Fibroblast growth factors (FGFs) are cell-signaling proteins with diverse functions in cell development, repair, and metabolism. The human FGF family consists of 22 structurally related members, which can be classified into three separate groups based on their action of mechanisms, namely: intracrine, paracrine/autocrine, and endocrine FGF subfamilies. FGF19, FGF21, and FGF23 belong to the hormone-like/endocrine FGF subfamily. These endocrine FGFs are mainly associated with the regulation of cell metabolic activities such as homeostasis of lipids, glucose, energy, bile acids, and minerals (phosphate/active vitamin D). Endocrine FGFs function through a unique protein family called klotho. Two members of this family, α-klotho, or β-klotho, act as main cofactors which can scaffold to tether FGF19/21/23 to their receptor(s) (FGFRs) to form an active complex. There are ongoing studies pertaining to the structure and mechanism of these individual ternary complexes. These studies aim to provide potential insights into the physiological and pathophysiological roles and therapeutic strategies for metabolic diseases. Herein, we provide a comprehensive review of the history, structure–function relationship(s), downstream signaling, physiological roles, and future perspectives on endocrine FGFs.

Duchene muscular dystrophy (DMD) is an X-linked neuromuscular disorder that affects about one in every 5000 live male births. DMD is caused by mutations in the gene that codes for dystrophin, which is required for muscle membrane stabilization. The loss of functional dystrophin causes muscle degradation that leads to weakness, loss of ambulation, cardiac and respiratory complications, and eventually, premature death. Therapies to treat DMD have advanced in the past decade, with treatments in clinical trials and four exon-skipping drugs receiving conditional Food and Drug Administration approval. However, to date, no treatment has provided long-term correction. Gene editing has emerged as a promising approach to treating DMD. There is a wide range of tools, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and, most notably, RNA-guided enzymes from the bacterial adaptive immune system clustered regularly interspaced short palindromic repeats (CRISPR). Although challenges in using CRISPR for gene therapy in humans still abound, including safety and efficiency of delivery, the future for CRISPR gene editing for DMD is promising. This review will summarize the progress in CRISPR gene editing for DMD including key summaries of current approaches, delivery methodologies, and the challenges that gene editing still faces as well as prospective solutions.

Since the onset of the COVID-19 pandemic, a variety of diagnostic approaches, including RT-qPCR, RAPID, and LFA, have been adopted, with RT-qPCR emerging as the gold standard. However, a significant challenge in COVID-19 diagnostics is the wide range of symptoms presented by patients, necessitating early and accurate diagnosis for effective management. Although RT-qPCR is a precise molecular technique, it is not immune to false-negative results. In contrast, CRISPR-based detection methods for SARS-CoV-2 offer several advantages: they are cost-effective, time-efficient, highly sensitive, and specific, and they do not require sophisticated instruments. These methods also show promise for scalability, enabling diagnostic tests. CRISPR technology can be customized to target any genomic region of interest, making it a versatile tool with applications beyond diagnostics, including therapeutic development. The CRISPR/Cas systems provide precise gene targeting with immense potential for creating next-generation diagnostics and therapeutics. One of the key advantages of CRISPR/Cas-based therapeutics is the ability to perform multiplexing, where different sgRNAs or crRNAs can target multiple sites within the same gene, reducing the likelihood of viral escape mutants. Among the various CRISPR systems, CRISPR/Cas13 and CARVER (Cas13-assisted restriction of viral expression and readout) are particularly promising. These systems can target a broad range of single-stranded RNA viruses, making them suitable for the diagnosis and treatment of various viral diseases, including SARS-CoV-2. However, the efficacy and safety of CRISPR-based therapeutics must be thoroughly evaluated in pre-clinical and clinical settings. While CRISPR biotechnologies have not yet been fully harnessed to control the current COVID-19 pandemic, there is an optimism that the limitations of the CRISPR/Cas system can be overcome soon. This review discusses how CRISPR-based strategies can revolutionize disease diagnosis and therapeutic development, better preparing us for future viral threats.

The CRISPR-Cas9 system is a revolutionary tool in genetic engineering, offering unprecedented precision and efficiency in genome editing. Cas9, an enzyme derived from bacteria, is guided by RNA to edit DNA sequences within cells precisely. However, while CRISPR-Cas9 presents notable benefits and encouraging outcomes as a molecular tool and a potential therapeutic agent, the process of producing and purifying recombinant Cas9 protein remains a formidable hurdle. In this study, we systematically investigated the expression of recombinant SpCas9-His in four distinct Escherichia coli (E. coli) strains (Rosetta2, BL21(DE3), BL21(DE3)-pLysS, and BL21(DE3)-Star). Through optimization of culture conditions, including temperature and post-induction time, the BL21(DE3)-pLysS strain demonstrated efficient SpCas9 protein expression. This study also presents a detailed protocol for the purification of recombinant SpCas9, along with detailed troubleshooting tips. Results indicate successful SpCas9 protein expression using E. coli BL21(DE3)-pLysS at 0.5 mM IPTG concentration. Furthermore, the findings suggest potential avenues for further enhancements, paving the way for large-scale Cas9 production. This research contributes valuable insights into optimizing E. coli strains and culture conditions for enhanced Cas9 expression, offering a step forward in the development of efficient genome editing tools and therapeutic proteins.

Attention-Deficit/Hyperactivity Disorder (ADHD) is a complex neurodevelopmental disorder with a multifactorial etiology involving genetic, epigenetic, and environmental factors. This review focuses on the current understanding of these contributing elements, examining how they interact to influence ADHD development. Genetic predispositions, environmental exposures, and epigenetic modifications collectively shape the risk and manifestation of the disorder. Despite advancements in research, significant challenges remain in identifying precise mechanisms and translating them into effective treatments. The variability of symptoms across individuals, influenced by factors such as age, gender, and cultural background, further complicates diagnosis and treatment. Addressing these challenges requires a deeper investigation into the underlying causes of ADHD and the development of tailored interventions. This review aims to highlight both the progress made in understanding ADHD etiology and the current gaps in treatment approaches, calling for more targeted research and personalized therapeutic strategies.

Human fibroblast growth factor (FGF) 1 or hFGF1 is a member of the FGF family that is involved in various vital processes such as cell proliferation, cell differentiation, angiogenesis, and wound healing. hFGF1, which is associated with low stability in vivo, is known to be stabilized by binding heparin sulfate, a glycosaminoglycan that aids the protein in the activation of its cell surface receptor. The poor thermal and proteolytic stability of hFGF1 and the stabilizing role of heparin have long been observed experimentally; however, the mechanistic details of these phenomena are not well understood. Here, we have used microsecond-level equilibrium molecular dynamics (MD) simulations to quantitatively characterize the structural dynamics of monomeric hFGF1 in the presence and absence of heparin hexasaccharide. We have observed a conformational change in the heparin-binding pocket of hFGF1 that occurs only in the absence of heparin. Several intramolecular interactions were also identified within the heparin-binding pocket that form only when hFGF1 interacts with heparin. The loss of both intermolecular and intramolecular interactions in the absence of heparin plausibly leads to the observed conformational change. This conformational transition results in increased flexibility of the heparin-binding pocket and provides an explanation for the susceptibility of apo hFGF1 to proteolytic degradation and thermal instability. This study provides a glimpse into mechanistic details of the heparin-mediated stabilization of hFGF1 and encourages the use of microsecond-level MD in studying the effect of binding on protein structure and dynamics. In addition, the observed differential behavior of hFGF1 in the absence and presence of heparin provides an example, where microsecond-level all-atom MD simulations are necessary to see functionally relevant biomolecular phenomena that otherwise will not be observed on sub-microsecond time scales.