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The global pandemic due to SARS-CoV2 virus, also known as COVID-19, has drastically increased the need for nasopharyngeal-swab-based testing resulting in shortages of commercially available nasopharyngeal (NP) swabs. One solution to overcome the national deficit of swabs is for medical device manufacturers and hospitals to generate NP swabs. Numerous entities are attempting to manufacture a direct from 3D printing NP swab but presented here is a validated two-part swab manufacturing protocol utilizing 3D printing and manual intervention (wrapping of nylon fibers). We recommend material extrusion or powder bed fusion technologies utilizing materials that can be sterilized using high level heat decontamination. Through the application of 3D printing and manual fabrication, we present an NP swab that can be created in a controlled environment. Coupled with CDC published viral transport media, the following swab can be used for COVID-19 testing or for testing for other respiratory viruses (eg, influenza, respiratory syncytial virus). The CDC viral transport medium has only four reagents which are readily available.

Healthcare workers (HCW) due to occupational exposure to patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a population at risk during the current coronavirus disease-2019 (COVID-19) pandemic. Pre-existing immunity induced by the exposition to several pathogens, including respiratory viruses, can shape susceptibility to infections but little is known about the presence of cross-reactive memory T cells that have the potential to recognize SARS-CoV-2. Herein we measured CD4+ T cell responses against common cold corona (CCC) viruses and SARS-CoV-2 in high-risk health care workers (HCW) and community controls. We observed higher levels of CCC reactive T cells in SARS-CoV-2 seronegative HCW compared to community donors, consistent with potential higher occupational exposure of HCW to CCC. We further show that SARS-CoV-2 reactivity of seronegative HCW was higher than community controls and correlation between CCC and SARS-CoV-2 responses shown to be consistent with cross-reactivity and not associated with recent in vivo activation. Surprisingly, CCC reactivity was consistently decreased in SARS-CoV-2 infected HCW, suggesting that exposure to SARS-CoV-2 might interfere with CCC responses, either directly or indirectly. This result was unexpected, but consistently detected in independent cohorts derived from Miami and San Diego.

Glucose-regulated protein 94 (GRP94) is one of the most abundant endoplasmic reticulum (ER) resident proteins and is the ER counterpart of the cytoplasmic heat shock protein 90 (HSP90). GRP94, a component of the GRP78 chaperone system in protein processing, has pro-survival properties with implicated function in cancer progression and autoimmune disease. Previous studies on the loss of GRP94 function showed that it is required for embryonic development, regulation of toll-like receptors and innate immunity of macrophages. Here we report the creation of mouse models targeting exon 2 of the Grp94 allele that allows both traditional and conditional knockout (KO) of Grp94. In this study, we utilized the viable Grp94+/+ and +/− mice, as well as primary mouse embryonic fibroblasts generated from them as experimental tools to study its role in ER chaperone balance and ER stress signaling. Our studies reveal that while Grp94 heterozygosity reduces GRP94 level it does not alter ER chaperone levels or the ER stress response. To study the effect of complete loss of GRP94 function, since homozygous GRP94 KO leads to embryonic lethality, we generated Grp94−/− embryonic stem cells. In contrast to Grp94 heterozygosity, complete knockout of GRP94 leads to compensatory upregulation of the ER chaperones GRP78, calnexin and calreticulin but not protein disulphide isomerase. Unexpectedly, loss of GRP94 leads to significant decrease in the level of ER-stress induced spliced form of XBP-1 protein, a downstream target of the IRE1 signaling pathway. Furthermore, from analysis of microarray database and immunohistochemical staining, we present predictions where GRP94 may play an important role in specific adult organ homeostasis and function.

We have examined the actions of the potent GnRH antagonist [N-acetyl-D beta Na11-D-pCl-Phe2-D-Phe3-D-Arg6,Phe7,Arg8-D-Ala10]NH2GnRH++ + (GnRHa) on basal and GnRH-stimulated LH secretion, inositol phospholipid turnover, and intracellular Ca2+ levels in dispersed rat anterior pituitary tissue. As expected, GnRHa was found to be a pure antagonist of secretion, but was paradoxically equipotent with GnRH in stimulating inositol phospholipid turnover. Examination of intracellular Ca2+ changes at the single cell level using digital video-enhanced fluorescence imaging demonstrated that dispersed rat pituitary cells appeared to contain three GnRH analog-responsive cell populations: those that increased intracellular Ca2+ in response to both GnRH and GnRHa, and those that responded to either GnRH or GnRHa only. These observations were extended to studies of the relatively homogeneous gonadotroph cell populations of endocrinologically inactive pituitary adenomas. Of five adenomas examined, one increased inositol phospholipid turnover in response to GnRHa plus GnRH, but not GnRH alone, three responded to GnRH only, and one responded to both GnRH and GnRHa. Our findings, therefore, suggest that three GnRH analog-responsive cell types are also present in human pituitary and that clonal expansion of any of these cell types may be responsible for tumor formation.

DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400-800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30x average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications.