Coxiella co-opts the Glutathione Peroxidase 4 to protect the host cell from oxidative stress-induced cell death.

The causative agent of human Q fever, Coxiella burnetii, is highly adapted to infect alveolar macrophages by inhibiting a range of host responses to infection. Despite the clinical and biological importance of this pathogen, the challenges related to genetic manipulation of both C. burnetii and macrophages have limited our knowledge of the mechanisms by which C. burnetii subverts macrophages functions. Here, we used the related bacterium Legionella pneumophila to perform a comprehensive screen of C. burnetii effectors that interfere with innate immune responses and host death using the greater wax moth Galleria mellonella and mouse bone marrow-derived macrophages. We identified MceF (Mitochondrial Coxiella effector protein F), a C. burnetii effector protein that localizes to mitochondria and contributes to host cell survival. MceF was shown to enhance mitochondrial function, delay membrane damage, and decrease mitochondrial ROS production induced by rotenone. Mechanistically, MceF recruits the host antioxidant protein Glutathione Peroxidase 4 (GPX4) to the mitochondria. The protective functions of MceF were absent in primary macrophages lacking GPX4, while overexpression of MceF in human cells protected against oxidative stress-induced cell death. C. burnetii lacking MceF was replication competent in mammalian cells but induced higher mortality in G. mellonella, indicating that MceF modulates the host response to infection. This study reveals an important C. burnetii strategy to subvert macrophage cell death and host immunity and demonstrates that modulation of the host antioxidant system is a viable strategy to promote the success of intracellular bacteria.

Perturbation of ATG16L1 function impairs the biogenesis of Salmonella and Coxiella replication vacuoles.

Anti-bacterial autophagy, known as xenophagy, is a host innate immune response that targets invading pathogens for degradation. Some intracellular bacteria, such as the enteric pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), utilize effector proteins to interfere with autophagy. One such S. Typhimurium effector, SopF, inhibits recruitment of ATG16L1 to damaged Salmonella-containing vacuoles (SCVs), thereby inhibiting the host xenophagic response. SopF is also required to maintain the integrity of the SCV during the early stages of infection. Here we show disruption of the SopF-ATG16L1 interaction leads to an increased proportion of cytosolic S. Typhimurium. Furthermore, SopF was utilized as a molecular tool to examine the requirement for ATG16L1 in the intracellular lifestyle of Coxiella burnetii, a bacterium that requires a functional autophagy pathway to replicate efficiently and form a single, spacious vacuole called the Coxiella-containing vacuole (CCV). ATG16L1 is required for CCV expansion and fusion but does not influence C. burnetii replication. In contrast, SopF did not affect CCV formation or replication, demonstrating that the contribution of ATG16L1 to CCV biogenesis is via its role in autophagy, not xenophagy. This study highlights the diverse capabilities of bacterial effector proteins to dissect the molecular details of host-pathogen interactions.

Microfluidic Particle Engineering of Hydrophobic Drug with Eudragit E100─Bridging the Amorphous and Crystalline Gap.

Co-processing active pharmaceutical ingredients (APIs) with excipients is a promising particle engineering technique to improve the API physical properties, which can lead to more robust downstream drug product manufacturing and improved drug product attributes. Excipients provide control over critical API attributes like particle size and solid-state outcomes. Eudragit E100 is a widely used polymeric excipient to modulate drug release. Being cationic, it is primarily employed as a precipitation inhibitor to stabilize amorphous solid dispersions. In this work, we demonstrate how co-processing of E100 with naproxen (NPX) (a model hydrophobic API) into monodisperse emulsions via droplet microfluidics followed by solidification via solvent evaporation allows the facile fabrication of compact, monodisperse, and spherical particles with an expanded range of solid-state outcomes spanning from amorphous to crystalline forms. Low E100 concentrations (≤26% w/w) yield crystalline microparticles with a stable NPX polymorph distributed uniformly across the matrix at a high drug loading (∼89% w/w). Structurally, E100 incorporation reduces the size of primary particles comprising the co-processed microparticles in comparison to neat API microparticles made using the same technique and the as-received API powder. This reduction in primary particle size translates into an increased internal porosity of the co-processed microparticles, with specific surface area and pore volume ∼9 times higher than the neat API microparticles. These E100-enabled structural modifications result in faster drug release in acidic media compared to neat API microparticles. Additionally, E100-NPX microparticles have a significantly improved flowability compared to neat API microparticles and as-received API powder. Overall, this study demonstrates a facile microfluidics-based co-processing method that broadly expands the range of solid-state outcomes obtainable with E100 as an excipient, with multiscale control over the key attributes and performance of hydrophobic API-laden microparticles.

Porous Polymerized High Internal Phase Emulsions Prepared Using Proteins and Essential Oils for Antimicrobial Applications.

High internal phase emulsions (HIPEs) provide a versatile platform for encapsulating large volumes of therapeutics that are immiscible in water. A stable scaffold is obtained by polymerizing the external phase, resulting in polyHIPEs. However, fabrication of polyHIPEs usually requires using a considerable quantity of surfactants along with nonbiocompatible components, which hinders their biological applications, e.g., drug-eluting devices. We describe here a straightforward method for generating porous biomaterials by using proteins as both the emulsifier and the building blocks for the fabrication of polyHIPEs. We demonstrate the versatility of this method by using different essential oils as the internal phase. After the gelation of protein building blocks is triggered by the addition of reducing agents, a stable protein hydrogel containing essential oils can be formed. These oils can be either extracted to obtain protein-based porous scaffolds or slowly released for antimicrobial applications.

Regulation of Proteins to the Cytosol Using Delivery Systems with Engineered Polymer Architecture.

Intracellular protein delivery enables selective regulation of cellular metabolism, signaling, and development through introduction of defined protein quantities into the cell. Most applications require that the delivered protein has access to the cytosol, either for protein activity or as a gateway to other organelles such as the nucleus. The vast majority of delivery vehicles employ an endosomal pathway however, and efficient release of entrapped protein cargo from the endosome remains a challenge. Recent research has made significant advances toward efficient cytosolic delivery of proteins using polymers, but the influence of polymer architecture on protein delivery is yet to be investigated. Here, we developed a family of dendronized polymers that enable systematic alterations of charge density and structure. We demonstrate that while modulation of surface functionality has a significant effect on overall delivery efficiency, the endosomal release rate can be highly regulated by manipulating polymer architecture. Notably, we show that large, multivalent structures cause slower sustained release, while rigid spherical structures result in rapid burst release.

Protein Delivery: If Your GFP (or Other Small Protein) Is in the Cytosol, It Will Also Be in the Nucleus.

Intracellular protein delivery is a transformative tool for biologics research and medicine. Delivery into the cytosol allows proteins to diffuse throughout the cell and access subcellular organelles. Inefficient delivery caused by endosomal entrapment is often misidentified as cytosolic delivery. This inaccuracy muddles what should be a key checkpoint in assessing delivery efficiency. Green fluorescent protein (GFP) is a robust cargo small enough to passively diffuse from the cytosol into the nucleus. Fluorescence of GFP in the nucleus is a direct readout for cytosolic access and effective delivery. Here, we highlight recent examples from the literature for the accurate assessment of cytosolic protein delivery using GFP fluorescence in the cytosol and nucleus.

Mucormycosis causing massive lower gastrointestinal bleeding: a case report.

BACKGROUND: Lower gastrointestinal bleeding (LGIB) is very common in the hospital setting. Most bleedings stop spontaneously, but rare infectious causes of LGIB may lead to rapid and serious complications if left untreated and are sometimes very difficult to diagnose preoperatively. CASE PRESENTATION: We described a young man with poorly controlled Type I diabetes mellitus and chronic alcohol abuse who presented with acute altered mental status. During his hospitalization for treatment of diabetic ketoacidosis, acute renal failure, and sepsis, he suddenly developed massive hematochezia of 1500 mL. Colonoscopy was performed and a deep ulcer covered with mucus with peripheral elevation was noted at the transverse colon. Biopsy of the ulcer later revealed nonpigmented, wide (5-20 µm in diameter), thin-walled, ribbon-like hyphae with few septations and right-angle branching suggestive of mucormycosis demonstrated by Periodic acid-Schiff stain. He received 2 months of antifungal treatment. Follow up colonoscopy post-treatment was normal with no ulcer visualized. CONCLUSIONS: Early diagnosis and treatment of gastrointestinal (GI) mucormycosis infection is critical but can be challenging, especially in the setting of massive hematochezia. Therefore, clinical awareness for immunocompromised patients and prompt antifungal prophylaxis in cases with high suspicion of infection are essential.

EirA Is a Novel Protein Essential for Intracellular Replication of Coxiella burnetii.

The zoonotic bacterial pathogen Coxiella burnetii is the causative agent of Q fever, a febrile illness which can cause a serious chronic infection. C. burnetii is a unique intracellular bacterium which replicates within host lysosome-derived vacuoles. The ability of C. burnetii to replicate within this normally hostile compartment is dependent on the activity of the Dot/Icm type 4B secretion system. In a previous study, a transposon mutagenesis screen suggested that the disruption of the gene encoding the novel protein CBU2072 rendered C. burnetii incapable of intracellular replication. This protein, subsequently named EirA (essential for intracellular replication A), is indispensable for intracellular replication and virulence, as demonstrated by infection of human cell lines and in vivo infection of Galleria mellonella The putative N-terminal signal peptide is essential for protein function but is not required for localization of EirA to the bacterial inner membrane compartment and axenic culture supernatant. In the absence of EirA, C. burnetii remains viable but nonreplicative within the host phagolysosome, as coinfection with C. burnetii expressing native EirA rescues the replicative defect in the mutant strain. In addition, while the bacterial ultrastructure appears to be intact, there is an altered metabolic profile shift in the absence of EirA, suggesting that EirA may impact overall metabolism. Most strikingly, in the absence of EirA, Dot/Icm effector translocation was inhibited even when EirA-deficient C. burnetii replicated in the wild type (WT)-supported Coxiella containing vacuoles. EirA may therefore have a novel role in the control of Dot/Icm activity and represent an important new therapeutic target.

Direct Cytosolic Delivery of Proteins through Coengineering of Proteins and Polymeric Delivery Vehicles.

Nanocarrier-mediated protein delivery is a promising strategy for fundamental research and therapeutic applications. However, the efficacy of the current platforms for delivery into cells is limited by endosomal entrapment of delivered protein cargo with concomitantly inefficient access to the cytosol and other organelles, including the nucleus. We report here a robust, versatile polymeric-protein nanocomposite (PPNC) platform capable of efficient (≥90%) delivery of proteins to the cytosol. We synthesized a library of guanidinium-functionalized poly(oxanorborneneimide) (PONI) homopolymers with varying molecular weights to stabilize and deliver engineered proteins featuring terminal oligoglutamate "E-tags". The polymers were screened for cytosolic delivery efficiency using imaging flow cytometry with cytosolic delivery validated using confocal microscopy and activity of the delivered proteins demonstrated through functional assays. These studies indicate that the PPNC platform provides highly effective and tunable cytosolic delivery over a wide range of formulations, making them robust agents for therapeutic protein delivery.

Effect of energy per atom (E/n) on the Ar gas cluster ion beam (Ar-GCIB) and O2+ cosputter process.

Gas cluster ion beam (GCIB) is a promising technique for preserving molecular structures during ion sputtering and successfully profiling biological and soft materials. However, although GCIB yields lower damage accumulation compared with C60+ and monoatomic ion beams, the inevitable alteration of the chemical structure can introduce artifacts into the resulting depth profile. To enhance the ionization yield and further mask damage, a low-energy O2+ (200-500 V) cosputter can be applied. While the energy per atom (E/n) of GCIB is known to be an important factor influencing the sputter process, the manner through which E/n affects the GCIB-O2+ cosputter process remains unclear. In this study, poly(ethylene terephthalate) (PET) was used as a model material to investigate the sputter process of 10-20 kV Ar1000-4000+ (E/n = 2.5-20 eV per atom) with and without O2+ cosputter at different energies and currents. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) with Bi32+ as the primary ion was used to examine surfaces sputtered at different fluences. The sputter craters were also measured by alpha-step and atomic force microscopy in quantitative imaging mode. The SIMS results showed that the steady-state cannot be obtained with E/n values of less than 5 eV per atom due to damage accumulation using single GCIB sputtering. With a moderate E/n value of 5-15 eV per atom, the steady-state can be obtained, but the ∼50% decay in intensity indicated that damage cannot be masked completely despite the higher sputter yield. Furthermore, the surface Young's modulus decreased with increasing E/n, suggesting that depolymerization occurred. At an E/n value of 20 eV per atom, a failed profile was obtained with rapidly decreased sputter rate and secondary ion intensity due to the ion-induced crosslink. With O2+ cosputtering and a moderate E/n value, the oxidized species generated by O2+ enhanced the ionization yield, which led to a higher ion intensity at steady-state in general. Because higher kinetic energy or current density of O2+ led to a larger interaction volume and more structural damage that suppressed molecular ion intensity, the enhancement from O2+ was most apparent with low-energy-high-current (200 V, 80 µA cm-2) or high-energy-low-current (500 V, 5 µA cm-2) O2+ cosputtering with 0.5 µA cm-2 GCIBs. In these cases, little or no intensity drop was observed at the steady-state.

Common pulmonary vein on the recurrence of atrial tachyarrhythmia after pulmonary vein isolation.

AIMS: Catheter ablation has become an effective treatment modality for atrial fibrillation (AF). However, the relationship between common pulmonary vein (PV) and recurrent atrial tachyarrhythmia (ATA) after PV isolation (PVI) remains controversial. This study aimed to explore the function of common PV on the risk of recurrent ATA after PVI. METHODS: We identified a total of 191 patients who received radiofrequency catheter ablation for paroxysmal AF at our hospital between July 2010 and December 2017 for retrospective chart review. We collected the following data for analysis: results of preprocedural computed tomography, including the anatomy of PV and left atrial (LA) volume; the incidence of early- and late-onset recurrence of ATA. We compared these characteristics between the two groups defined by the presence or absence of the late-onset recurrence of ATA. RESULTS: Compared to the no ATA recurrence group, the ATA recurrence group had larger LA size, larger LA end-diastolic and systolic volumes, larger maximal diameter of PV, higher prevalence of common PV, and higher incidence of early-onset recurrence of ATA. In multivariate logistic regression analyses, presence of common PV and early-onset recurrence were independently associated with late-onset recurrence of ATA. Compared to patients without common PV, patients with common PV had larger diameter of PV and higher incidence of late-onset recurrent ATA. CONCLUSION: In patients with paroxysmal AF, early-onset recurrence of ATA and the presence of common PV were independently associated with late-onset recurrent ATA after radiofrequency catheter ablation.

Protein delivery into cells using inorganic nanoparticle-protein supramolecular assemblies.

The delivery of proteins into cells is a potential game changer for a wide array of therapeutic purposes, including cancer therapy, immunomodulation and treatment of inherited diseases. In this review, we present recently developed nanoassemblies for protein delivery that utilize strategies that range from direct assembly, encapsulation and composite formation. We will discuss factors that affect the efficacy of nanoassemblies for delivery from the perspective of both nanoparticles and proteins. Challenges in the field, particularly achieving effective cytosolar protein delivery through endosomal escape or evasion are discussed.

Engineered Polymer Nanoparticles with Unprecedented Antimicrobial Efficacy and Therapeutic Indices against Multidrug-Resistant Bacteria and Biofilms.

The rapid emergence of antibiotic-resistant bacterial "superbugs" with concomitant treatment failure and high mortality rates presents a severe threat to global health. The superbug risk is further exacerbated by chronic infections generated from antibiotic-resistant biofilms that render them refractory to available treatments. We hypothesized that efficient antimicrobial agents could be generated through careful engineering of hydrophobic and cationic domains in a synthetic semirigid polymer scaffold, mirroring and amplifying attributes of antimicrobial peptides. We report the creation of polymeric nanoparticles with highly efficient antimicrobial properties. These nanoparticles eradicate biofilms with low toxicity to mammalian cells and feature unprecedented therapeutic indices against red blood cells. Most notably, bacterial resistance toward these nanoparticles was not observed after 20 serial passages, in stark contrast to clinically relevant antibiotics where significant resistance occurred after only a few passages.

Biodegradable Nanocomposite Antimicrobials for the Eradication of Multidrug-Resistant Bacterial Biofilms without Accumulated Resistance.

Infections caused by multidrug-resistant (MDR) bacteria are a rapidly growing threat to human health, in many cases exacerbated by their presence in biofilms. We report here a biocompatible oil-in-water cross-linked polymeric nanocomposite that degrades in the presence of physiologically relevant biomolecules. These degradable nanocomposites demonstrated broad-spectrum penetration and elimination of MDR bacteria, eliminating biofilms with no toxicity to cocultured mammalian fibroblast cells. Notably, serial passaging revealed that bacteria were unable to develop resistance toward these nanocomposites, highlighting the therapeutic promise of this platform.

CRISPRed Macrophages for Cell-Based Cancer Immunotherapy.

We present here an integrated nanotechnology/biology strategy for cancer immunotherapy that uses arginine nanoparticles (ArgNPs) to deliver CRISPR-Cas9 gene editing machinery into cells to generate SIRP-α knockout macrophages. The NP system efficiently codelivers single guide RNA (sgRNA) and Cas9 protein required for editing to knock out the "don't eat me signal" in macrophages that prevents phagocytosis of cancer cells. Turning off this signal increased the innate phagocytic capabilities of the macrophages by 4-fold. This improved attack and elimination of cancer cells makes this strategy promising for the creation of "weaponized" macrophages for cancer immunotherapy.

Exploring the expression bar code of SAA variants for gastric cancer detection.

We reported an integrated platform to explore serum protein variant pattern in cancer and its utility as a new class of biomarker panel for diagnosis. On the model study of serum amyloid A (SAA), we employed nanoprobe-based affinity mass spectrometry for enrichment, identification and quantitation of SAA variants from serum of 105 gastric cancer patients in comparison with 54 gastritis patients, 54 controls, and 120 patients from other cancer. The result revealed surprisingly heterogeneous and most comprehensive SAA bar code to date, which comprises 24 SAA variants including SAA1- and SAA2-encoded products, polymorphic isoforms, N-terminal-truncated forms, and three novel SAA oxidized isotypes, in which the variant-specific peptide sequence were also confirmed by LC-MS/MS. A diagnostic model was developed for dimension reduction and computational classification of the 24 SAA-variant bar code, providing good discrimination (AUC = 0.85 ± 3.2E-3) for differentiating gastric cancer group from gastritis and normal groups (sensitivity, 0.76; specificity, 0.81) and was validated with external validation cohort (sensitivity, 0.71; specificity, 0.74). Our platform not only shed light on the occurrence and modification extent of under-represented serum protein variants in cancer, but also suggested a new concept of diagnostic platform by serum protein variant profile.

In Vivo Delivery of CRISPR/Cas9 for Therapeutic Gene Editing: Progress and Challenges.

The successful use of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based gene editing for therapeutics requires efficient in vivo delivery of the CRISPR components. There are, however, major challenges on the delivery front. In this Topical Review, we will highlight recent developments in CRISPR delivery, and we will present hurdles that still need to be overcome to achieve effective in vivo editing.

Unusual body weight loss due to primary hyperparathyroidism: A case study with literature review.

Brown tumors (osteitis fibrosa cystica) are rare pathognomonic signs that occur in patients with primary hyperparathyroidism (PHPT). Brown tumors can exist in multiple bones and can easily be misdiagnosed as a metastatic tumor or multiple myeloma. It is also localized in the forearm, humerus, and leg. The symptoms of hypercalcemia, pathologic fracture, and bodyweight loss may increase the diagnostic difficulty of brown tumors because multiple myeloma and bone metastasis also show the same symptoms. We studied a 68-year-old woman who had experienced unusual bodyweight loss in the past 6 months (56kg-40kg) and bone pain. She went to the hospital after a fall with a complaint of bone pain. An X-ray revealed a left bubbly-like cystic change and multiple fractures at the left ulna midshaft. Upon investigation, the level of intact parathyroid hormone was ascertained to be 1800 (normal: 10-60) pg/ml. Microscopically, the tumor demonstrated a benign bone lesion and was compatible with osteitis fibrosa cystica due to PHPT. The parathyroid scan (Tc-99 m sestamibi) indicated right parathyroid hyperplasia, which was later confirmed by a parathyroidectomy. She was diagnosed with osteitis fibrosa cystica associated with PHPT due to a parathyroid adenoma. PHPT can be presented with multiple fractures, bone pain, and bodyweight loss. Therefore, if a patient presents these symptoms, PHPT should be considered.