Implementation of an Isolation Shelter for Individuals with COVID 19 Experiencing Homelessness.

The COVID-19 pandemic had a devastating impact on non-sheltered homeless and housing-insecure individuals. This report details the development of a Chicago-based isolation shelter designed for people experiencing homelessness and recovering from COVID-19. The model is informative concerning the rapid development of services for people marginalized by the health care system.

Synthesis, Hydrolysis, and Protonation-Promoted Intramolecular Reductive Breakdown of Potential NRTIs: Stavudine α-P-Borano-γ-P-N-L-tryptophanyltriphosphates.

Phosphorus-modified prodrugs of dideoxynucleoside triphosphates (ddNTPs) have shown promise as pronucleotide strategies for improving antiviral activity compared to their parent dideoxynucleosides. Borane modified NTPs offer a promising choice as nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs). However, the availability of α-P-borano-γ-P-substituted NTP analogs remains limited due to challenges with synthesis and purification. Here, we report the chemical synthesis and stability of a new potential class of NRTI prodrugs: stavudine (d4T) 5'-α-P-borano-γ-P-N-L-tryptophanyltriphosphates. One-pot synthesis of these compounds was achieved via a modified cyclic trimetaphosphate approach. Pure Rp and Sp diastereomers were obtained after HPLC separation. Based on LC-MS analysis, we report degradation pathways, half-lives (5-36 days) and mechanisms arising from structural differences to generate the corresponding borano tri- and di-phosphates, and H-phosphonate, via several parallel routes in buffer at physiologically relevant pH and temperature. Here, the major hydrolysis products, d4T α-P-boranotriphosphate Rp and Sp isomers, were isolated by HPLC and identified with spectral data. We first propose that one of the major degradation products, d4T H-phosphonate, was generated from the d4T pronucleotides via a protonation-promoted intramolecular reduction followed by a second step nucleophilic attack. This report could provide valuable information for pronucleotide-based drug design in terms of selective release of target nucleotides.

Practitioner perspectives on the National Institute of Justice's Violence Against Women Research and Evaluation Program.

Five practitioners who have worked with NIJ over recent years were asked to provide a critique of NIJ's VAW program based on their experience. Individuals from professions that the program seeks to assist or inform offered the following commentaries.

Inhibition of hepatitis C viral RNA-dependent RNA polymerase by α-P-boranophosphate nucleotides: exploring a potential strategy for mechanism-based HCV drug design.

Improved treatments for chronic HCV infections remain a challenge, and new chemical strategies are needed to expand the current paradigm. The HCV RNA polymerase (RdR(P)) has been a target for antiviral development. For the first time we show that the boranophosphate (BP) modification increases the substrate efficiency of ATP analogs into HCV NS5BΔ55 RdRP-catalyzed RNA. Boranophosphate nucleotides contain a borane (BH3) group substituted for a non-bridging phosphoryl oxygen of a normal phosphate group, resulting in a class of modified isoelectronic DNA and RNA mimics capable of modulating the reading and writing of genetic information. We determine that HCV NS5BΔ55, being a stereospecific enzyme, incorporates the Rp isomer of both ATPαB and the two boranophosphate analogs: 2'-O-methyladenosine 5'-(α-P-borano) triphosphate (2'-OMe ATPαB, 5a) and 3'-deoxyadenosine 5'-(α-P-borano) triphosphate (3'-dATPαB, 5b). The R(p) diastereomer of ATPαB (6), having no ribose modifications, was found to be a slightly better substrate than natural ATP, showing a 42% decrease in the apparent Michaelis-Menten constant (K(m)). The IC50 of both 2'-O-Me and 3'-deoxy ATP was decreased with the boranophosphate modification up to 16-fold. This "borano effect" was further confirmed by determining the steady-state inhibitory constant (K(i)), showing a comparable potency shift (21-fold). These experiments also indicate that the boranophosphate analogs 5a and 5b inhibit HCV NS5B through a competitive mode of inhibition. This evidence, together with previous crystal structure data, further supports the idea that HCV NS5B (in a similar manner to HIV-1 RT) discriminates against the 3'-deoxy modification via lost interactions between the 3'-OH on the ribose and the active site residues, or lost intramolecular hydrogen bonding interactions between the 3'-OH and the pyrophosphate leaving group during phosphoryl transfer. To our knowledge, these data represent the first time a phosphate modified NTP has been studied as a substrate for HCV NS5B RdRP.

Aptamer-mediated delivery of chemotherapy to pancreatic cancer cells.

Gemcitabine is a nucleoside analog that is currently the best available single-agent chemotherapeutic drug for pancreatic cancer. However, efficacy is limited by our inability to deliver sufficient active metabolite into cancer cells without toxic effects on normal tissues. Targeted delivery of gemcitabine into cancer cells could maximize effectiveness and concurrently minimize toxic side effects by reducing uptake into normal cells. Most pancreatic cancers overexpress epidermal growth factor receptor (EGFR), a trans-membrane receptor tyrosine kinase. We utilized a nuclease resistant RNA aptamer that binds and is internalized by EGFR on pancreatic cancer cells to deliver gemcitabine-containing polymers into EGFR-expressing cells and inhibit cell proliferation in vitro. This approach to cell type-specific therapy can be adapted to other targets and to other types of therapeutic cargo.

Stop the escalation before it begins by using the pediatric Behavior Response Team protocol.

In today's world, clinicians need to be prepared to care for challenging patients and families that are struggling with the stress of illness and hospitalization and have inadequate coping skills. The University of Michigan Health System (UMHS) has developed a protocol identifying a team with representatives from psychiatry, security, and risk management to provide a rapid response in situations that historically have resulted in, at worst, sentinel/adverse events and at best, service disasters. The pediatric BRT protocol formalizes the purpose of the team, how staff should access them, and the expectation for involved staff to debrief about the interventions at identified times. It has proven to be an effective intervention and allows clinicians to provide needed care to the patients.

Structural basis of the RNase H1 activity on stereo regular borano phosphonate DNA/RNA hybrids.

Numerous DNA chemistries for improving oligodeoxynucleotide (ODN)-based RNA targeting have been explored. The majority of the modifications render the ODN/RNA target insensitive to RNase H1. Borano phosphonate ODN's are among the few modifications that are tolerated by RNase H1. To understand the effect of the stereochemistry of the BH(3) modification on the nucleic acid structure and RNase H1 enzyme activity, we have investigated two DNA/RNA hybrids containing either a R(P) or S(P) BH(3) modification by nuclear magnetic resonance (NMR) spectroscopy. T(M) studies show that the stabilities of R(P) and S(P) modified DNA/RNA hybrids are essentially identical (313.8 K) and similar to that of an unmodified control (312.9 K). The similarity is also reflected in the imino proton spectra. To characterize such similar structures, we used a large number of NMR restraints (including dipolar couplings and backbone torsion angles) to determine structural features that were important for RNase H1 activity. The final NMR structures exhibit excellent agreement with the data (total R(x) values of <6%) with helical properties between those of an A and B helix. Subtle backbone variations are observed in the DNA near the modification, while the RNA strands are relatively unperturbed. In the case of the S(P) modification, for which more perturbations are recorded, a slightly narrower minor groove is also obtained. Unique NOE base contacts localize the S(P) BH(3) group in the major groove while the R(P) BH(3) group points away from the DNA. However, this creates a potential clash of the R(P) BH(3) groups with important RNase H1 residues in a complex, while the S(P) BH(3) groups could be tolerated. We therefore predict that on the basis of our NMR structures a fully R(P) BH(3) DNA/RNA hybrid would not be a substrate for RNase H1.

Synthesis and properties of (alpha-P-borano)-nucleoside 5'-triphosphate analogues as potential antiviral agents.

The alpha-P-borano modification, where one of the alpha-phosphate oxygens is replaced by borane, of chain terminating nucleoside triphosphates are currently being tested in cell culture and are showing promise as effective viral polymerase inhibitors. The goal of this project is to combine the alpha-P-borano and Nanogel drug delivery technology to increase the antiviral potency of chain terminating sugar and base modified purine nucleosides versus the Hepatitis C Viral RNA dependent RNA polymerase (HCV RdRp). Here we show the synthesis of Cordycepin and 2'-O-methyl alpha-P-borano triphosphate via a one-pot phosphorochloridite synthesis under mild conditions. These analogues will be used for future structure-activity relationship (SAR) studies.

Boranophosphate siRNA-aptamer chimeras for tumor-specific downregulation of cancer receptors and modulators.

There is a need for novel, effective, and cell- and gene-specific therapeutics for cancer. Modified oligonucleotides can be used to modulate specifically and potently the expression of several genes that are upregulated in breast and prostate cancer and have been found to be causal to the tumor phenotype. Synergistic downregulation of these genes may be a potent therapeutic intervention. We are investigating the use of boranophosphate (BP) analogues of RNA as promising candidates for enhancing the potential of three relatively new, gene-specific, anticancer strategies: (1) Tumor-targeted borane siRNA against a combination of genes that control metabolism and transduction; (2) Tumor-specific modified aptamers against prostate specific membrane antigen (PSMA) and ERB2 in breast cancer as delivery agents; and (3) Cancer cell obliteration by cell-specific radiation therapy: Boron-Neutron-Capture-Therapy.

Stereospecificity, substrate, and inhibitory properties of nucleoside diphosphate analogs for creatine and pyruvate kinases.

Antiviral alpha-P-borano substituted NTPs are promising chain terminators targeting HIV reverse transcriptase (RT). Activation of antiviral nucleoside diphosphates (NDPs) to NTPs may be carried out by pyruvate kinase (PK) and creatine kinase (CK). Herein, are presented the effects of nucleobase, ribose, and alpha-phosphate substitutions on substrate specificities of CK and PK. Both enzymes showed two binding modes and negative cooperativity with respect to substrate binding. The stereospecificity and inhibition of ADP phosphorylation by alpha-P-borano substituted NDP (NDPalphaB) stereoisomers were also investigated. The Sp-ADPalphaB isomer was a 70-fold better substrate for CK than the Rp isomer, whereas PK preferred the Rp isomer of NDPalphaBs. For CK, the Sp-ADPalphaB isomer was a competitive inhibitor; for PK, the Rp-ADPalphaB isomer was a poor competitive inhibitor and the Sp-ADPalphaB isomer was a poor non-competitive inhibitor. Taken together, these data suggest that, although the Rp-NDPalphaB isomer would be minimally phosphorylated by CK or PK, it should not inhibit either enzyme.

Synthesis of 9-fluorenemethyl boranophosphonodiphosphate via an H-phosphonate approach.

9-Fluorenemethyl boranophosphonate 6 and its boranophosphonodiphosphate 7 were synthesized via an H-phosphonate approach. The method is efficient for the synthesis of acyclic compounds 6 & 7, and can be explored for the synthesis of nucleoside 5'-deoxy boranophosphonodiphosphate.

Efficient synthesis of thymidine boranophosphoramidates conjugated with amino acids.

An efficient synthesis of a thymidine boranophosphoramidate prodrug was accomplished using a phosphoramidite approach in high yield. This new class of compounds is designed to have improved antiviral and anticancer advantages conferred by combining the boranophosphate and normal nucleoside amino acid phosphoramidate. Compounds were characterized by MS and 31P NMR.

Versatility of borane nucleic acids mimics for coding, decoding and modulating genetic information.

This presentation will focus on the targeted regulation of gene expression, effective siRNA silencing with boranophosphates, and suppression of drugresistant reverse transcriptase by boranophosphate nucleotide analogues.

High potency silencing by single-stranded boranophosphate siRNA.

In RNA interference (RNAi), double-stranded short interfering RNA (ds-siRNA) inhibits expression from complementary mRNAs. Recently, it was demonstrated that short, single-stranded antisense RNA (ss-siRNA) can also induce RNAi. While ss-siRNA may offer several advantages in both clinical and research applications, its overall poor activity compared with ds-siRNA has prevented its widespread use. In contrast to the poor gene silencing activity of native ss-siRNA, we found that the silencing activity of boranophosphate-modified ss-siRNA is comparable with that of unmodified ds-siRNA. Boranophosphate ss-siRNA has excellent maximum silencing activity and is highly effective at low concentrations. The silencing activity of boranophosphate ss-siRNA is also durable, with significant silencing up to 1 week after transfection. Thus, we have demonstrated that boranophosphate-modified ss-siRNA can silence gene expression as well as native ds-siRNA, suggesting that boranophosphate-modified ss-siRNAs should be investigated as a potential new class of therapeutic agents.

Synthesis of alpha-P-modified nucleoside diphosphates with ethylenediamine.

This report describes a one-pot synthesis of alpha-P-borano-, alpha-P-thio-, and alpha-P-seleno-modified nucleoside diphosphate analogues that are otherwise difficult to obtain. The key step involves the intramolecular nucleophilic attack by an amino group in 5 to remove the gamma-phosphate. The absolute configurations of P-diastereomers were confirmed by analysis of their 1H NMR. Affinity studies revealed that the nucleoside boranodiphosphates are potentially useful in antiviral research.

Incorporation of ribonucleoside 5'-(alpha-P-borano)triphosphates into a 20-mer RNA by T7 RNA polymerase.

The enzymatic synthesis of short boranophosphate RNA was studied by comparing the yield and pattern of abortive products of in vitro transcription at steady-state conditions with that of normal RNA. Boranophosphate short RNA can be readily synthesized by T7 RNA polymerase.

Synthesis of 5-(1-propynyl)-2'-deoxyuridine 5'-(alpha-P-borano)triphosphate and kinetic characterization as a substrate for mmlv reverse transcriptase.

In order to introduce pyrimidine C5-propynyl modification into boranophosphate oligodeoxyribonucleotides (BP- ODNs), 5-(1-propynyl)-2'-deoxyuridine 5'-(alpha-P-borano) triphosphate (d5PUTPalphaB) was synthesized. The two diastereomers were separated by reverse-phase HPLC. Kinetic studies showed that the Rp isomer was a slightly better substrate for MMLV reverse transcriptase than thymidine triphosphate or Rp-thymidine 5'-(alpha-P-borano)triphosphate. Using the Rp isomers of d5PUTPalphaB and the other three 5'-(alpha-P-borano) triphosphates, a DNA primer could be extended to the full length of the template.