A nanobody toolbox for recognizing distinct epitopes on Cas9.

Cas9s and fusions of Cas9s have emerged as powerful tools for genetic manipulations. Fusions of Cas9 with other DNA editing enzymes have led to variants capable of single base editing and catalytically dead Cas9s have emerged as tools to specifically target desired regions of a genome. Here we describe the generation of a panel of nanobodies directed against three unique epitopes on Streptococcus pyogenes Cas9. The nanobodies were identified from a nanobody library derived from an alpaca that had been immunized with Cas9. The most potent binders recognize Cas9 and RNA bound Cas9 equally well and do not inhibit Cas9 cleavage of target DNA. These nanobodies bind non-overlapping epitopes as determined by ELISA based epitope binning experiments and mass photometry. We present the sequences of these clones and supporting biochemical data so the broader scientific community can access these reagents.

Staphylococcus aureus adapts to exploit collagen-derived proline during chronic infection.

Staphylococcus aureus is a pulmonary pathogen associated with substantial human morbidity and mortality. As vaccines targeting virulence determinants have failed to be protective in humans, other factors are likely involved in pathogenesis. Here we analysed transcriptomic responses of human clinical isolates of S. aureus from initial and chronic infections. We observed upregulated collagenase and proline transporter gene expression in chronic infection isolates. Metabolomics of bronchiolar lavage fluid and fibroblast infection, growth assays and analysis of bacterial mutant strains showed that airway fibroblasts produce collagen during S. aureus infection. Host-adapted bacteria upregulate collagenase, which degrades collagen and releases proline. S. aureus then imports proline, which fuels oxidative metabolism via the tricarboxylic acid cycle. Proline metabolism provides host-adapted S. aureus with a metabolic benefit enabling out-competition of non-adapted strains. These data suggest that clinical settings characterized by airway repair processes and fibrosis provide a milieu that promotes S. aureus adaptation and supports infection.

A retrospective audit with subsequent cost and environmental analysis of a denosumab self-injection program.

The Metabolic Bone Health Department, Cardiff and Vale University Health Board, serves a local population of approximately 445 000 people. A retrospective audit of attendance data regarding the denosumab treatment clinic (the traditional treatment pathway) and the denosumab Self-Injection Program (SIP) was conducted to determine whether the SIP is both cost-effective and environmentally beneficial, compared to the traditional treatment pathway. Cost analysis was then conducted by the Finance Department. The audit was conducted over 3 years following the implementation of the service development; 233 patients had enrolled in the program at the time of the audit and 69 had completed 3 years of self-injected treatment. A control group of 497 patients were identified by the service. This group remained on the historical pathway and had consistent attendance activity over the 3-yr period from 2017 to 2019. Pre- and post-period activity of all patients on the program was compared, together with the activity for the independent control group. The SIP resulted in a reduction in clinical contacts, with financial analysis showing a total opportunity cost saving per patient of £420 per annum. There were obvious benefits to the patient of a reduced number of visits to a clinical site, which also resulted in an estimated carbon footprint reduction of 59 kg CO2 per patient per annum. The cost analysis is based on our organization's 2022 charges. The SIP demonstrates that by focusing on care "closer to home", it is possible to maximize resources, improve the patient experience through reduced travel, and reduce the environmental impact of healthcare.

The Glioblastoma Landscape: Hallmarks of Disease, Therapeutic Resistance, and Treatment Opportunities.

Malignant brain tumors are aggressive and difficult to treat. Glioblastoma is the most common and lethal form of primary brain tumor, often found in patients with no genetic predisposition. The median life expectancy for individuals diagnosed with this condition is 6 months to 2 years and there is no known cure. New paradigms in cancer biology implicate a small subset of tumor cells in initiating and sustaining these incurable brain tumors. Here, we discuss the heterogenous nature of glioblastoma and theories behind its capacity for therapy resistance and recurrence. Within the cancer landscape, cancer stem cells are thought to be both tumor initiators and major contributors to tumor heterogeneity and therapy evasion and such cells have been identified in glioblastoma. At the cellular level, disruptions in the delicate balance between differentiation and self-renewal spur transformation and support tumor growth. While rapidly dividing cells are more sensitive to elimination by traditional treatments, glioblastoma stem cells evade these measures through slow division and reversible exit from the cell cycle. At the molecular level, glioblastoma tumor cells exploit several signaling pathways to evade conventional therapies through improved DNA repair mechanisms and a flexible state of senescence. We examine these common evasion techniques while discussing potential molecular approaches to better target these deadly tumors. Equally important, the presented information encourages the idea of augmenting conventional treatments with novel glioblastoma stem cell-directed therapies, as eliminating these harmful progenitors holds great potential to modulate tumor recurrence.

Purine biosynthetic enzymes assemble into liquid-like condensates dependent on the activity of chaperone protein HSP90.

Enzymes within the de novo purine biosynthetic pathway spatially organize into dynamic intracellular assemblies called purinosomes. The formation of purinosomes has been correlated with growth conditions resulting in high purine demand, and therefore, the cellular advantage of complexation has been hypothesized to enhance metabolite flux through the pathway. However, the properties of this cellular structure are unclear. Here, we define the purinosome in a transient expression system as a biomolecular condensate using fluorescence microscopy. We show that purinosomes, as denoted by formylglycinamidine ribonucleotide synthase granules in purine-depleted HeLa cells, are spherical and appear to coalesce when two come into contact, all liquid-like characteristics that are consistent with previously reported condensates. We further explored the biophysical and biochemical means that drive the liquid-liquid phase separation of these structures. We found that the process of enzyme condensation into purinosomes is likely driven by the oligomeric state of the pathway enzymes and not a result of intrinsic disorder, the presence of low-complexity domains, the assistance of RNA scaffolds, or changes in intracellular pH. Finally, we demonstrate that the heat shock protein 90 KDa helps to regulate the physical properties of the condensate and maintain their liquid-like state inside HeLa cells. We show that disruption of heat shock protein 90 KDa activity induced the transformation of formylglycinamidine ribonucleotide synthase clusters into more irregularly shaped condensates, suggesting that its chaperone activity is essential for purinosomes to retain their liquid-like properties. This refined view of the purinosome offers new insight into how metabolic enzymes spatially organize into dynamic condensates within human cells.

Bio-CaRGOS: capture and release gels for optimized storage of hemoglobin.

Room temperature biospecimen storage for prolonged periods is essential to eliminate energy consumption by ultra-low freezing or refrigeration-based storage techniques. State of the art practices that sufficiently minimize the direct or hidden costs associated with cold-chain logistics include ambient temperature storage of biospecimens (i.e., DNA, RNA, proteins, lipids) in the dry state. However, the biospecimens are still well-exposed to the stress associated with drying and reconstitution cycles, which augments the pre-analytical degradation of biospecimens prior to their downstream processing. An aqueous storage solution that can eliminate these stresses which are correlated to several cycles of drying/rehydration or freezing of biospecimens, is yet to be achieved by any current technology. In our study, we have addressed this room temperature biospecimen-protection challenge using aqueous capture and release gels for optimized storage (Bio-CaRGOS) of biospecimens. Herein, we have demonstrated a single-step ∼95% recovery of a metalloprotein hemoglobin at room temperature using a cost-effective standard microwave-based aqueous formulation of Bio-CaRGOS. Although hemoglobin samples are currently stored at sub-zero or under refrigeration (4 °C) conditions to avoid loss of integrity and an unpredictable diagnosis during their downstream assays, our results have displayed an unprecedented room temperature integrity preservation of hemoglobin. Bio-CaRGOS formulations efficiently preserve hemoglobin in its native state, with single-step protein recovery of ∼95% at ambient conditions (1 month) and ∼96% (7 months) under refrigeration conditions. In contrast, two-thirds of the control samples degrade under ambient (1 month) and refrigeration (7 months) settings.