Understanding Three Hydration-Dependent Transitions of Zwitterionic Carboxybetaine Hydrogel by Molecular Dynamics Simulations

In this work, molecular dynamics simulations were performed to study a carboxybetaine methacrylate (CBMA) hydrogel under various swelling states. The water content in this study ranged from 28% to 91% of the total weight of the hydrogel. Three transitions of the CBMA hydrogel were observed as the water content increased. The first transition occurs when the water content increases from 33% to 37%. The observed kink in the self-diffusion coefficient of water indicates that the hydration of the polymer network of the hydrogel is saturated; the further added water is in a less confined state. The second transition was found to be related to the physical cross-links of the polymer network. As the water content rises to above 62%, the lifetime of the physical cross-links decreases significantly. This abrupt change in the lifetime indicates that the transition represents the equilibrium swelling state of the hydrogel. Finally, the third transition was observed when the water content goes above 81%. The significant increases in the bond and angle energies of the polymer network indicate that the hydrogel reaches its upper limit swelling state at this transition. These results are comparable to previously published experimental studies of similar zwitterionic hydrogels

MODERATE ACCUMULATION OF TDP-43 IN NEURONS IS SUFFICIENT TO CAUSE ADULT-ONSET MOTOR NEURON DISEASE

Cytoplasmic aggregation of TAR DNA-binding protein 43 (TDP-43), accompanied by its nuclear clearance, is a key common pathological hallmark of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Mutations in TARDBP (the gene encoding TDP-43) linked to familial and sporadic ALS established this essential RNA binding protein to play a central role in the pathogenesis of ALS and FTLD. While most mutations cluster within the C-terminal, prion-like domain of TDP-43, a few are found within the 3’ untranslated region (3’ UTR) where TDP-43 binds and regulates the level of its own mRNA. ALS cases harboring the 3’ UTR mutation exhibited modest elevation of TDP-43, possibly because the mutation compromised the ability of TDP-43 to downregulate its own transcript. To determine whether modest increase of TDP-43 is sufficient to cause motor neuron disease, two lines of transgenic mice that accumulate modest levels of TDP-43 in the nervous system are generated and characterized. One of these lines, S97, expressed TARDBP carrying the ALS associated missense mutation (corresponding to the mutant G298S protein), whereas the other line, W2, expressed wild-type human TARDBP. S97 and W2 transgenic mice survive to adulthood, gain weight appropriately before plateauing, exhibit a progressive loss of strength, and lose large motor axons in their adult life. Also, S97 and W2 transgenic mice display adult-onset muscle degeneration and neuromuscular junction and motor end-plate abnormalities. Significantly, a percentage of S97 and W2 transgenic mice progress to paralysis late in life. The S97 and W2 lines represent the first transgenic TDP-43 lines with mice that exhibit motor neuron degeneration leading to paralysis in late adulthood. As such, these mouse lines will be useful to further clarify disease mechanisms and for testing therapeutic strategies to attenuate neurodegeneration in ALS. Advisor: Dr. Philip C. Wong Readers: Dr. Philip C. Wong and Dr. Joseph L. Mankowsk

Ancillary Testing in Lung Cancer Diagnosis

The pathologic diagnosis of lung cancer historically has relied primarily on morphologic features of tumors in histologic sections. With the emergence of new targeted therapies, the pathologist is called upon increasingly to provide not only accurate typing of lung cancers, but also to provide prognostic and predictive information, based on a growing number of ancillary tests, that may have significant impact on patient management. This review provides an overview of ancillary tests currently used in the pathologic diagnosis of lung cancer, with a focus on immunohistochemistry and molecular diagnostics

Evolving Patterns of Metastasis in Renal Cell Carcinoma: Do We Need to Perform Routine Bone Imaging?

Advance diagnostic and treatment modalities have improved outcomes for renal cell carcinoma (RCC) patients, but the prognosis for those with metastatic disease (mRCC) remains poor. As given metastatic distribution is critical in guiding treatment decisions for mRCC patients, we evaluated evolving metastatic patterns to assess if our current practice standards effectively address patient needs. A systematic literature review was performed to identify all publicly available prospective clinical trials in metastatic renal cell carcinoma (mRCC) from 1990 to 2018. A total of 16,899 mRCC patients from 127 qualified phase I–III clinical trials with metastatic site documentations were included for analysis for incidence of metastases to lung, liver, bone, and lymph nodes (LNs) over time. Studies were categorized into three treatment eras based on the timing of regulatory approval: Cytokine Era (1990-2004), vascular endothelial growth factor/tyrosine kinase inhibitor (TKI) Era (2005-2016), and immune checkpoint inhibitor/TKI Era (ICI-TKI, 2017-2018) and also classified as first-line only (FLO) or second-line and beyond (SLB). Overall, an increase in the incidence of bone and LNs metastases in FLO and SLB, and lung metastases in FLO, was seen over the three treatment eras. Generally, the burden of disease is higher in SLB when compared with FLO. Importantly, in the ICI-TKI era, the incidences of bone metastasis are 28% in FLO and 29% in SLB settings. The disease burden in patients with mRCC has increased steadily over the past three decades. Given the unexpectedly high rate of bone metastasis, routine dedicated bone imaging should be considered in all patients with mRCC

LuxS Coexpression Enhances Yields of Recombinant Proteins in Escherichia coli in Part through Posttranscriptional Control of GroEL

Cell-to-cell communication, or quorum sensing (QS), enables cell density-dependent regulation of bacterial gene expression which can be exploited for the autonomous-signal-guided expression of recombinant proteins (C. Y. Tsao, S. Hooshangi, H. C. Wu, J. J. Valdes, and W. E. Bentley, Metab. Eng. 12:291-297, 2010). Earlier observations that the metabolic potential of Escherichia coli is conveyed via the QS signaling molecule autoinducer-2 (AI-2) suggested that the capacity for protein synthesis could also be affected by AI-2 signaling (M. P. DeLisa, J. J. Valdes, and W. E. Bentley, J. Bacteriol. 183:2918-2928, 2001). In this work, we found that simply adding conditioned medium containing high levels of AI-2 at the same time as inducing the synthesis of recombinant proteins doubled the yield of active product. We have hypothesized that AI-2 signaling “conditions” cells as a natural consequence of cell-to-cell communication and that this could tweak the signal transduction cascade to alter the protein synthesis landscape. We inserted luxS (AI-2 synthase) into vectors which cosynthesized proteins of interest (organophosphorus hydrolase [OPH], chloramphenicol acetyltransferase [CAT], or UV-variant green fluorescent protein [GFPuv]) and evaluated the protein expression in luxS-deficient hosts. In this way, we altered the level of luxS in the cells in order to “tune” the synthesis of AI-2. We found conditions in which the protein yield was dramatically increased. Further studies demonstrated coincident upregulation of the chaperone GroEL, which may have facilitated higher yields and is shown for the first time to be positively regulated at the posttranscriptional level by AI-2. This report is the first to demonstrate that the protein synthesis capacity of E. coli can be altered by rewiring quorum sensing circuitry

ANALYSIS AND FORMULATION OF COMBUSTIBLE COMPONENTS IN RUBBER SOLID WASTES

Wang, MHS, McGinnis, WC and Wang, LK (2022). Analysis and formulation of combustible components in rubber solid wastes. In: “Evolutionary Progress in Science, Technology, Engineering, Arts and Mathematics (STEAM)”, Wang, LK and Tsao, HP (eds.), 4 (9B), STEAM-VOL4-NUM9B-SEPT2022, 46 pages. Lenox Institute Press, Massachusetts, USA. ..........ABSTRACT: This publication is the result of a study of the inherent properties of solid wastes with special emphasis on rubber solid waste. An attempt was made to improve the ultimate analysis model values. A survey was undertaken to determine empirical chemical values for rubber products. Characteristics of the rubber industry's waste is discussed as well as some alternatives that have been tested or implemented. The ultimate analysis values of solid rubber wastes are concluded to be: Carbon 82.570%, Hydrogen 3.689%, Oxygen 0.496%, Nitrogen 0.166%, Sulfur 1.587%, and Ash 11.492%. The authors recommend that further similar research be conducted for the plastic solid waste as well as other solid wastes. This publication also introduces a general mathematical model for : (a) prediction of effect of the combustible solid waste on air pollution if the waste is to be used as a supplemental fuel; (b) solid waste characterization by burning the solid waste, effluent gases sampling and analysis, and reverse calculation using the model. This publication is one of the authors' memoirs. It is the authors' hope that this publication can be considered as a stepping stone by the readers for future solid waste research.

ANALYSIS AND FORMULATION OF COMBUSTIBLE COMPONENTS IN NON-HAZARDOUS SOLID WASTES

Wang, LK, McGinnis, WC and Wang, MHS (2022). Analysis and formulation of combustible components in non-hazardous solid wastes. In: “Evolutionary Progress in Science, Technology, Engineering, Arts and Mathematics (STEAM)”, Wang, LK and Tsao, HP (eds.), 4 (9A), STEAM-VOL4-NUM9A-SEPT2022. https://doi.org/10.17613/0p7c-wp38. 36 pages. September 2022., Lenox Institute Press, Massachusetts, USA. ......ABSTRACT: The non-hazardous solid wastes from industrial plants are similar to municipal solid wastes (MSW). An industrial plant is planning to reuse its own non-hazardous solid wastes as a supplemental source of fuel to its incinerator. The objectives of this research are: (a) to determine the energy values using both the modified Dulong Formula, and a traditional method; and (b) to determine the empirical chemical formula of the plant's own solid wastes using the values of ultimate analysis. This publication introduces the various solid wastes, (such as food, paper, cardboard, plastics, textiles, rubber, leather, garden trimming, wood, glass, tin cans, nonferrous metals, ferrous metals, dirt, and ashes), and their physical and chemical compositions (such as, percent by weight, dry weight, moisture content, inert residue, and energy content). Using the available typical MSW information, the energy values for the target industrial non-hazardous combustible solid wastes (excluding the wastes of glass, tin cans, nonferrous metals, ferrous metals, dirt, and ashes) were determined to be 5776 Btu/lb with the modified Dulong Formula and 6045 Btu/lb with the traditional method. The two calculated energy values are very close. The empirical chemical formula of the target solid waste was determined to be C664 H1741 O745 N14.4 S. This formula can be used by the plant's planners and managers in evaluating the feasibility of alternative uses of the target solid waste such as heat generation and materials recycling

A Redox-Based Autoinduction Strategy to Facilitate Expression of 5xCys-Tagged Proteins for Electrobiofabrication

Biofabrication utilizes biological materials and biological means, or mimics thereof, for assembly. When interfaced with microelectronics, electrobiofabricated assemblies enable exquisite sensing and reporting capabilities. We recently demonstrated that thiolated polyethylene glycol (PEG-SH) could be oxidatively assembled into a thin disulfide crosslinked hydrogel at an electrode surface; with sufficient oxidation, extra sulfenic acid groups are made available for covalent, disulfide coupling to sulfhydryl groups of proteins or peptides. We intentionally introduced a polycysteine tag (5xCys-tag) consisting of five consecutive cysteine residues at the C-terminus of a Streptococcal protein G to enable its covalent coupling to an electroassembled PEG-SH film. We found, however, that its expression and purification from E. coli was difficult, owing to the extra cysteine residues. We developed a redox-based autoinduction methodology that greatly enhanced the yield, especially in the soluble fraction of E. coli extracts. The redox component involved the deletion of oxyRS, a global regulator of the oxidative stress response and the autoinduction component integrated a quorum sensing (QS) switch that keys the secreted QS autoinducer-2 to induction. Interestingly, both methods helped when independently employed and further, when used in combination (i.e., autodinduced oxyRS mutant) the results were best—we found the highest total yield and highest yield in the soluble fraction. We hypothesize that the production host was less prone to severe metabolic perturbations that might reduce yield or drive sequestration of the -tagged protein into inclusion bodies. We expect this methodology will be useful for the expression of many such Cys-tagged proteins, ultimately enabling a diverse array of functionalized devices

Parsed synthesis of pyocyanin via co-culture enables context-dependent intercellular redox communication

Microbial co-cultures and consortia are of interest in cell-based molecular production and even as “smart” therapeutics in that one can take advantage of division of labor and specialization to expand both the range of available functions and mechanisms for control. The development of tools that enable coordination and modulation of consortia will be crucial for future application of multi-population cultures. In particular, these systems would benefit from an expanded toolset that enables orthogonal inter-strain communication. We created a co-culture for the synthesis of a redox-active phenazine signaling molecule, pyocyanin (PYO), by dividing its synthesis into the generation of its intermediate, phenazine carboxylic acid (PCA) from the first strain, followed by consumption of PCA and generation of PYO in a second strain. Interestingly, both PCA and PYO can be used to actuate gene expression in cells engineered with the soxRS oxidative stress regulon, although importantly this signaling activity was found to depend on growth media. That is, like other signaling motifs in bacterial systems, the signaling activity is context dependent. We then used this co-culture’s phenazine signals in a tri-culture to modulate gene expression and production of three model products: quorum sensing molecule autoinducer-1 and two fluorescent marker proteins, eGFP and DsRed. We also showed how these redox-based signals could be intermingled with other quorum-sensing (QS) signals which are more commonly used in synthetic biology, to control complex behaviors. To provide control over product synthesis in the tri-cultures, we also showed how a QS-induced growth control module could guide metabolic flux in one population and at the same time guide overall tri-culture function. Specifically, we showed that phenazine signal recognition, enabled through the oxidative stress response regulon soxRS, was dependent on media composition such that signal propagation within our parsed synthetic system could guide different desired outcomes based on the prevailing environment. In doing so, we expanded the range of signaling molecules available for coordination and the modes by which they can be utilized to influence overall function of a multi-population culture. Our results show that redox-based signaling can be intermingled with other quorum sensing signaling in ways that enable user-defined control of microbial consortia yielding various outcomes defined by culture medium. Further, we demonstrated the utility of our previously designed growth control module in influencing signal propagation and metabolic activity is unimpeded by orthogonal redox-based signaling. By exploring novel multi-modal strategies for guiding communication and consortia outcome, the concepts introduced here may prove to be useful for coordination of multiple populations within complex microbial systems.https://doi.org/10.1186/s12934-021-01703-