The foodborne pathogenic bacterium Campylobacter jejuni utilizes chemotaxis to assist in the colonization of host niches. A key to revealing the relationship among chemotaxis and pathogenicity is the discovery of signaling molecules perceived by the chemoreceptors. The C. jejuni chemoreceptor Tlp11 is encoded by the highly infective C. jejuni strains. In the present study, we report that the dCache-type ligand-binding domain (LBD) of C. jejuni ATCC 33560 Tlp11 binds directly to novel ligands methyl pyruvate, toluene, and quinoline using the same pocket. Methyl pyruvate elicits a strong chemoattractant response, while toluene and quinoline function as the antagonists without triggering chemotaxis. The sensory LBD was used to control heterologous proteins by constructing chimeras, indicating that the signal induced by methyl pyruvate is transmitted across the membrane. In addition, bioinformatics and experiments revealed that the dCache domains with methyl pyruvate-binding sites and ability are widely distributed in the order Campylobacterales. This is the first report to identify the class of dCache chemoreceptors that bind to attractant methyl pyruvate and antagonists toluene and quinoline. Our research provides a foundation for understanding the chemotaxis and virulence of C. jejuni and lays a basis for the control of this foodborne pathogen.

RNA has diverse cellular functionality, including regulating gene expression, protein translation, and cellular response to stimuli, due to its intricate structures. Over the past decade, small molecules have been discovered that target functional structures within cellular RNAs and modulate their function. Simple binding, however, is often insufficient, resulting in low or even no biological activity. To overcome this challenge, heterobifunctional compounds have been developed that can covalently bind to the RNA target, alter RNA sequence, or induce its cleavage. Herein, we review the recent progress in the field of RNA-targeted heterobifunctional compounds using representative case studies. We identify critical gaps and limitations and propose a strategic pathway for future developments of RNA-targeted molecules with augmented functionalities.

Trypanosoma cruzi is the causative agent of Chagas disease, a global public health problem. New therapeutic drugs and biologics are needed. The TSA-1 recombinant protein of T. cruzi is one such promising antigen for developing a therapeutic vaccine. However, it is overexpressed in E. coli as inclusion bodies, requiring an additional refolding step. As an alternative, in this study, we propose the endogenous cysteine protease inhibitor chagasin as a molecular scaffold to generate chimeric proteins. These proteins will contain combinations of two of the five conserved epitopes (E1 to E5) of TSA-1 in the L4 and L6 chagasin loops. Twenty chimeras (Q1-Q20) were designed, and their solubility was predicted using bioinformatics tools. Nine chimeras with different degrees of solubility were selected and expressed in E. coli BL21 (DE3). Western blot assays with anti-6x-His and anti-chagasin antibodies confirmed the expression of soluble recombinant chimeras. Both theoretically and experimentally, the Q12 (E5-E3) chimera was the most soluble, and the Q20 (E4-E5) the most insoluble protein. Q4 (E5-E1) and Q8 (E5-E2) chimeras were classified as proteins with medium solubility that exhibited the highest yield in the soluble fraction. Notably, Q4 has a yield of 239 mg/L, well above the yield of recombinant chagasin (16.5 mg/L) expressed in a soluble form. The expression of the Q4 chimera was scaled up to a 7 L fermenter obtaining a yield of 490 mg/L. These data show that chagasin can serve as a molecular scaffold for the expression of TSA-1 epitopes in the form of soluble chimeras.

Orthotopic liver transplantation (OLT) currently serves as the sole definitive treatment for thousands of patients suffering from end-stage liver disease; and the existing supply of donor livers for OLT is drastically outpaced by the increasing demand. To alleviate this significant gap in treatment, several experimental approaches have been devised with the aim of either offering interim support to patients waiting on the transplant list or bioengineering complete livers for OLT by infusing them with fresh hepatic cells. Recently, interspecies blastocyst complementation has emerged as a promising method for generating complete organs in utero over a short timeframe. When coupled with gene editing technology, it has brought about a potentially revolutionary transformation in regenerative medicine. Blastocyst complementation harbors notable potential for generating complete human livers in large animals, which could be used for xenotransplantation in humans, addressing the scarcity of livers for OLT. Nevertheless, substantial experimental and ethical challenges still need to be overcome to produce human livers in larger domestic animals like pigs. This review compiles the current understanding of interspecies blastocyst complementation and outlines future possibilities for liver xenotransplantation in humans.

Deciphering the origins of life on a molecular level includes unravelling the numerous interactions that could occur between the most important biomolecules being the lipids, peptides and nucleotides. They were likely all present on the early Earth and all necessary for the emergence of cellular life. In this study, we intended to explore conditions that were at the same time conducive to chemical reactions critical for the origins of life (peptide-oligonucleotide couplings and templated ligation of oligonucleotides) and compatible with the presence of prebiotic lipid vesicles. For that, random peptides were generated from activated amino acids and analysed using NMR and MS, whereas short oligonucleotides were produced through solid-support synthesis, manually deprotected and purified using HPLC. After chemical activation in prebiotic conditions, the resulting mixtures were analysed using LC-MS. Vesicles could be produced through gentle hydration in similar conditions and observed using epifluorescence microscopy. Despite the absence of coupling or ligation, our results help to pave the way for future investigations on the origins of life that may gather all three types of biomolecules rather than studying them separately, as it is still too often the case.

In this paper, we outline how one might conduct a comprehensive ethical evaluation of human brain organoid transplantation in animals. Thus far, ethical concerns regarding this type of research have been assumed to be similar to those associated with other transplants of human cells in animals, and have therefore not received significant attention. The focus has been only on the welfare, moral status, or mental capacities of the host animal. However, the transplantation of human brain organoids introduces several new ethical issues. Many of these are related to uncertainty regarding whether or not brain organoids might be conscious. While these concerns might not be immediately relevant, they warrant closer scrutiny. We discuss how various ethical issues are relevant to different stages of human brain organoid transplantation and can guide the ethical evaluation of research. Our examination would broaden the horizons of the debate on the transplantation of brain organoids.

Organ transplantation is a highly utilized treatment for many medical conditions, yet the number of patients waiting for organs far exceeds the number available. The challenges and limitations currently associated with organ transplantation and technological advances in gene editing techniques have led scientists to pursue alternate solutions to the donor organ shortage. Growing human organs in animals and harvesting those organs for transplantation into humans is one such solution. These chimeric animals usually have certain genes necessary for a specific organ\'s development inhibited at an early developmental stage, followed by the addition of cultured pluripotent human cells to fill that developmental niche. The result is a chimeric animal that contains human organs which are available for transplant into a patient, circumventing some of the limitations currently involved in donor organ transplantation. In this review, we will discuss both the current scientific and legal landscape of human-animal chimera (HAC) research. We present an overview of the technological advances that allow for the creation of HACs, the patents that currently exist on these methods, as well as current public attitude and understanding that can influence HAC research policy. We complement our scientific and public attitude discussion with a regulatory overview of chimera research at both the national and state level, while also contrasting current U.S. legislation with regulations in other countries. Overall, we provide a comprehensive analysis of the legal and scientific barriers to conducting research on HACs for the generation of transplantable human organs, as well as provide recommendations for the future.

The identification of new effective therapeutic options for non-small-cell lung cancer (NSCLC) represents a crucial challenge in oncology. Recent studies indicate that cancer-associated fibroblasts (CAFs) participate in tumor progression by establishing a favorable microenvironment that promotes cancer progression. Therefore, the development of strategies inhibiting the interplay between CAFs and cancer cells is considered a winning approach for the development of effective anti-cancer drugs. Among other factors, the signal transducer and activator of transcription-3 (STAT3) has been reported as a key mediator of CAF oncogenic actions, representing a promising therapeutic target. Here, we applied an aptamer-based conjugate (named Gint4.T-STAT3), containing a STAT3 siRNA linked to an aptamer binding and inhibiting the platelet-derived growth factor receptor (PDGFR)β, to obtain STAT3-specific silencing and interfere with CAF pro-tumorigenic functions. We demonstrated that this molecule effectively delivers the STAT3 siRNA in NSCLC cells, and blocks CAF-induced cancer cell growth and migration and reduced spheroid dimension. In addition, we found that Gint4.T-STAT3 alters CAF phenotype, thus functioning as a double-acting molecule able to inhibit the entire tumor bulk. Our data provide a proof of principle for the targeting of CAF pro-tumor functions through an aptamer-based drug, and can open innovative horizons in NSCLC therapy.

Multiple displacement amplification (MDA) based on isothermal random priming and high fidelity phi29 DNA polymerase-mediated processive extension has revolutionized the field of whole genome amplification by enabling the amplification of minute amounts of DNA, such as from a single cell, generating vast amounts of DNA with high genome coverage. Despite its advantages, MDA has its own challenges, one of the grandest being the formation of chimeric sequences (chimeras), which presents in all MDA products and seriously disturbs the downstream analysis. In this review, we provide a comprehensive overview of current research on MDA chimeras. We first reviewed the mechanisms of chimera formation and chimera detection methods. We then systematically summarized the characteristics of chimeras, including overlap, chimeric distance, chimeric density, and chimeric rate, as found in independently published sequencing data. Finally, we reviewed the methods used to process chimeric sequences and their impacts on the improvement of data utilization efficiency. The information presented in this review will be useful for those interested in understanding the challenges with MDA and in improving its performance.

Cerebral organoid models in-of-themselves are considered as an alternative to research animal models. But their developmental and biological limitations currently inhibit the probability that organoids can fully replace animal models. Furthermore, these organoid limitations have, somewhat ironically, brought researchers back to the animal model via xenotransplantation, thus creating hybrids and chimeras. In addition to attempting to study and overcome cerebral organoid limitations, transplanting cerebral organoids into animal models brings an opportunity to observe behavioral changes in the animal itself. Traditional animal ethics frameworks, such as the well-known three Rs (reduce, refine, and replace), have previously addressed chimeras and xenotransplantation of tissue. But these frameworks have yet to completely assess the neural-chimeric possibilities. And while the three Rs framework was a historical landmark in animal ethics, there are identifiable gaps in the framework that require attention. The authors propose to utilize an expanded three Rs framework initially developed by David DeGrazia and Tom L. Beauchamp, known as the Six Principles (6Ps). This framework aims to expand upon the three Rs, fill in the gaps, and be a practical means for assessing animal ethical issues like that of neural-chimeras and cerebral organoid xenotransplantation. The scope of this 6Ps application will focus on two separate but recent studies, which were published in 2019 and 2020. First, they consider a study wherein cerebral organoids were grown from donors with Down syndrome and from neurotypical donors. After these organoids were grown and studied, they were then surgically implanted into mouse models to observe the physiological effects and any behavioral change in the chimera. Second, they consider a separate study wherein neurotypical human embryonic stem cell-derived cerebral organoids were grown and transplanted into mouse and macaque models. The aim was to observe if such a transplantation method would contribute to therapies for brain injury or stroke. The authors place both studies under the lens of the 6Ps framework, assess the relevant contexts of each case, and provide relevant normative conclusions. In this way, they demonstrate how the 6Ps could be applied in future cases of neural-chimeras and cerebral organoid xenotransplantation.