDS) is an infectious disease caused by Human Immunodeficiency Virus (HIV) infection and its replication requires the Reverse Transcriptase (RT) enzyme. RT plays a key role in the HIV life cycle, making it one of the most important targets for designing new drugs. Thus, in order to increase therapeutic options against AIDS, halolactone derivatives (D-halolactone) that have been showed as potential non-nucleoside inhibitors of the RT enzyme were studied. In the present work, a series of D-halolactone were investigated by molecular modeling studies, combining Three-dimensional Quantitative Structure-Activity Relationship (3 D-QSAR), molecular docking and Molecular Dynamics (MD) techniques, to understand the molecular characteristics that promote biological activity. The internal and external validation parameters indicated that the 3 D-QSAR model has good predictive capacity and statistical significance. Contour maps provided useful information on the structural characteristics of compounds for anti-HIV-1 activity. The docking results showed that D-halolactone present good complementarity by the RT allosteric site. In MD simulations it was observed that the formation of enzyme-ligand complexes were favorable, and from the free energy decomposition it was found that Leu100, Val106, Tyr181, Try188, and Trp229 are key residues for stabilization in the enzymatic site. Thus, the results showed that the proposed models can be used to design promising HIV-1 RT inhibitors. Communicated by Ramaswamy H. Sarma

Pyridine/Pyrimidine Substituted Imidazol-5-one Analogs as HIV-1 RT Inhibitors: Design, Synthesis, Docking and Molecular Dynamic Simulation Studies.

This paper reports the synthesis, Non-nucleoside reverse transcriptase inhibitory (NNRTIs) activity and computational studies of 2-((4-chloro-2-subtitutedphenoxy) methyl)-4-(furan-2-ylmethylene)-1-substituted Pyridine/-pyrimidine-1H-imidazol-5(4H)-ones. The imidazol-5-one analogs were synthesized by conventional method and characterized by FT-IR, NMR and mass spectral data. All compounds were evaluated for in-vitro NNRTI activity by using reverse transcriptase (RT) assay kit (Roche). The in-silico docking studies were conducted on RT enzyme to investigate binding site interactions of synthesized compounds. The MMGBSA method was also used to calculate the binding free energy between the inhibitors and RT enzyme. The MD simulation was further performed for the apo form of the RT enzyme and docked complex of compound A6-RT enzyme to better understand the stability of the protein-ligand complex. The bioactivity analysis revealed that most of the synthesized compounds showed significant inhibitory activity against RT enzyme and the IC50 value was found to be in the range of 1.76-3.88 μM. The computational studies suggest that the docked compounds form the H-bonding with amino acid residue Lys101 and hydrophobic interactions with amino acid residues Tyr188, Tyr181, Trp229, and Tyr318, which act as the primary driving forces for protein-ligand interaction. The reported imidazol-5-one analogs can act as lead for further development of prospective RT inhibitors.

HIV-Infection and AIDS

HIV (human immunodeficiency virus) is a retrovirus that infects CD4+ T cells of the human immune system. If the infection is not treated, these cells are destroyed, resulting in an acquired immunodeficiency, i.e., “AIDS” (acquired immunodeficiency syndrome). HIV owns a reverse transcriptase enzyme to convert its RNA into DNA, which is then integrated into the human genome – then undetectable by the immune system. Today, sexual transmission is the major route of HIV infection, while parenteral transmission (sharing needles among drug addicts; rarely blood transfusion) and perinatal transmission are also possible. Acute HIV infection is accompanied by infectious mononucleosis-like symptoms (fevers, rash, lymphadenopathy, sore throat, fatigue), followed by a chronic asymptomatic stage, with viral replication at low levels, followed years later by AIDS, characterized by a plethora of possible opportunistic infections and cancers that result from T-cell deficiency and finally in death within about 2–3 years. Antiretroviral treatment (ART) includes 6 main classes of medicines that affect different steps of viral activities. While no cure is possible, ART – and particularly “Highly active antiretroviral therapy” (HAART) – has made HIV infections a chronic disease and therapy also results in a reduction of transmission. A large variety of vaccine candidates have been assessed – including phase 3 studies – but for many reasons, none of them have been successful to date.

Detection of Viroids Using RT-qPCR.

Viroids are the smallest known infectious pathogens. They are nonprotein-encoding, single-stranded, circular, naked RNA molecules that can cause several diseases in economically important crops. With the advent of thermal cyclers incorporating fluorescent detection, reverse transcription coupled to the quantitative polymerase chain reaction (RT-qPCR) has transformed the way the viroids are detected. The method involves using sequence-specific primers that anneal to the viroid RNA of interest. The viroid RNA serves as a template during reverse transcription, in which the enzyme reverse transcriptase generates a cDNA copy of a portion of the target RNA molecule. After first-strand cDNA synthesis, RNA template from cDNA:RNA hybrid molecule is removed by digestion with RNase H to improve the sensitivity of PCR step. This cDNA is then be used as a template for amplification of viroid sequence in PCR.

A new RNA-DNA interaction required for integration of group II intron retrotransposons into DNA targets.

Mobile group II introns are site-specific retrotransposable elements abundant in bacterial and organellar genomes. They are composed of a large and highly structured ribozyme and an intron-encoded reverse transcriptase that binds tightly to its intron to yield a ribonucleoprotein (RNP) particle. During the first stage of the mobility pathway, the intron RNA catalyses its own insertion directly into the DNA target site. Recognition of the proper target rests primarily on multiple base-pairing interactions between the intron RNA and the target DNA, while the protein makes contacts with only a few target positions by yet-unidentified mechanisms. Using a combination of comparative sequence analyses and in vivo mobility assays we demonstrate the existence of a new base-pairing interaction named EBS2a–IBS2a between the intron RNA and its DNA target site. This pairing adopts a Watson–Crick geometry and is essential for intron mobility, most probably by driving unwinding of the DNA duplex. Importantly, formation of EBS2a–IBS2a also requires the reverse transcriptase enzyme which stabilizes the pairing in a non-sequence-specific manner. In addition to bringing to light a new structural device that allows subgroup IIB1 and IIB2 introns to invade their targets with high efficiency and specificity our work has important implications for the biotechnological applications of group II introns in bacterial gene targeting.

Development of a Bicistronic Anti-CD19/CD20 CAR Construct Including Optimization to Abrogate Retroviral Recombination Events

Anti-CD19 chimeric antigen receptor (CAR) T cells can eliminate lymphoma, but CD19 loss from lymphoma can cause anti-CD19 CAR T-cell treatment failure. To address CD19 loss, we designed a bicistronic construct encoding 2 CARs. The first CAR in the bicistronic construct was the anti-CD19 CAR Hu19-CD828Z, which incorporated a human anti-CD19 single-chain variable fragment (scFv) along with CD28 and CD3ζ domains (Brudno et al. Nature Medicine 2020). The 2nd CAR in the construct was a novel anti-CD20 CAR incorporating a human anti-CD20 scFv, plus 4-1BB and CD3ζ domains. This bicistronic CAR construct, designated Hu1928-Hu20BB-original, was encoded by the mouse stem cell virus-based splice-gag (MSGV1) gamma-retroviral vector.Repeated segments of identical DNA sequence can lead to recombination events when retroviral vectors are utilized. Recombination events driven by repeated identical nucleotide segments can occur during retroviral vector production in packaging cells. Recombination events can also occur in transduced T cells during reverse transcription when repeated segments of identical nucleotides on the same nucleotide strand anneal, which leads to strand switching by the reverse transcriptase enzyme and deletion of part of the sequence.We assessed T cells transduced with Hu1928-Hu20BB-original for evidence of recombination. Southern blots of DNA from T cells transduced with Hu1928-Hu20BB-original showed a dominant band of the expected CAR transgene length and no aberrant bands after 21 hours of exposure; however, with extended exposure of 8 days, an unexpected band indicating shorter transgene length became visible. This aberrant band was consistent with deletion of part of the transgene sequence due to recombination. To investigate possible recombination by a more sensitive method, we assessed RNA from transduced T cells by single-molecule real-time RNA sequencing (SMRT-RNAseq, PacBio). Deletions of Hu1928-Hu20BB-original RNA sequence between segments of identical sequence were detected by SMRT-RNAseq. These findings were consistent with recombination. The most common recombination events were between the CD8α hinge and transmembrane (HT) domains of the 2 CARs of Hu1928-Hu20BB-original. These HT domains shared regions of identical nucleic acid sequence. Less commonly, recombination events occurred between segments of identical sequence shared by the heavy-chain domains and the light-chain domains of the two CARs in the construct. The attached table summarizes deletions consistent with recombination in Hu1928-Hu20BB-original RNA from transduced T cells of one patient. Similar results were obtained in T cells of 3 other patients. We confirmed deletions consistent with recombination in CAR RNA transcripts by short-read RNA sequencing (Illumina). Digital droplet PCR analysis of DNA from transduced T cells showed that recombination events between the HT domains of Hu1928-Hu20BB-original were present in DNA of transduced T cells.We modified the DNA sequence of Hu1928-Hu20BB-original to reduce repeated segments of identical DNA sequence to form a new CAR designated Hu1928-Hu20BB-standard. When T cells transduced with Hu1928-Hu20BB-standard were studied by SMRT RNAseq, deletions of RNA sequence consistent with recombination were substantially reduced (Table).We modified Hu1928-Hu20BB-standard by lengthening the linker in the anti-CD20 scFv to form a construct designated Hu1928-Hu20BB-long. We compared Hu1928-Hu20BB-standard to Hu1928-Hu20BB-long in vitro, and both specifically recognized CD19 and CD20 in assays of degranulation, cytokine release, and proliferation. In vitro assays showed higher interleukin-2 release by T cells transduced with Hu1928-Hu20BB-long versus Hu1928-Hu20BB-standard in response to 3 different CD20+ target cells (P value range 0.001 to 0.036). We tested Hu1928-Hu20BB-long against tumors established in nod-scid common gamma-chain knockout (NSG) mice. Human T cells expressing Hu1928-Hu20BB-long eliminated tumors of the following tumor cell lines: st486 (CD19+, CD20+), st486-CD19-negative (CD20+, CD19 low to negative expression), and NALM6 (CD19+, CD20 low expression). One million Hu1928-Hu20BB-long T cells per mouse eradicated st486 tumors. These optimized anti-CD19/CD20 bicistronic CAR constructs are promising for clinical treatment of B-cell malignancies. [Display omitted] DisclosuresLam: Kite, a Gilead Company: Patents & Royalties. Kochenderfer: Bristol Myers Squibb: Research Funding; Kite, a Gilead Company: Patents & Royalties: on anti-CD19 CARs, Research Funding.

HIV-1 gp120 Antagonists Also Inhibit HIV-1 Reverse Transcriptase by Bridging the NNRTI and NRTI Sites.

HIV-1 infection is typically treated using ≥2 drugs, including at least one HIV-1 reverse transcriptase (RT) inhibitor. Drugs targeting RT comprise nucleos(t)ide RT inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). NRTI-triphosphates bind at the polymerase active site and, following incorporation, inhibit DNA elongation. NNRTIs bind at an allosteric pocket ∼10 Å away from the polymerase active site. This study focuses on compounds ("NBD derivatives") originally developed to bind to HIV-1 gp120, some of which inhibit RT. We have determined crystal structures of three NBD compounds in complex with HIV-1 RT, correlating with RT enzyme inhibition and antiviral activity, to develop structure-activity relationships. Intriguingly, these compounds bridge the dNTP and NNRTI-binding sites and inhibit the polymerase activity of RT in the enzymatic assays (IC50 < 5 μM). Two of the lead compounds, NBD-14189 and NBD-14270, show potent antiviral activity (EC50 < 200 nM), and NBD-14270 shows low cytotoxicity (CC50 > 100 μM).

Nose to brain delivery of Efavirenz nanosuspension for effective neuro AIDS therapy: in-vitro, in-vivo and pharmacokinetic assessment

Efavirenz is inhibitor of non-nucleoside reverse transcriptase enzyme; BCS class II drug. The objective of the present research was to prepare and evaluate nanosuspension of Efavirenz for the treatment of neuro-AIDS. Efavirenz is the substrate for drug resistant proteins at BBB prone to efflux and could not reach brain with effective levels. Current need of the therapy is to develop drug delivery systems targeting viral reservoirs at effective concentration in the brain. With this need we developed Efavirenz nanosuspension for nose to brain drug transport to bypass blood brain barrier. Nanosuspension prepared with high-pressure homogenization had a mean particle size of 223 nm, PDI of 0.2 and -21.2 mV zeta potential. Histopathology study on goat nasal mucosa showed no adverse effects of formulation on nasal tissues. Gamma scintigraphy study and in-vivo study on Wistar rat model reveals drug transport to the CNS after nasal administration. Pharmacokinetic parameters and drug targeting potential of 99.46 % suggest direct nose to brain transport of Efavirenz nanoparticle. Results reveal that nose to brain delivery of Efavirenz is the best possible alternative for neuro –AIDS treatment.

T lymphocyte senescence is attenuated in Parkinson\u2019s disease

BackgroundImmune involvement is well-described in Parkinson’s disease (PD), including an adaptive T lymphocyte response. Given the increasing prevalence of Parkinson’s disease in older age, age-related dysregulation of T lymphocytes may be relevant in this disorder, and we have previously observed changes in age-associated CD8+ T cell subsets in mid-stage PD. This study aimed to further characterise T cell immunosenescence in newly diagnosed PD patients, including shifts in CD4+ and CD8+ subpopulations, and changes in markers of cellular ageing in CD8+ T lymphocytes.MethodsPeripheral blood mononuclear cells were extracted from the blood of 61 newly diagnosed PD patients and 63 age- and sex-matched controls. Flow cytometric analysis was used for immunophenotyping of CD8+ and CD4+ lymphocyte subsets, and analysis of recent thymic emigrant cells. Telomere length within CD8+ T lymphocytes was assessed, as well as the expression of the telomerase reverse transcriptase enzyme (hTERT), and the cell-ageing markers p16INK4a and p21CIP1/Waf1.ResultsThe number of CD8+ TEMRA T cells was found to be significantly reduced in PD patients compared to controls. The expression of p16INK4a in CD8+ lymphocytes was also lower in patients versus controls. Chronic latent CMV infection was associated with increased senescent CD8+ lymphocytes in healthy controls, but this shift was less apparent in PD patients.ConclusionsTaken together, our data demonstrate a reduction in CD8+ T cell replicative senescence which is present at the earliest stages of Parkinson’s disease.

HIV-1 Reverse Transcriptase Inhibition by Major Compounds in a Kenyan Multi-Herbal Composition (CareVid™): In Vitro and In Silico Contrast.

CareVid is a multi-herbal product used in southwest Kenya as an immune booster and health tonic and has been anecdotally described as improving the condition of HIV-positive patients. The product is made up of roots, barks and whole plant of 14 African medicinal plants: Acacia nilotica (L.) Willd. ex Delile (currently, Vachelia nilotica (L.) P.J.H Hurter & Mabb.), Adenia gummifera (Harv.) Harms, Anthocleista grandiflora Gilg, Asparagus africanus Lam., Bersama abyssinica Fresen., Clematis hirsuta Guill. & Perr., Croton macrostachyus Hochst. ex Delile, Clutia robusta Pax (accepted as Clutia kilimandscharica Engl.), Dovyalis abyssinica (A. Rich.) Warb, Ekebergia capensis Sparm., Periploca linearifolia Quart.-Dill. & A. Rich., Plantago palmata Hook.f., Prunus africana Hook.f. Kalkman and Rhamnus prinoides L’Her. The objective of this study was to determine the major chemical constituents of CareVid solvent extracts and screen them for in vitro and in silico activity against the HIV-1 reverse transcriptase enzyme. To achieve this, CareVid was separately extracted using CH2Cl2, MeOH, 80% EtOH in H2O, cold H2O, hot H2O and acidified H2O (pH 1.5–3.5). The extracts were analysed using HPLC–MS equipped with UV diode array detection. HIV-1 reverse transcriptase inhibition was performed in vitro and compared to in silico HIV-1 reverse transcriptase inhibition, with the latter carried out using MOE software, placing the docking on the hydrophobic pocket in the subdomain of p66, the NNRTI pocket. The MeOH and 80% EtOH extracts showed strong in vitro HIV-1 reverse transcriptase inhibition, with an EC50 of 7 μg·mL−1. The major components were identified as sucrose, citric acid, ellagic acid, catechin 3-hexoside, epicatechin 3-hexoside, procyanidin B, hesperetin O-rutinoside, pellitorine, mangiferin, isomangiferin, 4-O-coumaroulquinic acid, ellagic acid, ellagic acid O-pentoside, crotepoxide, oleuropein, magnoflorine, tremulacin and an isomer of dammarane tetrol. Ellagic acid and procyanidin B inhibited the HIV-1 reverse transcription process at 15 and 3.2 µg/mL−1, respectively. Docking studies did not agree with in vitro results because the best scoring ligand was crotepoxide (ΔG = −8.55 kcal/mol), followed by magnoflorine (ΔG = −8.39 kcal/mol). This study showed that CareVid has contrasting in vitro and in silico activity against HIV-1 reverse transcriptase. However, the strongest in vitro inhibitors were ellagic acid and procyanidin B.

COVID-19 Vaccines for HIV-Infected Patients.

Nearly 40 years have passed since the initial cases of infection with the human mmunodeficiency virus (HIV) were identified as a new disease entity and the cause of acquired immunodeficiency disease (AIDS). This virus, unlike any other, is capable of causing severe suppression of our adaptive immune defense mechanisms by directly infecting and destroying helper T cells leading to increased susceptibility to a wide variety of microbial pathogens, especially those considered to be intracellular or opportunistic. After T cells are infected, HIV reproduces itself via a somewhat unique mechanism involving various metabolic steps, which includes the use of a reverse transcriptase enzyme that enables the viral RNA to produce copies of its complementary DNA. Subsequent physiologic steps lead to the production of new virus progeny and the eventual death of the invaded T cell. Fortunately, both serologic and molecular tests (such as PCR) can be used to confirm the diagnosis of an HIV infection. In the wake of the current COVID-19 pandemic, it appears that people living with HIV/AIDS are equally or slightly more susceptible to the etiologic agent, SARS-CoV-2, than the general population having intact immune systems, but they may have more serious outcomes. Limited clinical trials have also shown that the currently available COVID-19 vaccines are both safe and effective in affording protection to HIV/AIDS patients. In this review, we further explore the unique dynamic of HIV/AIDS in the context of the worldwide COVID-19 pandemic and the implementation of vaccines as a protective measure against COVID-19, as well as what immune parameters and safeguards should be monitored in this immunocompromised group following vaccination.