Addressing food insecurity in HIV care: perspectives from healthcare and social service providers in New York state.

Ending the HIV epidemic in the United States will require addressing social determinants contributing to poor care engagement among people living with HIV (PLH), such as food insecurity. Food insecurity is associated with poor care engagement among PLH. Yet, few studies have examined the perspectives of healthcare and social services providers on addressing food insecurity in HIV care. Guided by the Social Ecological Model, we conducted semi-structured interviews with 18 providers in New York State to understand barriers and facilitators to addressing food insecurity in HIV care. Thematic analysis illustrated eight themes across various levels of the Social Ecological Model. At the patient-level, providers perceived patients' feelings of embarrassment, shame, and judgement, and low health literacy as barriers. At the provider-level, challenges included limited time. Facilitators included fostering strong, patient-provider relationships. Barriers at the clinic-level included limited funding, while clinic resources served as facilitators. At the community-level, challenges included intersecting stigmas arising from community norms towards PLH and people who receive food assistance and limited access to healthy food. Findings suggest the need to incorporate their insights into the development of interventions that address food insecurity in HIV care.

Standards for Cell Line Authentication and Beyond.

Different genomic technologies have been applied to cell line authentication, but only one method (short tandem repeat [STR] profiling) has been the subject of a comprehensive and definitive standard (ASN-0002). Here we discuss the power of this document and why standards such as this are so critical for establishing the consensus technical criteria and practices that can enable progress in the fields of research that use cell lines. We also examine other methods that could be used for authentication and discuss how a combination of methods could be used in a holistic fashion to assess various critical aspects of the quality of cell lines.

Development and interlaboratory evaluation of a NIST Reference Material RM 8366 for EGFR and MET gene copy number measurements.

Background The National Institute of Standards and Technology (NIST) Reference Material RM 8366 was developed to improve the quality of gene copy measurements of EGFR (epidermal growth factor receptor) and MET (proto-oncogene, receptor tyrosine kinase), important targets for cancer diagnostics and treatment. The reference material is composed of genomic DNA prepared from six human cancer cell lines with different levels of amplification of the target genes. Methods The reference values for the ratios of the EGFR and MET gene copy numbers to the copy numbers of reference genes were measured using digital PCR. The digital PCR measurements were confirmed by two additional laboratories. The samples were also characterized using Next Generation Sequencing (NGS) methods including whole genome sequencing (WGS) at three levels of coverage (approximately 1 ×, 5 × and greater than 30 ×), whole exome sequencing (WES), and two different pan-cancer gene panels. The WES data were analyzed using three different bioinformatic algorithms. Results The certified values (digital PCR) for EGFR and MET were in good agreement (within 20%) with the values obtained from the different NGS methods and algorithms for five of the six components; one component had lower NGS values. Conclusions This study shows that NIST RM 8366 is a valuable reference material to evaluate the performance of assays that assess EGFR and MET gene copy number measurements.

Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine.

BACKGROUND: Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use. METHODS: We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (KRAS c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement. RESULTS: Concentration values for samples of KRAS G12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%-8% and 5%-10%, respectively). CONCLUSIONS: This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.

NIST Spectroscopic Measurement Standards.

Ultraviolet (UV) absorbance measurements provide a rapid and reliable method to determine protein concentrations. the National Institute of standards and technology (NIST) has developed Standard Reference Material (SRM) 2082 as a pathlength standard for UV absorbance measurements for use with the new generation of microvolume spectrophotometers and short-pathlength cuvettes. short pathlengths are used with high-concentration targets to ensure that absorbance values are within the optimal range. the short-pathlength instruments and cuvettes also reduce the required volumes to conserve valuable samples. the authors compared the results obtained with high-quality dual-beam spectrophotometers and short-pathlength cuvettes to the results obtained from a microvolume spectrophotometer and a microvolume plate reader. SRM 2082 can be used to accurately calculate pathlength values, thereby increasing the accuracy in subsequent measurements using the short-pathlength cuvettes and microvolume absorbance instruments. RM 8671 (reference material, the NISTmAb) can then be used to ensure the accuracy and reproducibility of protein concentration measurements by providing an industrially relevant reference material, a well-characterized monoclonal antibody.

Development of NIST Standard Reference Material 2082, a Pathlength Standard for Measurements in the Ultraviolet Spectrum.

New spectrophotometers and cuvettes have been designed to allow the measurement of absorbance values from samples using microliter volume sizes. These measurements are done using short pathlengths to decrease the sample volumes required. The major applications for these spectrophotometers and cuvettes are samples that are difficult to obtain in large amounts, such as proteins and nucleic acids that absorb light in the ultraviolet range. Existing ultraviolet absorbance standards have been designed for longer pathlength measurements. Standard Reference Material (SRM) 2082 was developed to validate the pathlengths of short-pathlength cuvettes and instruments using materials with absorbance spectra that are similar to the most commonly used samples. SRM 2082 consists of three individual components: a blank buffer solution, a solution of the amino acid tryptophan in the buffer, and a solution of the nucleobase uracil in the buffer. The tryptophan solution has an absorbance spectrum (peak at 280 nm) similar to proteins, and the uracil has an absorbance spectrum (peak at 260 nm) similar to nucleic acids. The absorbance values of these solutions were determined using a series of cuvettes with pathlengths from 0.1 mm to 2 mm. The pathlengths of the cuvettes used for the absorbance measurements were determined at the National Institute of Standards and Technology by physical and optical measurements. The effects of temperature and spectral bandwidth variations on the absorbance values of SRM 2082 were also investigated.

Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry.

BACKGROUND: In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells. METHOD: Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry. RESULTS: The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10(5) and (0.85 ± 0.11) × 10(5), respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC. CONCLUSION: Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

Perspectives on Injectable HIV Pre-Exposure Prophylaxis: A Qualitative Study of Health Care Providers in the United States.

The introduction of injectable HIV pre-exposure prophylaxis (PrEP) has the potential to significantly change the biomedical HIV prevention landscape. However, effective implementation will require health care providers to adopt, prescribe, and administer injectable PrEP within clinical settings. This study qualitatively examined challenges and benefit of injectable PrEP implementation from the perspective of health care providers. From April to August 2022, we conducted 19 in-depth interviews with current PrEP-prescribing health care providers in New York State, including 3 physician assistants, 5 physicians, and 11 nurse practitioners. Interviews were audio-recorded, transcribed verbatim, and thematically analyzed to report semantic-level themes regarding injectable PrEP implementation. More than half of participants (61%) were aware of injectable PrEP; only 21% had experience prescribing it. Qualitative findings highlighted five themes. Three themes represented implementation challenges, including speculative concerns about side effects, appointment compliance, and practical and logistical considerations. The remaining two themes described benefits of injectable PrEP relative to oral PrEP, which included greater convenience and enhanced privacy. Findings from this qualitative study make significant applied contributions to the sparse knowledge on health care provider perspectives of injectable PrEP post-US Food and Drug Administration approval and their concerns and considerations regarding implementation in real-world clinical settings.

Quantitation and integrity evaluation of RNA genome in lentiviral vectors by direct reverse transcription-droplet digital PCR (direct RT-ddPCR).

Lentiviral vectors (LV) have proven to be powerful tools for stable gene delivery in both dividing and non-dividing cells. Approval of these LVs for use in clinical applications has been achieved by improvements in LV design. Critically important characteristics concerning quality control are LV titer quantification and the detection of impurities. However, increasing evidence concerning high variability in titration assays indicates poor harmonization of the methods undertaken to date. In this study, we developed a direct reverse transcription droplet digital PCR (Direct RT-ddPCR) approach without RNA extraction and purification for estimation of LV titer and RNA genome integrity. The RNA genome integrity was assessed by RT-ddPCR assays targeted to four distant regions of the LV genome. Results of the analyses showed that direct RT-ddPCR without RNA extraction and purification performs similarly to RT-ddPCR on purified RNA from 3 different LV samples, in terms of robustness and assay variance. Interestingly, these RNA titer results were comparable to physical titers by p24 antigen ELISA (enzyme-linked immunosorbent assay). Moreover, we confirmed the partial degradation or the incomplete RNA genomes in the prepared 3 LV samples. These results may partially explain the discrepancy of the LV particle titers to functional titers. This work not only demonstrates the feasibility of direct RT-ddPCR in determining LV titers, but also provides a method that can be easily adapted for RNA integrity assessment.

Human CD4+ lymphocytes for antigen quantification: characterization using conventional flow cytometry and mass cytometry.

To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto-Trol and PBMC-National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto-Trol is lower and the value on PBMC-NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOF™ mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD4(+) cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4(+) cells from lyophilized PBMC-NIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach.

Plasma contains ultrashort single-stranded DNA in addition to nucleosomal cell-free DNA.

Plasma cell-free DNA is being widely explored as a biomarker for clinical screening. Currently, methods are optimized for the extraction and detection of double-stranded mononucleosomal cell-free DNA of ∼160bp in length. We introduce uscfDNA-seq, a single-stranded cell-free DNA next-generation sequencing pipeline, which bypasses previous limitations to reveal a population of ultrashort single-stranded cell-free DNA in human plasma. This species has a modal size of 50nt and is distinctly separate from mononucleosomal cell-free DNA. Treatment with single-stranded and double-stranded specific nucleases suggests that ultrashort cell-free DNA is primarily single-stranded. It is distributed evenly across chromosomes and has a similar distribution profile over functional elements as the genome, albeit with an enrichment over promoters, exons, and introns, which may be suggestive of a terminal state of genome degradation. The examination of this cfDNA species could reveal new features of cell death pathways or it can be used for cell-free DNA biomarker discovery.

Authentication of African green monkey cell lines using human short tandem repeat markers.

BACKGROUND: Tools for authenticating cell lines are critical for quality control in cell-based biological experiments. Currently there are methods to authenticate human cell lines using short tandem repeat (STR) markers based on the technology and procedures successfully used in the forensic community for human identification, but there are no STR based methods for authenticating nonhuman cell lines to date. There is significant homology between the human and vervet monkey genome and we utilized these similarities to design the first multiplex assay based on human STR markers for vervet cell line identification. RESULTS: The following STR markers were incorporated into the vervet multiplex PCR assay: D17S1304, D5S1467, D19S245, D1S518, D8S1106, D4S2408, D6S1017, and DYS389. The eight markers were successful in uniquely identifying sixty-two vervet monkey DNA samples and confirmed that Vero76 cells and COS-7 cells were derived from Vero and CV-1 cells, respectively. The multiplex assay shows specificity for vervet DNA within the determined allele range for vervet monkeys; however, the primers will also amplify human DNA for each marker resulting in amplicons outside the vervet allele range in several of the loci. The STR markers showed genetic stability in over sixty-nine passages of Vero cells, suggesting low mutation rates in the targeted STR sequences in the Vero cell line. CONCLUSIONS: A functional vervet multiplex assay consisting of eight human STR markers with heterozygosity values ranging from 0.53-0.79 was successful in uniquely identifying sixty-two vervet monkey samples. The probability of a random match using these eight markers between any two vervet samples is approximately 1 in 1.9 million. While authenticating a vervet cell line, the multiplex assay may also be a useful indicator for human cell line contamination since the assay is based on human STR markers.

Past and ongoing shifts in Joshua tree distribution support future modeled range contraction.

The future distribution of the Joshua tree (Yucca brevifolia) is projected by combining a geostatistical analysis of 20th-century climates over its current range, future modeled climates, and paleoecological data showing its response to a past similar climate change. As climate rapidly warmed approximately 11 700 years ago, the range of Joshua tree contracted, leaving only the populations near what had been its northernmost limit. Its ability to spread northward into new suitable habitats after this time may have been inhibited by the somewhat earlier extinction of megafaunal dispersers, especially the Shasta ground sloth. We applied a model of climate suitability for Joshua tree, developed from its 20th-century range and climates, to future climates modeled through a set of six individual general circulation models (GCM) and one suite of 22 models for the late 21st century. All distribution data, observed climate data, and future GCM results were scaled to spatial grids of approximately 1 km and approximately 4 km in order to facilitate application within this topographically Complex region. All of the models project the future elimination of Joshua tree throughout most of the southern portions of its current range. Although estimates of future monthly precipitation differ between the models, these changes are outweighed by large increases in temperature common to all the models. Only a few populations within the current range are predicted to be sustainable. Several models project significant potential future expansion into new areas beyond the current range, but the species' historical and current rates of dispersal would seem to prevent natural expansion into these new areas. Several areas are predicted to be potential sites for relocation/ assisted migration. This project demonstrates how information from paleoecology and modern ecology can be integrated in order to understand ongoing processes and fuiture distributions.

Reference standards for accurate validation and optimization of assays that determine integrated lentiviral vector copy number in transduced cells.

Lentiviral vectors (LV) have emerged as a robust technology for therapeutic gene delivery into human cells as advanced medicinal products. As these products are increasingly commercialized, there are concomitant demands for their characterization to ensure safety, efficacy and consistency. Standards are essential for accurately measuring parameters for such product characterization. A critical parameter is the vector copy number (VCN) which measures the genetic dose of a transgene present in gene-modified cells. Here we describe a set of clonal Jurkat cell lines with defined copy numbers of a reference lentiviral vector integrated into their genomes. Genomic DNA was characterized for copy number, genomic integrity and integration coordinates and showed uniform performance across independent quantitative PCR assays. Stability studies during continuous long-term culture demonstrated sustained renewability of the reference standard source material. DNA from the Jurkat VCN standards would be useful for control of quantitative PCR assays for VCN determination in LV gene-modified cellular products and clinical samples.

Validation of ctDNA Quality Control Materials Through a Precompetitive Collaboration of the Foundation for the National Institutes of Health.

We report the results from a Foundation for the National Institutes of Health Biomarkers Consortium project to address the absence of well-validated quality control materials (QCMs) for circulating tumor DNA (ctDNA) testing. This absence is considered a cause of variance and inconsistencies in translating ctDNA results into clinical actions. METHODS: In this phase I study, QCMs with 14 clinically relevant mutations representing single nucleotide variants, insertions or deletions (indels), translocations, and copy number variants were sourced from three commercial manufacturers with variant allele frequencies (VAFs) of 5%, 2.5%, 1%, 0.1%, and 0%. Four laboratories tested samples in quadruplicate using two allele-specific droplet digital polymerase chain reaction and three (amplicon and hybrid capture) next-generation sequencing (NGS) panels. RESULTS: The two droplet digital polymerase chain reaction assays reported VAF values very close to the manufacturers' claimed concentrations for all QCMs. NGS assays reported most single nucleotide variants and indels, but not translocations, close to the expected VAF values. Notably, two NGS assays reported lower VAF than expected for all translocations in all QCM mixtures, possibly related to technical challenges detecting these variants. The ability to call ERBB2 copy number amplifications varied across assays. All three QCMs provided valuable insight into assay precision. Each assay across all variant types demonstrated dropouts at 0.1%, suggesting that the QCM can serve for testing of an assay's limit of detection with confidence claims for specific variants. CONCLUSION: These results support the utility of the QCM in testing ctDNA assay analytical performance. However, unique designs and manufacturing methods for the QCM, and variations in a laboratory's testing configuration, may require testing of multiple QCMs to find the best reagents for accurate result interpretation.

Vegetation response to early holocene warming as an analog for current and future changes.

Temperatures in southwestern North America are projected to increase 3.5-4 degrees C over the next 60-90 years. This will precipitate ecological shifts as the ranges of species change in response to new climates. During this shift, rapid-colonizing species should increase, whereas slow-colonizing species will at first decrease, but eventually become reestablished in their new range. This successional process has been estimated to require from 100 to over 300 years in small areas, under a stable climate, with a nearby seed source. How much longer will it require on a continental scale, under a changing climate, without a nearby seed source? I considered this question through an examination of the response of fossil plant assemblages from the Grand Canyon, Arizona, to the most recent rapid warming of similar magnitude that occurred at the start of the Holocene, 11,700 years ago. At that time, temperatures in southwestern North America increased about 4 degrees C over less than a century. Grand Canyon plant species responded at different rates to this warming climate. Early-successional species rapidly increased, whereas late-successional species decreased. This shift persisted throughout the next 2700 years. I found two earlier, less-extreme species shifts following rapid warming events around 14,700 and 16,800 years ago. Late-successional species predominated only after 4000 years or more of relatively stable temperature. These results suggest the potential magnitude, duration, and nature of future ecological changes and have implications for conservation plans, especially those incorporating equilibrium assumptions or reconstituting past conditions. When these concepts are extended to include the most rapid early-successional colonizers, they imply that the recent increases in invasive exotics may be only the most noticeable part of a new resurgence of early-successional vegetation. Additionally, my results challenge the reliability of models of future vegetation and carbon balance that project conditions on the basis of assumptions of equilibrium within only a century.

Development of a candidate secondary reference procedure (immunoassay based measurement procedure of higher metrological order) for cardiac troponin I: I. Antibody characterization and preliminary validation.

In this study, the first steps in the development of a secondary reference measurement procedure (RMP) 'higher metrological order measurement procedure' to support the cardiac troponin I (cTnI) standardization initiative is described. The RMP should be used to assign values to serum-based secondary reference materials (RMs) without analytical artifacts causing bias. A multiplexed bead-based assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to identify the optimum monoclonal antibody pair (clones 560 and 19C7) for the RMP. Using these antibodies, an ELISA-based procedure was developed to accurately measure the main cTnI forms present in blood. The proposed RMP appears to show no bias when tested on samples containing various troponin complexes, phosphorylated and dephosphorylated forms, and heparin. The candidate assay displayed suitable linearity and sensitivity (limit of detection, 0.052 µg/L) for the measurement of the proposed cTnI secondary RMs. Preliminary comparison data on patient samples with a commercial cTnI assay are also provided to support the suitability of RMP for value assignment to RMs. Full validation and final assessment of the RMP will be performed through transferability and inter-comparison studies.

U.S. Government engagement in support of global disease surveillance.

Global cooperation is essential for coordinated planning and response to public health emergencies, as well as for building sufficient capacity around the world to detect, assess and respond to health events. The United States is committed to, and actively engaged in, supporting disease surveillance capacity building around the world. We recognize that there are many agencies involved in this effort, which can become confusing to partner countries and other public health entities. This paper aims to describe the agencies and offices working directly on global disease surveillance capacity building in order to clarify the United States Government interagency efforts in this space.

Multilaboratory Assessment of a New Reference Material for Quality Assurance of Cell-Free Tumor DNA Measurements.

We conducted a multilaboratory assessment to determine the suitability of a new commercially available reference material with 40 cancer variants in a background of wild-type DNA at four different variant allele frequencies (VAFs): 2%, 0.50%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected for their clinical importance and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. DNA was extracted from these samples and characterized with different methods and multiple laboratories. The various extraction methods had differences in yield, perhaps because of differences in chemistry. Digital PCR assays were used to measure VAFs to compare results from different NGS methods. Comparable VAFs were observed across the different NGS methods. This multilaboratory assessment demonstrates that the new reference material is an appropriate tool to determine the analytical parameters of different measurement methods and to ensure their quality assurance.

Ancient DNA provides evidence of 27,000-year-old papillomavirus infection and long-term codivergence with rodents.

The long-term evolutionary history of many viral lineages is poorly understood. Novel sources of ancient DNA combined with phylogenetic analyses can provide insight into the time scale of virus evolution. Here we report viral sequences from ancient North American packrat middens. We screened samples up to 27,000-years old and found evidence of papillomavirus (PV) infection in Neotoma cinerea (Bushy-tailed packrat). Phylogenetic analysis placed the PV sequences in a clade with other previously published PV sequences isolated from rodents. Concordance between the host and virus tree topologies along with a correlation in branch lengths suggests a shared evolutionary history between rodents and PVs. Based on host divergence times, PVs have likely been circulating in rodents for at least 17 million years. These results have implications for our understanding of PV evolution and for further research with ancient DNA from Neotoma middens.