Silk is a protein of interest to both biological and industrial sciences. The silkworm, Bombyx mori, forms this protein into strong threads starting from soluble silk proteins using a number of biochemical and physical cues to allow the transition from liquid to fibrous silk. A pH gradient has been measured along the gland, but the methodology employed was not able to precisely determine the pH at specific regions of interest in the silk gland. Furthermore, the physiological mechanisms responsible for the generation of this pH gradient are unknown. In this study, concentric ion selective microelectrodes were used to determine the luminal pH of Bombyx mori silk glands. A gradient from pH 8.2 to 7.2 was measured in the posterior silk gland, with a pH 7 throughout the middle silk gland, and a gradient from pH 6.8 to 6.2 in the beginning of the anterior silk gland where silk processing into fibers occurs. The small diameter of the most anterior region of the anterior silk gland prevented microelectrode access in this region. Using a histochemical method, the presence of active carbonic anhydrase was identified in the funnel and anterior silk gland of fifth instar larvae. The observed pH gradient collapsed upon addition of the carbonic anhydrase inhibitor methazolamide, confirming an essential role for this enzyme in pH regulation in the Bombyx mori silk gland. Plastic embedding of whole silk glands allowed clear visualization of the morphology, including the identification of four distinct epithelial cell types in the gland and allowed correlations between silk gland morphology and silk stages of assembly related to the pH gradient. Bombyx mori silk glands have four different epithelial cell types, one of which produces carbonic anhydrase. Carbonic anhydrase is necessary for the mechanism that generates an intraluminal pH gradient, which likely regulates the assembly of silk proteins and then the formation of fibers from soluble silk proteins. These new insights into native silk formation may lead to a more efficient production of artificial or regenerated silkworm silk fibers.

Cell-cultured meat offers the potential for a more sustainable, ethical, resilient, and healthy food system. However, research and development has been hindered by the lack of suitable serum-free media that enable the robust expansion of relevant cells (e.g., muscle satellite cells) over multiple passages. Recently, a low-cost serum-free media (B8) was described for induced pluripotent stem cells. Here, we adapt this media for bovine satellite cells and show that the addition of a single component, recombinant albumin, renders B8 suitable for the long-term expansion of cells without sacrificing myogenicity. We show that this new media (Beefy-9) maintains robust cell growth over the entire culture period tested (seven passages) with an average growth rate of 39 hours per population doubling). Along with demonstrated efficacy for bovine cells, this work provides a promising starting-point for developing serum-free media for cultures from other meat-relevant species. Ultimately, this work offers a promising foundation for escaping the reliance on serum in cultured meat research, thereby accelerating the field.

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Enterovirus 71 (EV71), a human viral pathogen, is a member of the Enterovirus genus of the Picornavirus family and causes blisters and ulcers in the mouth and rashes on the hands and feet, commonly known as human hand, foot, and mouth disease (HFMD). The major target populations are children under five years old, who account for 92.23% of the total cases reported [1]. EV71 can also cause severe clinical symptoms of polio-like paralysis, meningitis, and fatal encephalitis with pulmonary edema and cardiac failure in extreme cases, especially in the age group under one year [1, 2]. Because there are no welldefined parameters to predict the mild, self-limiting form of the disease from the severe form of HFMD, extreme precautions are necessary when administering care to pediatric HFMD patients [3-5]. Over the last decade, largescale outbreaks of EV71 infection-associated HFMD have occurred in the Asia-Pacific regions [6-9]. The cost of public health care to manage EV71 outbreaks was significant. Since no effective antiviral agents are readily available, developing EV71 vaccines for primary prevention among the very young is considered to be the best control strategy against EV71 [2, 5, 10]. Inactivated whole-virus vaccines are a favorable choice for EV71 vaccine development [11-13]. Due to the

<p>Hierarchical molecular assembly directed by cell-regulated aqueous solvent is a fundamental strategy for manufacturing various proteinaceous structures that are of intense interest for nanotechnology, sustainable manufacturing and regenerative medicine. However, to translate the natural strategy into advanced digital manufacturing like three-dimensional (3D) printing remains a tremendous technical and theoretical challenge. This work presents a 3D printing technique of a particular protein, silk fibroin, by rationally designing an<i> de novo </i>aqueous salt bath capable of directing the hierarchical assembly of the protein molecules. This technique, conducted under aqueous and ambient conditions, results in 3D proteinaceous architectures characterized by intrinsic biocompatibility/biodegradability and remarkable mechanical performance. The versatility of this method is shown in a diversity of 3D shapes and a range of functional components integrated into the 3D prints. Exceptional manufacturing capability and one promising application is exemplified by the single-step construction of perfusable microfluidic chips, also an analogy of small-diameter vascular grafts, which eliminates the use of supporting or sacrificial materials owing to optimized crosslinking dynamics and compartmentalized printing parameters. The 3D shaping capability of the protein material can benefit a multitude of biomedical devices, from drug delivery to surgical implants to tissue scaffolds.</p>