Bioinformatics Insights on Viral Gene Expression Transactivation: From HIV-1 to SARS-CoV-2.

Viruses provide vital insights into gene expression control. Viral transactivators, with other viral and cellular proteins, regulate expression of self, other viruses, and host genes with profound effects on infected cells, underlying inflammation, control of immune responses, and pathogenesis. The multifunctional Tat proteins of lentiviruses (HIV-1, HIV-2, and SIV) transactivate gene expression by recruiting host proteins and binding to transacting responsive regions (TARs) in viral and host RNAs. SARS-CoV-2 nucleocapsid participates in early viral transcription, recruits similar cellular proteins, and shares intracellular, surface, and extracellular distribution with Tat. SARS-CoV-2 nucleocapsid interacting with the replication-transcription complex might, therefore, transactivate viral and cellular RNAs in the transcription and reactivation of self and other viruses, acute and chronic pathogenesis, immune evasion, and viral evolution. Here, we show, by using primary and secondary structural comparisons, that the leaders of SARS-CoV-2 and other coronaviruses contain TAR-like sequences in stem-loops 2 and 3. The coronaviral nucleocapsid C-terminal domains harbor a region of similarity to TAR-binding regions of lentiviral Tat proteins, and coronaviral nonstructural protein 12 has a cysteine-rich metal binding, dimerization domain, as do lentiviral Tat proteins. Although SARS-CoV-1 nucleocapsid transactivated gene expression in a replicon-based study, further experimental evidence for coronaviral transactivation and its possible implications is warranted.

Intragenomic rearrangements involving 5'-untranslated region segments in SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses.

BACKGROUND: Variation of the betacoronavirus SARS-CoV-2 has been the bane of COVID-19 control. Documented variation includes point mutations, deletions, insertions, and recombination among closely or distantly related coronaviruses. Here, we describe yet another aspect of genome variation by beta- and alphacoronaviruses that was first documented in an infectious isolate of the betacoronavirus SARS-CoV-2, obtained from 3 patients in Hong Kong that had a 5'-untranslated region segment at the end of the ORF6 gene that in its new location translated into an ORF6 protein with a predicted modified carboxyl terminus. While comparing the amino acid sequences of translated ORF8 genes in the GenBank database, we found a subsegment of the same 5'-UTR-derived amino acid sequence modifying the distal end of ORF8 of an isolate from the United States and decided to carry out a systematic search. METHODS: Using the nucleotide and in the case of SARS-CoV-2 also the translated amino acid sequence in three reading frames of the genomic termini of coronaviruses as query sequences, we searched for 5'-UTR sequences in regions other than the 5'-UTR in SARS-CoV-2 and reference strains of alpha-, beta-, gamma-, and delta-coronaviruses. RESULTS: We here report numerous genomic insertions of 5'-untranslated region sequences into coding regions of SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses, but not delta- or gammacoronaviruses. To our knowledge this is the first systematic description of such insertions. In many cases, these insertions would change viral protein sequences and further foster genomic flexibility and viral adaptability through insertion of transcription regulatory sequences in novel positions within the genome. Among human Embecorivus betacoronaviruses, for instance, from 65% to all of the surveyed sequences in publicly available databases contain inserted 5'-UTR sequences. CONCLUSION: The intragenomic rearrangements involving 5'-untranslated region sequences described here, which in several cases affect highly conserved genes with a low propensity for recombination, may underlie the generation of variants homotypic with those of concern or interest and with potentially differing pathogenic profiles. Intragenomic rearrangements thus add to our appreciation of how variants of SARS-CoV-2 and other beta- and alphacoronaviruses may arise.

Experimental encephalomyelitis at age 90, still relevant and elucidating how viruses trigger disease.

20 yr ago, a tribute appeared in this journal on the 70th anniversary of an animal model of disseminated encephalomyelitis, abbreviated EAE for experimental autoimmune encephalomyelitis. "Observations on Attempts to Produce Disseminated Encephalomyelitis in Monkeys" appeared in the Journal of Experimental Medicine on February 21, 1933. Rivers and colleagues were trying to understand what caused neurological reactions to viral infections like smallpox, vaccinia, and measles, and what triggered rare instances of encephalomyelitis to smallpox vaccines. The animal model known as EAE continues to display its remarkable utility. Recent research, since the 70th-anniversary tribute, helps explain how Epstein-Barr virus triggers multiple sclerosis via molecular mimicry to a protein known as GlialCAM. Proteins with multiple domains similar to GlialCAM, tenascin, neuregulin, contactin, and protease kinase C inhibitors are present in the poxvirus family. These observations take us a full circle back to Rivers' first paper on EAE, 90 yr ago.

Physician Modified Low Profile Endograft for Endovascular Repair of Juxtarenal Abdominal Aortic Aneurysms in Patients with Small Access Vessels.

INTRODUCTION: Urgent or emergency treatment of patients with abdominal aortic aneurysms that are anatomically unsuitable for conventional repair because of short proximal necks, small diameters and access vessel calcification, and high risk for open repair can be performed with commercially available branched or fenestrated aortic endografts or physician modified stent grafts. REPORT: A technique is described for modification and successful implantation of a commercially available standard aortic stent graft with a low profile main body in two patients at high risk for open repair, with small access vessels and requiring uni- or bilateral renal artery fenestration for juxtarenal aneurysm repair. DISCUSSION: Based on two case experiences, the use of physician modified off the shelf endografts appears to be a feasible and effective alternative to fenestrated endovascular repair in patients with juxtarenal abdominal aortic aneurysms at high risk for open surgical repair. Studies comparing effectiveness of the different options, including chimney/snorkel technique and debranching, are warranted.

Long-term clinical and quality of life outcomes after stenting of femoropopliteal artery stenosis: 3-year results from the STROLL study.

OBJECTIVES: To evaluate the clinical and health status outcomes of patients undergoing superficial femoral artery (SFA) revascularization using the Shape Memory Alloy Recoverable Technology (S.M.A.R.T.®) nitinol self-expanding stent through 3 years of follow-up. BACKGROUND: Limited long-term data are available describing the durability of benefits after femoropopliteal revascularization. METHODS: In a multicenter, prospective, core-lab adjudicated study, 250 subjects with de novo or restenotic femoropopliteal arterial lesions were treated with the S.M.A.R.T.® stent. The primary endpoint of target vessel patency, a composite of ultrasound-assessed patency and freedom from clinically driven target lesion revascularization (TLR), was evaluated through 3 years. Secondary endpoints included stent fracture and health status. Health status was measured using generic and disease-specific instruments, including the Peripheral Artery Questionnaire (PAQ). RESULTS: At 3-year follow-up, Kaplan-Meier estimated target vessel patency was 72.7%, freedom from clinically driven TLR was 78.5%, and the incidence of stent fracture was 3.6%. The PAQ summary score was markedly impaired at baseline (mean 37.3 ± 19.6 points) and improved substantially at 1 month (mean change from baseline of 31.4 points, 95% CI: 28.5-34.3; P < 0.001). Disease-specific health status benefits assessed by the PAQ were largely preserved through 3 years of follow-up (mean change from baseline, 28.0 points, 95% CI: 24.3-31.7; P < 0.0001). CONCLUSIONS: In patients undergoing revascularization for moderately complex SFA disease, use of the self-expanding S.M.A.R.T® stent was associated with a high rate of target vessel patency through 3 years and led to substantial and sustained health status benefits.

S.M.A.R.T. self-expanding nitinol stent for the treatment of atherosclerotic lesions in the superficial femoral artery (STROLL): 1-year outcomes.

PURPOSE: To assess safety and efficacy of the S.M.A.R.T. Vascular Stent System (Cordis Corp, Fremont, California) in obstructive superficial femoral artery (SFA) disease. MATERIALS AND METHODS: The single-arm, multicenter STROLL study (S.M.A.R.T. Nitinol Self-Expanding Stent in the Treatment of Obstructive Superficial Femoral Artery Disease) included 250 patients (250 lesions in SFA or proximal popliteal artery). The efficacy endpoint was primary patency defined by freedom from binary restenosis (peak systolic velocity ratio > 2.5) as derived by duplex ultrasound plus clinically driven target lesion revascularization (TLR) at 12 months. RESULTS: Mean age of patients was 67.7 years ± 10.3; 47.2% of patients had diabetes; distribution of Rutherford/Becker classes 2, 3, and 4 was 45.8%, 51.4%, and 2.8%. Mean lesion length and reference vessel diameter were 77.3 mm ± 35.3 and 4.9 mm ± 0.7, respectively (23.6% cases with total occlusions). The 30-day freedom from major adverse events (death, index limb amputation, clinically driven TLR) was 100%. The 1-year primary patency was 81.7% by Kaplan-Meier estimate. The presence of diabetes or total occlusion had no effect on primary patency. Ankle-brachial index was 0.4-0.8 in 84.6% of patients at baseline and improved to > 0.8 in 81.0% of patients at 12 months. The proportion of patients in Rutherford/Becker class 3-4 was reduced from 54.2% at baseline to 8.0% at 12 months. Four patients (2.0%) experienced single-stent strut fracture (type I) at 1 year, without associated loss of stent patency. CONCLUSIONS: The S.M.A.R.T. Vascular Stent System proved to be safe and effective for endovascular treatment of obstructive SFA and proximal popliteal artery disease, based on 1-year vessel patency and associated hemodynamic and clinical improvements.

Regenerative dental medicine: stem cells and tissue engineering in dentistry.

The dawn of this century is brightened by the growing understanding and experimentation with stem cells as primary tools in the expanding regenerative medicine and tissue engineering revolution. The tradition of using prosthetic artificial implants to restore lost or damaged dental tissue will gradually be supplanted by more natural alternatives, including biological tooth replacement or induction. The practice of dentistry is likely to be revolutionized by biological therapies based on growth and differentiation factors that accelerate and/or induce a natural biological regeneration. This prospect has flourished from the gained knowledge provided by the molecular biological characterization of the genetic makeup of human cells and from a growing understanding of the effect of environmental factors. Prevention of dental diseases will also gain new ground as more insight is gained into the genetic makeup of microbial pathogens, their interactions with the host, and the host repair mechanisms. This review summarizes current knowledge, barriers, and challenges in the clinical use of stem cells with an emphasis on applications in dentistry.

Human immunodeficiency virus type 1 pharmacogenomics in clinical practice: relevance of HIV-1 drug resistance testing (Part 2).

Throughout most of the past century, physicians could offer patients no treatments for infections caused by viruses. The experience with treatment of infection by human immunodeficiency virus (HIV) has changed the way healthcare workers deal with viral infections and has triggered a growing rate of discovery and use of antiviral agents, the first fruits of the expanding genomics revolution. HIV treatment also provides an informative paradigm for pharmacogenomics because control of infection and its consequences is limited by the development of viral drug resistance and by host factors. This report summarizes studies published to date on the significance of testing of HIV-1 resistance to antiretroviral drugs. The only Food and Drug Administration-approved kit is commercially available through Visible Genetics, Inc., for HIV drug resistance testing by genotypic sequencing. Genotyping sequencing alone is most likely an adequate test to assist in the therapeutic decision-making process in cases of previous regimen failure, treatment-naïve patients in areas of high prevalence of transmitted resistant virus, and pregnant women. However, in exceptional cases of highly complex mutation patterns and extensive cross-resistance, it may be useful to obtain a phenotype test, because that result may more easily identify drugs to which the virus is least resistant. There are no published clinical trial results on the usefulness of the so-called virtual phenotype over genotypic sequencing alone. The paradigm of viral pharmacogenomics in the form of HIV genotypic sequencing has been not only useful to the treatment of other viral diseases but also important to the real-life implementation of the growing discipline of genomics or molecular medicine. The application of this paradigm to the thousands of potential therapeutic targets that have become available through the various human genome projects will certainly gradually change the landscape of diagnosis and management of many diseases, including cancer.

Human immunodeficiency virus type 1 pharmacogenomics in clinical practice: relevance of HIV-1 drug resistance testing (Part 1).

Throughout most of the past century, physicians could offer patients no treatments for infections caused by viruses. The experience with treatment of infection by human immunodeficiency virus (HIV) has changed the way healthcare workers deal with viral infections and has triggered a growing rate of discovery and use of antiviral agents, the first fruits of the expanding genomics revolution. HIV treatment also provides an informative paradigm for pharmacogenomics because control of infection and its consequences is limited by the development of viral drug resistance and by host factors. This report summarizes studies published to date on the significance of testing of HIV-1 resistance to antiretroviral drugs. The only Food and Drug Administration-approved kit for HIV drug resistance testing by genotypic sequencing is commercially available through Visible Genetics, Inc. Genotyping sequencing alone is most likely an adequate test to assist in the therapeutic decision-making process for previous regimen failure, for treatment-naïve patients in areas of high prevalence of transmitted resistant virus, and for pregnant women. However, in exceptional cases of highly complex mutation patterns and extensive cross-resistance, it may be useful to obtain a phenotype test, because that result may more easily identify drugs to which virus is least resistant. There are no published clinical trials results on the usefulness of the so-called virtual phenotype over genotypic sequencing alone. Not only has the paradigm of viral pharmacogenomics in the form of HIV genotypic sequencing been useful in treating other viral diseases, but it is also important to the real-life implementation of the growing discipline ofgenomics or molecular medicine. The application of this paradigm to the thousands of potential therapeutic targets that have become available through the various human genome projects will certainly gradually change the landscape of diagnosis and management of many diseases, including cancer.

The role of pteridines in physiology and pathology.

Disorders or persistent noxious stimulation of the neuroimmunological circuitry can lead to neoplastic, neurological, immunological, psychiatric, or multiorgan pathology. The cause/effect relationship has encouraged a search for neuroimmunological markers possessing functional or pathological correlates. Among the molecules studied, those associated with the biopterin biosynthetic pathway (including neopterin) have proven to be useful and informative. This review addresses the relevance of the latter mediators to our understanding of the pathophysiology of neoplastic, neurological, psychiatric, and immunological disease processes.

Fundamentos de la inmunoterapia adoptiva con células T CD8+ en pacientes con SIDA y Sarcoma de Kaposi / Basis of the adoptive immunotherapy with T CD8+ cells in AIDS and Kaposi's sarcoma

El presente artículo resume los fundamentos científico y clínicos que conllevaron a dos ensayos clínicos de inmunoterapia adoptiva con células T CD8+ en pacientes con síndrome de inmunodeficiencia adquirida (SIDA) y sarcoma de Kaposi (KS). En el primer ensayo, pacientes con SIDA y KS o leucoplaquia vellosa oral (OHL) recibieron cinco rondas de reinfusiones de 10-10 células T CD8+ autólogas las cuales habían sido expandidas y activadas ex vivo. Se coadministró interleuquina-2 recombinante (rIL-2) sólo con la quinta infusión final. Esta intervención resultó en mejora, y en algunos casos resolución sin efectos secundarios, de OHL, KS y candidiasis. La observación de que la mejora clínica del KS era más pronunciada cuando la reinfusión de células T CD8+ era seguida por enfusión de rIL-2 conllevó a un segundo ensayo clínico, que fue diseñado para examinar el efecto sobre el KS asociado a SIDA, de infusiones repetidas de células T CD8+ autólogas con administración concomitante de rIL-2. Se observó mejora del grado de KS en cuatro de los ocho pacientes estudiados (tres con respuesta parcial y uno con respuesta total). El protocolo de inmunoterapia con células T CD8+ también proporcionó la oportunidad de estudiar comparativamente los programas genéticos asociados a células a células CD8+ en SIDA y controles sin infección. Se observó que los patrones de expresión basal de marcadores inmunes solubles y de superficie de células CD8+ provenientes de pacientes con SIDA y de controles sin infección eran predominantemente del tipo 1 y diferían principalmente a nivel cuantitativo o cinético. Otro hallazgo en los ensayos clínicos fue la disipación, con la progresión a través del protocolo, de las deficiencias en la expresión de mediadores inmunes por parte de células T CD8+ de pacientes con SIDA. Estas observaciones son analizadas en el contexto del conocimiento actual y de las implicancias terapéuticas de la función de células CD8+ en el SIDA y la neoplasia.

Efectos del bromuro de benzalconio sobre el sistema inmune y su relevancia a la inhibición del virus del SIDA / Effect of benzalkonium bromide on the inmune system and its relevance to the inhibition of AIDS virus

El bromuro de laurildimetilbenzilamonio es una sal de benzalconio o alquilamina biocida que interfiere con la transducción de señales en una variedad de tipos y procesos celulares a través de su actividad sobre membranas biológicas y su acción sobre proteínas que interactúan con el nucleótido de guanina trifosfato (proteínas G). En el presente trabajo se exploraron los efectos del bromuro de laurildimetilbenzilamonio sobre el sistema inmune y su relevancia al ciclo de vida del virus de inmunodeficiencia adquirida. Se encontró que este biocida inhibe de manera dosis-dependiente la actividad proliferativa estimulada por mitógenos de linfocitos T y B. A pesar de que no afecta la expresión de marcadores de superficie en células inmunes en reposo o en proliferación, el bromuro de laurildimetilbenzilamonio afecta diferencialmente la expresión de genes de citoquinas, probablemente como como reflejo de una dependencia variable de la expresión de estas últimas sobre procesos mediados por proteínas G. Además el bromuro de laurildimetilbenzilamonio no afecta la actividad citotóxica de células asesinas naturales intactas contra celulas K562, una actividad que ha sido reportada como insensible a anticuerpos anti-G. Las observaciones presentadas en este trabajo junto con reportes previos de actividad viricida y espermicida directa, favorecen el uso de las sales benzalconio en preparaciones de uso tópico para la prevención de infecciones venereas virales como, por ejemplo, aquellas usadas en condones.