Introduction: Fluorescent-activated cell sorting (FACS) is often the most appropriate technique to obtain pure populations of a cell type of interest for downstream analysis. However, aerosol droplets can be generated during the sort, which poses a biosafety risk when working with samples containing risk group 3 pathogens such as Francisella tularensis, Mycobacterium tuberculosis, Yersinia pestis, and severe acute respiratory syndrome coronavirus 2. For many researchers, placing the equipment required for FACS at biosafety level 3 (BSL-3) is often not possible due to expense, space, or expertise available. Methods: We performed aerosol testing as part of the biosafety evaluation of the MACSQuant Tyto, a completely closed, cartridge-based cell sorter. We also established quality control procedures to routinely evaluate instrument performance. Results: The MACSQuant Tyto does not produce aerosols as part of the sort procedure. Discussion: These data serve as guidance for other facilities with containment laboratories wishing to use the MACSQuant Tyto for cell sorting. Potential users should consult with their Institutional Biosafety Committees to perform in-house risk assessments of this equipment. Conclusion: The MACSQuant Tyto can safely be used on the benchtop to sort samples at BSL-3.
The distribution of new HIV infections among key populations, including female sex workers (FSWs), gay men and other men who have sex with men (MSM), and people who inject drugs (PWID) are essential information to guide an HIV response, but data are limited in sub-Saharan Africa (SSA). We analyzed empirically derived and mathematical model-based estimates of HIV incidence among key populations and compared with the Joint United Nations Programme on HIV/AIDS (UNAIDS) estimates.
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Exome sequencing is a powerful tool for discovery of the Mendelian disease genes. Previously, we reported a novel locus for autosomal recessive non-syndromic mental retardation (NSMR) in a consanguineous family [Nolan, D.K., Chen, P., Das, S., Ober, C. and Waggoner, D. (2008) Fine mapping of a locus for nonsyndromic mental retardation on chromosome 19p13. Am. J. Med. Genet. A, 146A, 1414–1422]. Using linkage and homozygosity mapping, we previously localized the gene to chromosome 19p13. The parents of this sibship were recently included in an exome sequencing project. Using a series of filters, we narrowed the putative causal mutation to a single variant site that segregated with NSMR: the mutation was homozygous in five affected siblings but in none of eight unaffected siblings. This mutation causes a substitution of a leucine for a highly conserved proline at amino acid 182 in TECR (trans-2,3-enoyl-CoA reductase), a synaptic glycoprotein. Our results reveal the value of massively parallel sequencing for identification of novel disease genes that could not be found using traditional approaches and identifies only the seventh causal mutation for autosomal recessive NSMR.
Background: Unmyelinated primary afferent nociceptors are commonly classified into two main functional types: those expressing neuropeptides, and non-peptidergic fibers that bind the lectin IB4. However, many small diameter primary afferent neurons neither contain any known neuropeptides nor bind IB4. Most express high levels of vesicular glutamate transporter 2 (VGluT2) and are assumed to be glutamatergic nociceptors but their terminations within the spinal cord are unknown. We used in vitro anterograde axonal tracing with Neurobiotin to identify the central projections of these putative glutamatergic nociceptors. We also quantitatively characterised the spatial arrangement of these terminals with respect to those that expressed the neuropeptide, calcitonin gene-related peptide (CGRP). Results: Neurobiotin-labeled VGluT2-immunoreactive (IR) terminals were restricted to lamina I, with a medial-tolateral distribution similar to CGRP-IR terminals. Most VGluT2-IR terminals in lateral lamina I were not labeled by Neurobiotin implying that they arose mainly from central neurons. 38 ± 4% of Neurobiotin-labeled VGluT2-IR terminals contained CGRP-IR. Conversely, only 17 ±4% of Neurobiotin-labeled CGRP-IR terminals expressed detectable VGluT2-IR. Neurobiotin-labeled VGluT2-IR or CGRP-IR terminals often aggregated into small clusters or microdomains partially surrounding intrinsic lamina I neurons. Conclusions: The central terminals of primary afferents which express high levels of VGluT2-IR but not CGRP-IR terminate mainly in lamina I. The spatial arrangement of VGluT2-IR and CGRP-IR terminals suggest that lamina I neurons receive convergent inputs from presumptive nociceptors that are primarily glutamatergic or peptidergic. This reveals a previously unrecognized level of organization in lamina I consistent with the presence of multiple nociceptive processing pathways.
Prospective studies are needed to examine the temporal relationship between oral human papillomavirus (HPV) detection and risk of head and neck squamous cell carcinoma (HNSCC). Moreover, the oral cavity contains a wide spectrum of α-, β-, and γ-HPV types, but their association with risk of HNSCC is unknown.To prospectively examine associations between α-, β-, and γ-HPV detection in the oral cavity and incident HNSCC.A nested case-control study was carried out among 96 650 participants, cancer free at baseline, with available mouthwash samples in 2 prospective cohort studies: (1) the American Cancer Society Cancer Prevention Study II Nutrition Cohort and (2) the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Incident cases of HNSCC (n = 132) were identified during an average 3.9 years of follow-up in both cohorts. Three controls per case (n = 396) were selected through incidence density sampling and matched on age, sex, race/ethnicity, and time since mouthwash collection.Through a next-generation sequencing assay, DNA from α-, β-, and γ-HPV types were detected. Conditional logistic regression models were used to estimate odds ratios (ORs) and 95% CIs, adjusting for smoking history, alcohol consumption, and detection of HPV-16 for β- and γ-HPVs.Incident HNSCC, which includes cancers of the oropharynx, oral cavity, and larynx.A total of 132 participants developed HNSCC during the follow-up period (103 men and 29 women; average age at baseline, 66.5 years). Oral HPV-16 detection was associated with incident HNSCC (OR, 7.1; 95% CI, 2.2-22.6), with positive association for oropharyngeal SCC (OR, 22.4; 95% CI, 1.8-276.7), but not for oral cavity (OR, 4.5; 95% CI, 0.6-34.7) or laryngeal SCCs (OR, 0.11; 95% CI, 0.01-834.80). Detection of β1-HPV-5 and β2-HPV-38 types, as well as γ-11 and γ-12 species, had ORs for HNSCC that ranged from 2.64 to 5.45 (P < .01 for all comparisons). Detection of β1-HPV-5 type was associated with oropharyngeal (OR, 7.42; 95% CI, 0.98-56.82; P = .054), oral cavity (OR, 5.34; 95% CI, 1.51-18.80; P = .01), and laryngeal SCCs (OR, 2.71; 95% CI, 1.00-7.43; P = .05), whereas γ11- and γ12-HPV species were associated with both oral cavity (OR, 7.47; 95% CI, 1.21-46.17; P = .03; and OR, 6.71; 95% CI, 1.47-30.75; P = .01, respectively) and laryngeal SCCs (OR, 7.49; 95% CI, 1.10-51.04; P = .04 and OR, 5.31; 95% CI, 1.13-24.95; P = .03, respectively).This study demonstrates that HPV-16 detection precedes the incidence of oropharyngeal SCC. Associations of other HPVs, including γ11- and γ12-HPV species and β1-HPV-5 type suggest a broader role for HPVs in HNSCC etiology.
Key populations (KPs), including female sex workers (FSWs), gay men and other men who have sex with men (MSM), people who inject drugs (PWID), and transgender women (TGW) experience disproportionate risks of HIV acquisition. The UNAIDS Global AIDS 2022 Update reported that one-quarter of all new HIV infections occurred among their non-KP sexual partners. However, this fraction relied on heuristics regarding the ratio of new infections that KPs transmitted to their non-KP partners to the new infections acquired among KPs (herein referred to as "infection ratios"). We recalculated these ratios using dynamic transmission models.
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