Mark M. Banaszak Holl

Polyhydroxyalkanoates (PHAs) are biopolyesters that accumulate as cytosolic cell inclusions in many bacteria, enhancing resistance to environmental stresses. However, the structure, spatial localization, plasticization effects, and mechanical properties of PHA granules in vivo remain underexplored. Here, Cupriavidus necator H16 grown under nutrient-rich and limited conditions was investigated across a 72 hour accumulation cycle using orthogonal techniques including atomic force microscopy infrared (AFM-IR) spectroscopy, contact resonance mechanical analysis, optical photothermal infrared spectroscopy, nano-thermal analysis, and proteomics to map polyhydroxybutyrate (PHB) granule evolution. Across the PHB production cycle, the PHB content increased from 34 to 69% after 72 hours in minimal media, while in vivo crystallinity reached a maximum of 20% compared to 42–63% when extracted. Ester carbonyl band shifts (1720–1740 cm–1) indicated mostly amorphous PHB in vivo, while contact resonance mapping showed that the granules were mechanically softer than the surrounding cellular matrix. Notably, AFM-IR revealed membrane patterning consistent with spinodal decomposition, representing the first reported observation of spinodal-like membrane organization in C. necator. Proteomics identified increased production of PHB biosynthesis, regulation, and phasin proteins during the stationary phase, supporting phasin-assisted suppression of crystallization. Whole-pathway analysis further revealed stronger perturbations in formate oxidation and polyphosphate metabolism than in PHB biosynthesis alone, highlighting broader metabolic shifts. This study establishes a link between PHB accumulation, crystallinity, mechanical softness, and granule-associated protein production in C. necator, yielding a plasticized intracellular polymer state relevant to flexible bioplastic and biomedical material design.

Poly(hydroxyalkanoates) (PHAs) are biobased, compostable polyesters that offer a circular materials pathway for thin film applications, however their high crystallinity and mechanical brittleness limit their suitability for flexible material applications. In this study, 100 – 200 µm thick cast films were produced by blending poly(butylene adipate-co-terephthalate) (PBAT) with poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-3- hydroxyvalerate-co-4-hydroxybutyrate) (PHBV-co-P4HB) to improve flexibility. Three 50/50 wt% binary blends of PHA/PBAT were produced via cast film melt extrusion, while films containing 1 – 50 wt% PHBV-co-P4HB with PBAT and 10 wt% CaCO3 were produced to investigate filler effects. PBAT enabled successful melt processing of PHA based thin films. PHBV-co-P4HB/PBAT films exhibited lower crystallinity (12%) than PHB/PBAT (49%) and PHBV/PBAT (51%) blends. After one and a half year of storage, PHB/PBAT and PHBV/PBAT films became more brittle, with Young’s modulus increasing to 15 GPa and 25 GPa, respectively, accompanied by a loss of ductility (elongation at break ≤ 5%). PHBV-co-P4HB/PBAT blends retained flexibility over time, with elongation at break remaining stable (317 ± 66%) and Young’s modulus increasing from 0.36 GPa to 0.69 GPa. Addition of 10% CaCO3 to the PHBV-co-P4HB/PBAT blend increased the Young's modulus and crystallisation temperature, while reducing ductility. Optical photothermal infrared (O-PTIR) spectra obtained from 500 nm spot sizes revealed nanoscale structural heterogeneity not discernible by FTIR, including domain variations and filler effects. P4HB units in PHA copolymers reduced crystallinity and increased elongation at break relative to PHB based materials, with PBAT blending increased flexibility. This work establishes processing–structure–property relationships in cast film extruded PHA/PBAT blends, demonstrating that thin-film bioplastics can achieve mechanical performance and stability comparable to or exceeding polyethylene and polypropylene films.

A recent study on folate binding protein (FBP), the soluble form of a folate receptor (FR), revealed surprising inhibition of in vivo growth of KB cell-derived xenograft tumors in mice and the induction of in vitro caspase-mediated apoptosis in KB, HeLa, and A549 cells in culture. In this research, we investigated the mechanism(s) underlying FBP-induced cytotoxicity and apoptosis in FR-overexpressing KB cancer cells using next-generation sequencing (NGS), a high-throughput technology for RNA sequencing and gene expression profiling, followed by bioinformatics analysis. The transcriptomics data, in addition to immunofluorescence analyses, indicated over and under expression of many proteins and transcription factors, such as Snail1, MCP-1 (CCL2), ZFP36, and HOXA9 in KB cells after treatment with 50 μM FBP for 6, 12, and 24 h. The alteration of an expression level in these genes, which are involved in epithelial to mesenchymal transition (EMT), inflammatory, and apoptosis pathways, is consistent with the induction of folate deficiency in FBP-exposed KB cells. The temporal effect of 50 μM FBP on DNA breakage and the induction of apoptosis, examined by the TUNEL assay, also supports the hypothesis of FBP-mediated folate deprivation of KB cells. The evidence reported in this study sheds light on the mechanism of FBP cytotoxicity in FR-overexpressing KB cancer cells in culture and suggests possible pathways for the observation of inhibited growth for KB xenograft tumors in vivo.

Atomic force microscopy-infrared spectroscopy can simultaneously acquire topographical, chemical, and stiffness information with nanoscale resolution. One approach to measuring relative stiffness is to use the phase-locked loop frequency channel to assess the probe-sample contact resonance. A stiffer sample domain results in higher contact resonance frequency, and more compliant domains result in lower frequency contact resonance; however, in practice, these data are difficult to compare between samples and can even be problematic from tip-engagement to tip-engagement on the same sample. This challenge is due to cantilever and tip variations that give rise to contact resonance frequency offsets, which can be large compared to the width of the frequency manifold associated with the surface. To overcome this challenge, we present a standard procedure for contact resonance frequency comparison between samples that circumvents the problem of the absolute frequency variation. Application of the technique is demonstrated on a carbon-epoxy composite, an enzyme-functionalized cellulose amide compressed disk, and human cadaver ligament samples.

Antimicrobial resistance (AMR) poses a significant global health threat, highlighting the need for effective bacteriophage (phage) therapy formulations. Understanding phenotypic and genotypic changes is essential for optimizing phage formulation and delivery. Here, we examine the chemical composition changes of Pseudomonas aeruginosa-specific phages subjected to external stresses, including heat and acidification, using atomic force microscopy-infrared spectroscopy (AFM-IR), with results confirmed by transmission electron microscopy (TEM) and biological assays. We assessed two structurally distinct phages─long-tailed myovirus PEV20 and short-tailed podovirus PEV31─to evaluate their differential resilience. We observed variations in signal intensity and peak positions in the amide I and II regions, indicating protein degradation on phage capsids, as well as changes in the nucleic acid content related to capsid stability and integrity. TEM analysis confirmed structural alterations such as ruptured capsids and tail detachment, which were more pronounced in PEV31, correlating with a greater loss of viability observed in biological assays. These findings enhance our understanding of phage resilience to stress and support the development of stable phage-based therapies for combating multidrug-resistant bacterial infections.

Introduction: Dysregulated calcium homeostasis and consequentially aberrant Ca²⁺ signalling could enhance survival, proliferation and metastasis in various cancers. Despite rapid development in exploring the ion channel functions in relation to cancer, most of the mechanisms accounting for the impact of ion channel modulators have yet to be fully clarified. Although harnessing small interfering RNA (siRNA) to specifically silence gene expression has the potential to be a pivotal approach, its success in therapeutic intervention is dependent on an efficient delivery system. Nanoparticles have the capacity to strongly bind siRNAs. They remain in the circulation and eventually deliver the siRNA payload to the target organ. Afterward, they interact with the cell surface and enter the cell via endocytosis. Finally, they help escape the endo-lysosomal degradation system prior to unload the siRNAs into cytosol. Carbonate apatite (CA) nanocrystals primarily is composed of Ca²⁺, carbonate and phosphate. CA possesses both anion and cation binding domains to target negatively charged siRNA molecules. Methods: Hybrid CA was synthesized by complexing CA NPs with a hydrophilic polysaccharide – hyaluronic acid (HA). The average diameter of the composite particles was determined using Zetasizer and FE-SEM and their zeta potential values were also measured. Results and Discussion: The stronger binding affinity and cellular uptake of a fluorescent siRNA were observed for HA-CA NPs as compared to plain CA NPs. Hybrid CA was electrostatically bound individually and combined with three different siRNAs to silence expression of calcium ion channel and transporter genes, TRPC6, TRPM8 and SLC41A1 in a human breast cancer cell line (MCF-7) and evaluate their potential for treating breast cancer. Hybrid NPs carrying TRPC6, TRPM8 and SLC41A1 siRNAs could significantly enhance cytotoxicity both in vitro and in vivo. The resultant composite CA influenced biodistribution of the delivered siRNA, facilitating reduced off target distribution and enhanced breast tumor targetability.

Nanosheet-based membranes have shown enormous potential for energy-efficient molecular transport and separation applications, but designing these membranes for specific separations remains a great challenge due to the lack of good understanding of fluid transport mechanisms in complex nanochannels. We synthesized reduced MXene/graphene hetero-channel membranes with sub–1-nm pores for experimental measurements and theoretical modeling of their structures and fluid transport rates. Our experiments showed that upon complete rejection of salt and organic dyes, these membranes with subnanometer channels exhibit remarkably high solvent fluxes, and their solvent transport behavior is very different from their homo-structured counterparts. We proposed a subcontinuum flow model that enables accurate prediction of solvent flux in sub–1-nm slit-pore membranes by building a direct relationship between the solvent molecule–channel wall interaction and flux from the confined physical properties of a liquid and the structural parameters of the membranes. This work provides a basis for the rational design of nanosheet-based membranes for advanced separation and emerging nanofluidics.