Caged Cumate Enables Proximity-Dependent Control Over Gene Expression.

Cell-cell interactions underlie diverse physiological processes yet remain challenging to examine with conventional imaging tools. Here we report a novel strategy to illuminate cell proximity using transcriptional activators. We repurposed cumate, a small molecule inducer of gene expression, by caging its key carboxylate group with a nitrile. Nitrilase-expressing activator cells released the cage, liberating cumate for consumption by reporter cells. Reporter cells comprising a cumate-responsive switch expressed a target gene when in close proximity to the activator cells. Overall, this strategy provides a versatile platform to image and potentially manipulate cellular interactions over time.

Characteristics of coronavirus disease 19 convalescent plasma donors and donations in the New York metropolitan area.

BACKGROUND: Convalescent plasma (CP) is an important initial treatment in pandemics and the New York (NY) metropolitan area is likely to remain a hotspot for collection and distribution of such units. This study reports characteristics of coronavirus disease 19 CP (CCP) donors and their donations to the New York Blood Center (NYBC). STUDY DESIGN AND METHODS: All CCP data from our first day of collection on March 26th through July 7th, 2020 are included in this retrospective analysis. Donor and donation data were extracted from NYBC electronic databases. SARS-CoV-2 antibody testing was initially performed by the NY State Department of Health, and later by NYBC using Ortho and Abbott platforms. RESULTS: CCP donor age and ABO distributions were consistent with reported lower COVID-19 susceptibility in O blood types. CCP versus whole blood donors had similar on-site deferrals, but higher post-donation deferral rates. CCP versus routine plasmapheresis donations had higher vasovagal reactions but similar product rejection rates. Changes in antibody (Ab) test platforms resulted in significant changes in the percent of donors regarded as antibody positive. Donor correlates with higher anti-spike total Ig S/CO ratios were Hispanic ethnicity, overweight body mass index, and longer symptom duration; and with higher anti-nucleocapsid IgG S/CO ratios were male gender, older age, Hispanic ethnicity, and fewer days between symptom onset and first donation. DISCUSSION: We identify donor characteristics not previously reported to correlate with Ab titer. Our analysis should assist with donor outreach strategies, blood center operating logistics, and recruitment of high titer donors.

Genome sequence, metabolic properties and cyanobacterial attachment of Porphyrobacter sp. HT-58-2 isolated from a filamentous cyanobacterium-microbial consortium.

Tolyporphins are structurally diverse tetrapyrrole macrocycles produced by the cyanobacterial culture HT-58-2. Although tolyporphins were discovered over 25 years ago, little was known about the microbiology of the culture. The studies reported herein expand the description of the community of predominantly alphaproteobacteria associated with the filamentous HT-58-2 cyanobacterium and isolate a dominant bacterium, Porphyrobacter sp. HT-58-2, for which the complete genome is established and growth properties are examined. Fluorescence in situ hybridization (FISH) analysis of the cyanobacterium-microbial community with a probe targeting the 16S rRNA of Porphyrobacter sp. HT-58-2 showed fluorescence emanating from the cyanobacterial sheath. Although genes for the biosynthesis of bacteriochlorophyll a (BChl a) are present in the Porphyrobacter sp. HT-58-2 genome, the pigment was not detected under the conditions examined, implying the absence of phototrophic growth. Comparative analysis of four Porphyrobacter spp. genomes from worldwide collection sites showed significant collinear gene blocks, with two inversions and three deletion regions. Taken together, the results enrich our understanding of the HT-58-2 cyanobacterium-microbial culture.

A Review of the Risk of Cow's Milk Protein Allergy in Oral Medication.

Immunoglobulin E (IgE)-mediated cow's milk allergy (CMA) is a common food reaction resulting from the consumption of cow's milk protein (CMP). The clinical manifestations of CMA include mild to severe urticaria, skin-manifested hypersensitivity reactions, and anaphylaxis. Food allergies may affect 8% of children and 10% of adults. The Federal Food, Drug, and Cosmetic Act (FD&C Act) requires that the label of a food must declare the presence of a "major food allergen" (MFA) contained in the food or ingredient. The Food and Drug Administration (FDA) generally regards milk protein concentrate (MPC) as safe for human consumption and use. The increasing use of MPC in formulations raises the need for its revelation in prescription and on labels of over-the-counter drugs. This review investigates oral and topical (including mucosal) preparations containing MPC for dermatologic and other uses and their therapeutic impact. Our findings suggest that for the adult population, the risk of serious cow's milk protein allergy (CMPA) from medications is minimal.

Myoblast deactivation within engineered human skeletal muscle creates a transcriptionally heterogeneous population of quiescent satellite-like cells.

Satellite cells (SCs), the adult Pax7-expressing stem cells of skeletal muscle, are essential for muscle repair. However, in vitro investigations of SC function are challenging due to isolation-induced SC activation, loss of native quiescent state, and differentiation to myoblasts. In the present study, we optimized methods to deactivate in vitro expanded human myoblasts within a 3D culture environment of engineered human skeletal muscle tissues ("myobundles"). Immunostaining and gene expression analyses revealed that a fraction of myoblasts within myobundles adopted a quiescent phenotype (3D-SCs) characterized by increased Pax7 expression, cell cycle exit, and activation of Notch signaling. Similar to native SCs, 3D-SC quiescence is regulated by Notch and Wnt signaling while loss of quiescence and reactivation of 3D-SCs can be induced by growth factors including bFGF. Myobundle injury with a bee toxin, melittin, induces robust myofiber fragmentation, functional decline, and 3D-SC proliferation. By applying single cell RNA-sequencing (scRNA-seq), we discover the existence of two 3D-SC subpopulations (quiescent and activated), identify deactivation-associated gene signature using trajectory inference between 2D myoblasts and 3D-SCs, and characterize the transcriptomic changes within reactivated 3D-SCs in response to melittin-induced injury. These results demonstrate the ability of an in vitro engineered 3D human skeletal muscle environment to support the formation of a quiescent and heterogeneous SC population recapitulating several aspects of the native SC phenotype, and provide a platform for future studies of human muscle regeneration and disease-associated SC dysfunction.

Three-dimensional tissue-engineered human skeletal muscle model of Pompe disease.

In Pompe disease, the deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) causes skeletal and cardiac muscle weakness, respiratory failure, and premature death. While enzyme replacement therapy using recombinant human GAA (rhGAA) can significantly improve patient outcomes, detailed disease mechanisms and incomplete therapeutic effects require further studies. Here we report a three-dimensional primary human skeletal muscle ("myobundle") model of infantile-onset Pompe disease (IOPD) that recapitulates hallmark pathological features including reduced GAA enzyme activity, elevated glycogen content and lysosome abundance, and increased sensitivity of muscle contractile function to metabolic stress. In vitro treatment of IOPD myobundles with rhGAA or adeno-associated virus (AAV)-mediated hGAA expression yields increased GAA activity and robust glycogen clearance, but no improvements in stress-induced functional deficits. We also apply RNA sequencing analysis to the quadriceps of untreated and AAV-treated GAA-/- mice and wild-type controls to establish a Pompe disease-specific transcriptional signature and reveal novel disease pathways. The mouse-derived signature is enriched in the transcriptomic profile of IOPD vs. healthy myobundles and partially reversed by in vitro rhGAA treatment, further confirming the utility of the human myobundle model for studies of Pompe disease and therapy.