Application of droplet digital PCR in minimal residual disease monitoring of rare fusion transcripts and mutations in haematological malignancies.

Leukaemia of various subtypes are driven by distinct chromosomal rearrangement or genetic abnormalities. The leukaemogenic fusion transcripts or genetic mutations serve as molecular markers for minimal residual disease (MRD) monitoring. The current study evaluated the applicability of several droplet digital PCR assays for the detection of these targets at RNA and DNA levels (atypical BCR::ABL1 e19a2, e23a2ins52, e13a2ins74, rare types of CBFB::MYH11 (G and I), PCM1::JAK2, KMT2A::ELL2, PICALM::MLLT10 fusion transcripts and CEBPA frame-shift and insertion/duplication mutations) with high sensitivity. The analytical performances were assessed by the limit of blanks, limit of detection, limit of quantification and linear regression. Our data demonstrated serial MRD monitoring for patients at molecular level could become "digitalized", which was deemed important to guide clinicians in treatment decision for better patient care.

SYNERGY-Everolimus-Eluting Stent With a Bioabsorbable Polymer in ST-Elevation Myocardial Infarction: CLEAR SYNERGY OASIS-9 Registry.

Our objective was to evaluate the clinical effectiveness of the SYNERGY stent (Boston Scientific Corporation, Marlborough, Massachusetts) in patients with ST-elevation myocardial infarction (STEMI). The only drug-eluting stent approved for treatment of STEMI by the Food and Drug Administration is the Taxus stent (Boston Scientific) which is no longer commercially available, so further data are needed. The CLEAR (Colchicine and spironolactone in patients with myocardial infarction) SYNERGY stent registry was embedded into a larger randomized trial of patients with STEMI (n = 7,000), comparing colchicine versus placebo and spironolactone versus placebo. The primary outcome for the SYNERGY stent registry is major adverse cardiac events (MACE) as defined by cardiovascular death, recurrent MI, or unplanned ischemia-driven target vessel revascularization within 12 months. We estimated a MACE rate of 6.3% at 12 months after primary percutaneous coronary intervention for STEMI based on the Thrombectomy vs percutaneous coronary intervention alone in STEMI (TOTAL) trial. Success was defined as upper bound of confidence interval (CI) to be less than the performance goal of 9.45%. Overall, 733 patients were enrolled from 8 countries with a mean age 60 years, 19.4% diabetes mellitus, 41.3% anterior MI, and median door-to-balloon time of 72 minutes. The MACE rate was 4.8% (95% CI 3.2 to 6.3%) at 12 months which met the success criteria against performance goal of 9.45%. The rates of cardiovascular death, recurrent MI, or target vessel revascularization were 2.7%, 1.9%, 1.0%, respectively. The rates of acute definite stent thrombosis were 0.3%, subacute 0.4%, late 0.4%, and cumulative stent thrombosis of 1.1% at 12 months. In conclusion, the SYNERGY stent in STEMI performed well and was successful compared with the performance goal based on previous trials.

Intravesicular Solute Delivery and Surface Area Regulation in Giant Unilamellar Vesicles Driven by Cycles of Osmotic Stresses.

Phospholipid bilayers are dynamic cellular components that undergo constant changes in their topology, facilitating a broad diversity of physiological functions including endo- and exocytosis, cell division, and intracellular trafficking. These shape transformations consume energy, supplied invariably by the activity of proteins. Here, we show that cycles of oppositely directed osmotic stresses─unassisted by any protein activity─can induce well-defined remodeling of giant unilamellar vesicles, minimally recapitulating the phenomenologies of surface area homeostasis and macropinocytosis. We find that a stress cycle consisting of deflationary hypertonic stress followed by an inflationary hypotonic one prompts an elaborate sequence of membrane shape changes ultimately transporting molecular cargo from the outside into the intravesicular milieu. The initial osmotic deflation produces microscopic spherical invaginations. During the subsequent inflation, the first subpopulation contributes area to the swelling membrane, thereby providing a means for surface area regulation and tensional homeostasis. The second subpopulation vesiculates into the lumens of the mother vesicles, producing pinocytic vesicles. Remarkably, the gradients of solute concentrations between the GUV and the daughter pinocytic vesicles create cascades of water current, inducing pulsatory transient poration that enable solute exchange between the buds and the GUV interior. This results in an efficient water-flux-mediated delivery of molecular cargo across the membrane boundary. Our findings suggest a primitive physical mechanism for communication and transport across protocellular compartments driven only by osmotic stresses. They also suggest plausible physical routes for intravesicular, and possibly intracellular, delivery of ions, solutes, and molecular cargo stimulated simply by cycles of osmotic currents of water.

Kinetic control of shape deformations and membrane phase separation inside giant vesicles.

A variety of cellular processes use liquid-liquid phase separation (LLPS) to create functional levels of organization, but the kinetic pathways by which it proceeds remain incompletely understood. Here in real time, we monitor the dynamics of LLPS of mixtures of segregatively phase-separating polymers inside all-synthetic, giant unilamellar vesicles. After dynamically triggering phase separation, we find that the ensuing relaxation-en route to the new equilibrium-is non-trivially modulated by a dynamic interplay between the coarsening of the evolving droplet phase and the interactive membrane boundary. The membrane boundary is preferentially wetted by one of the incipient phases, dynamically arresting the progression of coarsening and deforming the membrane. When the vesicles are composed of phase-separating mixtures of common lipids, LLPS within the vesicular interior becomes coupled to the membrane's compositional degrees of freedom, producing microphase-separated membrane textures. This coupling of bulk and surface phase-separation processes suggests a physical principle by which LLPS inside living cells might be dynamically regulated and communicated to the cellular boundaries.

Comparison of hamate versus second or third toe osteochondral graft using magnetic resonance imaging for reconstruction of proximal interphalangeal fracture-dislocations.

Hemi-hamate arthroplasty is a method used to reconstruct complex fracture-dislocations of the proximal interphalangeal joint of the fingers. Other graft sites, including the toe second and third phalanges, have been proposed as alternatives to hemi-hamate arthroplasty due to variable clinical outcomes and anatomy. Through a prospective magnetic resonance imaging (MRI)-based study in asymptomatic individuals, we aimed to characterize the anatomy of the proximal interphalangeal joint and compare this with the hamate, second and third toes to determine the closest anatomical match using pre-determined measurements. Our results show that the second and third toes have greater anatomical similarity to the proximal interphalangeal joint of the fingers compared to the hamate. High-resolution MRI is a reliable method of characterizing the anatomy of these structures and could be a useful clinical tool in determining reconstructive options in the management of this challenging injury.Level of evidence: II.

Biomimetic Stratum Corneum Liposome Models: Lamellar Organization and Permeability Studies.

The stratum corneum (SC), the outer layer of the skin, plays a crucial role as a barrier protecting the underlying cells from external stress. The SC comprises three key components: ceramide (CER), free fatty acid (FFA), and cholesterol, along with small fractions of cholesterol sulfate and cholesterol ester. In order to gain a deeper understanding about the interdependence of the two major components, CER and FFA, on the organizational, structural, and functional properties of the SC layer, a library of SC lipid liposome (SCLL) models was developed by mixing CER (phytosphingosine or sphingosine), FFA (oleic acid, palmitic acid, or stearic acid), cholesterol, and cholesterol sulfate. Self-assembly of the SC lipids into lamellar phases was first confirmed by small-angle X-ray scattering. Short periodicity and long periodicity phases were identified for SCLLs containing phytosphingosines and sphingosine CERs, respectively. Furthermore, unsaturation in the CER acyl and FFA chains reduced the lipid conformational ordering and packing density of the liposomal bilayer, which were measured by differential scanning calorimetry and Fourier transform infrared spectroscopy. The introduction of unsaturation in the CER and/or FFA chains also impacted the lamellar integrity and permeability. This extensive library of SCLL models exhibiting physiologically relevant lamellar phases with defined structural and functional properties may potentially be used as a model system for screening pharmaceuticals or cosmetic agents.

Mechanisms of a Mycobacterium tuberculosis Active Peptide.

Multidrug-resistant tuberculosis (MDR) continues to pose a threat to public health. Previously, we identified a cationic host defense peptide with activity against Mycobacterium tuberculosis in vivo and with a bactericidal effect against MDR M. tuberculosis at therapeutic concentrations. To understand the mechanisms of this peptide, we investigated its interactions with live M. tuberculosis and liposomes as a model. Peptide interactions with M. tuberculosis inner membranes induced tube-shaped membranous structures and massive vesicle formation, thus leading to bubbling cell death and ghost cell formation. Liposomal studies revealed that peptide insertion into inner membranes induced changes in the peptides' secondary structure and that the membranes were pulled such that they aggregated without permeabilization, suggesting that the peptide has a strong inner membrane affinity. Finally, the peptide targeted essential proteins in M. tuberculosis, such as 60 kDa chaperonins and elongation factor Tu, that are involved in mycolic acid synthesis and protein folding, which had an impact on bacterial proliferation. The observed multifaceted targeting provides additional support for the therapeutic potential of this peptide.

Surfactant-Mediated Solubilization of Myelin Figures: A Multistep Morphological Cascade.

Lamellar mesophases of insoluble lipids are readily solubilized by the micellar mesophases of soluble surfactants. This simple process underscores a broad array of biochemical methodologies, including purification, reconstitution, and crystallization of membrane proteins, as well as the isolation of detergent-resistant membrane fractions. Although much is now known about the thermodynamic driving forces of membrane solubilization, the kinetic pathways by which the surfactant alters vesicular mesophases are only beginning to be appreciated. Little is known about how these interactions affect the solubilization of more complex, multilamellar mesophases. Here, we investigate how a common zwitterionic detergent affects the solubilization of a smectic, multilamellar, cylindrical mesophase of lipids, called the myelin figure. Our results reveal that myelin solubilization occurs in a multistep manner, producing a well-defined sequence of morphologically distinct intermediates en route to complete solubilization. The kinetic processes producing these intermediates include (1) coiling, which encompasses the formation, propagation, and tightening of extended helices; (2) thinning, which reflects the unbinding of lamellae in the smectic stacks; and (3) detachment or retraction, which either dissociates the myelinic protrusion from the source lipid mass or returns the myelinic protrusion to the source lipid mass─all in transit toward complete solubilization. These occasionally overlapping steps are most pronounced in single-lipid component myelins, while compositionally graded multicomponent myelins inhibit the coiling step and detach more frequently. Taken together, the appearance of these intermediates during the solubilization of myelins suggests a complex free-energy landscape characterizing myelin solubilization populated by metastable, morphological intermediates correlated with locally minimized changes in energy dependent upon the mesophase's composition. This then predicts the accessibility of structurally distinct, kinetic intermediates─such as loose and tight coiled helices, peeled myelins, retracted tubes, and detached protrusions─before reaching the stable ground state corresponding to a dissolved suspension of mixed surfactant-lipid micelles.

Lipid bilayer composition as a determinant of cancer cell sensitivity to tumoricidal protein-lipid complexes.

Complexes formed by the alpha1 N-terminal peptide of alpha-lactalbumin and oleic acid (alpha1-oleate) interact with lipid bilayers. Plasma membrane perturbations trigger tumor cell death but normal differentiated cells are more resistant, and their plasma membranes are less strongly affected. This study examined membrane lipid composition as a determinant of tumor cell reactivity. Bladder cancer tissue showed a higher abundance of unsaturated lipids enriched in phosphatidylcholine, PC (36:4) and PC (38:4), and sphingomyelin, SM (36:1) than healthy bladder tissue, where saturated lipids predominated and the lipid extracts from bladder cancer tissue inhibited the tumoricidal effect of the complex more effectively than healthy tissue extracts. Furthermore, unsaturated PC in solution inhibited tumor cell death, and the complex interacted with giant unilamellar vesicles formed by PC, confirming the affinity of alpha1-oleate for fluid membranes enriched in PC. Quartz Crystal Microbalance with dissipation monitoring (QCM-D) detected a preference of the complex for the liquid-disordered phase, suggesting that the insertion into PC-based membranes and the resulting membrane perturbations are influenced by membrane lipid saturation. The results suggest that the membrane lipid composition is functionally important and that specific unsaturated membrane lipids may serve as "recognition motifs" for broad-spectrum tumoricidal molecules such as alpha1-oleate.

The arterialised saphenous venous flow-through flap for managing the radial forearm free flap donor site.

BACKGROUND: The radial forearm fasciocutaneous flap (RFFF) is a workhorse flap, however concerns with donor site morbidity include tendon exposure, delayed wound healing, impaired sensitivity, and poor cosmesis, have seen it fall out of favor. We present a method of using an arterialised saphenous flow through flap to reconstruct the RFFF donor site. METHOD: A cohort study of six patients (five male, one female; mean age 59 [range 19-90]) who had their RFFF donor site reconstructed with an arterialised saphenous flow through flap is presented. The use of multiple peripheral efferent venous anastomoses, flap rotation 180 degrees prior to inset, and the ligation of intra-flap connecting veins were three modifications employed. Primary outcomes include complication rates. Secondary outcomes were patient reported outcome measures via the Michigan Hand Outcomes Questionnaire, and patency and flow through the flap. RESULTS: In all six cases, there was flap survival. RFFF dimensions ranging from lengths of 6-15 cm (mean 11.5 cm) and widths of 4-6 cm (mean 5.3 cm), with an average flap area of 58 cm2 (range 24-90). There were no total flap losses, one partial superficial flap loss and one minor donor site delayed healing, over a mean follow-up of 6 months (4-24 months). The average overall patient satisfaction was 91 on Michigan Hand Outcomes Questionnaire. Pain was well tolerated with a low average pain score of 15. CONCLUSION: The modified arterialised saphenous flow through flap is a useful option for reconstructing the soft tissue defect and reconstituting the radial artery after RFFF harvest.

Nonequilibrium Self-Organization of Lipids into Hierarchically Ordered and Compositionally Graded Cylindrical Smectics.

When a dry mass of certain amphiphiles encounters water, a spectacular interfacial instability ensues: It gives rise to the formation of ensembles of fingerlike tubular protrusions called myelin figures─tens of micrometers wide and tens to hundreds of micrometers long─representing a novel class of nonequilibrium higher-order self-organization. Here, we report that when phase-separating mixtures of unsaturated lipid, cholesterol, and sphingomyelin are hydrated, the resulting myelins break symmetry and couple their compositional degrees of freedom with the extended myelinic morphology: They produce complementary, interlamellar radial gradients of concentrations of cholesterol (and sphingomyelin) and unsaturated lipid, which stands in stark contrast to interlamellar, lateral phase separation in equilibrated morphologies. Furthermore, the corresponding gradients of molecule-specific chemistries (i.e., cholesterol extraction by methyl-ß-cyclodextrin and GM1 binding by cholera toxin) produce unusual morphologies comprising compositionally graded vesicles and buckled tubes. We propose that kinetic differences in the information processing of hydration characteristics of individual molecules while expending energy dictate this novel behavior of lipid mixtures undergoing hydration.

Amphiphilic Membrane Environments Regulate Enzymatic Behaviors of Salmonella Outer Membrane Protease.

The role of an amphiphilic environment in the functional regulation of integral membrane proteins is well appreciated but how specific amphiphilic surrounding influences the conformational plasticity and function of a protein is less obvious. We focus on the Salmonella phosphoglycerate transport system (pgt)-encoded outer membrane protease E (PgtE), which plays an important role in tissue infiltration and survival of Salmonella enterica. Despite our understanding of its physiological functions, elucidation of its enzymatic behavior in response to the immediate amphiphilic surrounding is lacking. We monitor the proteolytic activity of PgtE reconstituted in Zwittergent 3-12 detergent micelles or a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayer and examine factors that influence its activity. We find, to our surprise, that PgtE, which is thought to elicit a rapid response toward various substrates, showed hysteretic enzymatic behavior, characterized by a prominent lag phase prior to achieving the exponential steady state in its detergent-stabilized form as well as in the outer membrane embedded native state in live bacteria. The lag phase was abolished under three conditions: preformation of an inactive detergent-stabilized PgtE-substrate complex without lipopolysaccharide (LPS), LPS-bound detergent-stabilized PgtE that had reached steady state velocity, or PgtE reconstituted into a POPC bilayer environment. Interestingly, detergent- and bilayer-stabilized PgtE showed comparable steady-state activity. And strikingly, lipopolysaccharide (LPS) becomes nonessential for the activation of PgtE when the protein is reconstituted in the phospholipid bilayer, contrasting a long-standing notion that LPS is required for proteases belonging to the omptin family to be proteolytically active. These findings suggest intriguing biological nuances for the proteolytic function of PgtE that were not well appreciated previously and offer new perspectives that may generally be applicable for omptins.

A scientific journey from discovery to validation of efficacy in cancer patients: HAMLET and alpha1-oleate.

The protein-lipid complex alpha1-oleate, derived from HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), is identified as a molecular entity with significant therapeutic potential. Structural characterization of the complex and results of a successful placebo-controlled clinical trial are presented.

Intravascular Lithotripsy for Calcium Modification in Chronic Total Occlusion Percutaneous Coronary Intervention.

Intravascular lithotripsy (IVL) has been shown to be safe and effective for calcium modification in nonocclusive coronary artery disease (CAD), but there are only case reports of its use in calcified chronic total occlusions (CTO). We report data from an international multicenter registry of IVL use during CTO percutaneous coronary intervention (PCI) and provide provisional data regarding its efficacy and safety. During the study period, IVL was used in 55 of 1053 (5.2%) CTO PCI procedures. IVL was used within the occluded segment after successful CTO crossing in 53 procedures and during incomplete CTO crossing in 2 cases. The mean J-CTO score was 3.1. CTO PCI technical and procedural success was achieved in 53 (96%) and 51 (93%) cases. Six patients had a procedural complication, with 3 main vessel perforations (5%). Two had covered stent implantation, one required pericardiocentesis, and one was managed conservatively. All had combination therapy with another calcium modification device. Two patients had a procedural myocardial infarction (PMI) (4%), and two others had a major adverse cardiovascular event (MACE) (4%) at a median follow-up of 13 (4-21) months. IVL can effectively facilitate calcium modification during CTO PCI. More data are required to establish the efficacy and safety of IVL and other calcium modification devices when used extraplaque or in combination during CTO PCI.

Bladder cancer therapy using a conformationally fluid tumoricidal peptide complex.

Partially unfolded alpha-lactalbumin forms the oleic acid complex HAMLET, with potent tumoricidal activity. Here we define a peptide-based molecular approach for targeting and killing tumor cells, and evidence of its clinical potential (ClinicalTrials.gov NCT03560479). A 39-residue alpha-helical peptide from alpha-lactalbumin is shown to gain lethality for tumor cells by forming oleic acid complexes (alpha1-oleate). Nuclear magnetic resonance measurements and computational simulations reveal a lipid core surrounded by conformationally fluid, alpha-helical peptide motifs. In a single center, placebo controlled, double blinded Phase I/II interventional clinical trial of non-muscle invasive bladder cancer, all primary end points of safety and efficacy of alpha1-oleate treatment are reached, as evaluated in an interim analysis. Intra-vesical instillations of alpha1-oleate triggers massive shedding of tumor cells and the tumor size is reduced but no drug-related side effects are detected (primary endpoints). Shed cells contain alpha1-oleate, treated tumors show evidence of apoptosis and the expression of cancer-related genes is inhibited (secondary endpoints). The results are especially encouraging for bladder cancer, where therapeutic failures and high recurrence rates create a great, unmet medical need.

Both maternal and embryonic exposure to mild hypoxia influence embryonic development of the intertidal gastropod Littorina littorea.

There is growing evidence that maternal exposure to environmental stressors can alter offspring phenotype and increase fitness. Here, we investigate the relative and combined effects of maternal and developmental exposure to mild hypoxia (65 and 74% air saturation, respectively) on the growth and development of embryos of the marine gastropod Littorina littorea Differences in embryo morphological traits were driven by the developmental environment, whereas the maternal environment and interactive effects of maternal and developmental environment were the main driver of differences in the timing of developmental events. While developmental exposure to mild hypoxia significantly increased the area of an important respiratory organ, the velum, it significantly delayed hatching of veliger larvae and reduced their size at hatching and overall survival. Maternal exposure had a significant effect on these traits, and interacted with developmental exposure to influence the time of appearance of morphological characters, suggesting that both are important in affecting developmental trajectories. A comparison between embryos that successfully hatched and those that died in mild hypoxia revealed that survivors exhibited hypertrophy in the velum and associated pre-oral cilia, suggesting that these traits are linked with survival in low-oxygen environments. We conclude that both maternal and developmental environments shape offspring phenotype in a species with a complex developmental life history, and that plasticity in embryo morphology arising from exposure to even small reductions in oxygen tensions affects the hatching success of these embryos.

Peptide-Oleate Complexes Create Novel Membrane-Bound Compartments.

A challenging question in evolutionary theory is the origin of cell division and plausible molecular mechanisms involved. Here, we made the surprising observation that complexes formed by short alpha-helical peptides and oleic acid can create multiple membrane-enclosed spaces from a single lipid vesicle. The findings suggest that such complexes may contain the molecular information necessary to initiate and sustain this process. Based on these observations, we propose a new molecular model to understand protocell division.

Facile Mixing of Phospholipids Promotes Self-Assembly of Low-Molecular-Weight Biodegradable Block Co-Polymers into Functional Vesicular Architectures.

In this work, we have used low-molecular-weight (PEG12-b-PCL6, PEG12-b-PCL9 or PEG16-b-PLA38; MW, 1.25-3.45 kDa) biodegradable block co-polymers to construct nano- and micron-scaled hybrid (polymer/lipid) vesicles, by solvent dispersion and electroformation methods, respectively. The hybrid vesicles exhibit physical properties (size, bilayer thickness and small molecule encapsulation) of a vesicular boundary, confirmed by cryogenic transmission electron microscopy, calcein leakage assay and dynamic light scattering. Importantly, we find that these low MW polymers, on their own, do not self-assemble into polymersomes at nano and micron scales. Using giant unilamellar vesicles (GUVs) model, their surface topographies are homogeneous, independent of cholesterol, suggesting more energetically favorable mixing of lipid and polymer. Despite this mixed topography with a bilayer thickness similar to that of a lipid bilayer, variation in surface topology is demonstrated using the interfacial sensitive phospholipase A2 (sPLA2). The biodegradable hybrid vesicles are less sensitive to the phospholipase digestion, reminiscent of PEGylated vesicles, and the degree of sensitivity is polymer-dependent, implying that the nano-scale surface topology can further be tuned by its chemical composition. Our results reveal and emphasize the role of phospholipids in promoting low MW polymers for spontaneous vesicular self-assembly, generating a functional hybrid lipid-polymer interface.