Distal Sites Research Articles

Extended Depth of Focus Two-Photon Light-Sheet Microscopy for In Vivo Fluorescence Imaging of Large Multicellular Organisms at Cellular Resolution.

Two-photon excitation in light-sheet microscopy advances applications to live imaging of multicellular organisms. In a previous study, we developed a two-photon Bessel beam light-sheet microscope with a nearly 1-mm field of view and less than 4-μm axial resolution, using a low magnification (10×), middle numerical aperture (NA 0.5) detection objective. In this study, we aimed to construct a light-sheet microscope with higher resolution imaging while maintaining the large field of view, using low magnification (16×) with a high NA 0.8 objective. To address potential illumination and detection mismatch, we investigated the use of a depth of focus (DOF) extension method. Specifically, we used a stair-step device composed of five-layer annular zones that extended DOF two-fold, enough to cover the light-sheet thickness. Resolution measurements using fluorescent beads showed that the reduction in resolutions was small. We then applied this system to in vivo imaging of medaka fish and found that image quality degradation at the distal site of the beam injection could be compensated. This demonstrates that the extended DOF system combined with wide-field two-photon light-sheet microscopy offers a simple and easy setup for live imaging application of large multicellular organism specimens with sub-cellular resolution.

The 3D emergence profile on implant-supported restorations: A method for evaluating restorative angles.

Emerging evidence indicates that the emergence profile and subsequent restorative angle play a crucial role in clinical outcomes and can potentially affect the development and progression of peri-implant diseases. However, the traditional evaluation of the emergence profile and angle has been limited to mesial and distal sites using periapical x-rays, without considering the buccal sites. To describe a novel 3D method to estimate the emergence profile and restorative angles around single implant-supported crowns including buccal sites. A total of 30 implant-supported crowns (11 molars, 8 premolars, 8 central incisors and 1 canine) were extra-orally scanned using an intraoral scanner and the STL files produced were imported into a 3D software. The crown/abutment interface of each crown was delineated, and apico-coronal lines were automatically drawn following the shape of the crown. Three reference points were defined on the apico-coronal lines at the transition edge of the biological (BC) and the esthetic zone (EC) and the resulting angles were then calculated. The reliability of the measurements (2D and 3D) were assessed using the intraclass correlation coefficient (ICC). In anterior restorations, the mean angle of the esthetic zone amounted to 162 ± 14° at mesial sites, to 140 ± 10° at buccal sites and to 163 ± 11° at distal sites. The corresponding angles at the biological zones, amounted to 155 ± 13° at mesial sites, 139 ± 15° at buccal sites and 157 ± 5° at distal sites. In posterior restorations, the mean angle of the esthetic zone amounted to 162 ± 12° at mesial sites, to 157 ± 13 at buccal sites and to 162 ± 11 at distal sites. The corresponding angles at the biological zone, amounted to 158 ± 8 at mesial sites, 150 ± 15° at buccal sites and 156 ± 10 at distal sites. The ICC for all measurements ranged between 0.77 and 0.99 indicating a good intra-examiner reliability. Within the limitations of the present study, the 3D analysis seems to be a reliable and applicable method for the quantitative evaluation of the emergence profile in daily practice. Future randomized clinical trials are needed to assess whether a 3D analysis with the ensuing the emergence profile serves as a predictor for clinical outcomes. The development and implementation of a 3D workflow will provide technicians and dentists with the ability to assess the restorative angle of implant-supported restorations during the provisional phase and the final restoration. This approach may help achieve an aesthetically pleasing restoration while minimizing potential clinical complications.

Structural and dynamic characterization of the hexa-coordinated globin from Spisula solidissima

High energy consumption in the nervous system requires a continuous supply of O2. This role is assisted by proteins from the globin super-family in the nerve cells of invertebrates, where ‘nerve hemoglobins’ (nHbs) are mainly present at mM concentrations and exhibit oxygen affinities comparable to those of vertebrate myoglobins. To gain insight into the structural bases of this function, we report the crystal structure of nHb from the Atlantic surf clam Spisula solidissima (SsHb), previously suggested to display a bis-histidyl hexa-coordinated heme in the deoxy state, high O2 affinity, and ligand binding cooperativity when assayed in situ. The crystallized protein forms a dimer through packing of a 4-helix bundle involving helices E and F of each subunit. The SsHb ‘classic’ globin fold displays bis-histidyl (His71(E7) and His103(F8)) hexa-coordination of the heme-Fe atom, with structural and dynamics variations found in the inter-helix hinge regions. Molecular Dynamics simulations of both monomeric and dimeric species in the bis-histidyl hexa-coordinated, deoxy penta-coordinated, and O2-bound hexa-coordinated states reveal distinct structural rearrangements at the interface between subunits in the dimer; these would affect the magnitude of the conformational fluctuations observed between monomer and dimer, and the topology of cavities within the protein matrix and at the interface. These results point to a distal site opening mechanism allowing access of the exogenous ligand to the heme and cast hypotheses on the dimer interface structural and dynamic properties that may support ligand binding cooperativity in dimeric SsHb.-

The "Bubble Test" in the Arthroscopic Diagnosis of Triangular Fibrocartilage Central Lesion: Description and Relevance to Treatment.

Background Ulnarly sided wrist pain is a common and challenging symptom. Arthroscopy has become a safe and effective tool for the correct diagnosis and treatment, proving to be better than all other clinical and imaging diagnostic methods. Some tests have been described for the diagnosis of triangular fibrocartilage (TFC) lesions, such as the trampoline test for peripheral lesions and the hook, ghost, and suction tests for foveal detachments. In tears at the disc level, when they affect all layers of the TFC (complete lesion) at the same point, testing with the probe will usually suffice for the diagnosis. However, in some apparently partial lesions, the probe cannot pass through all layers because the proximal and distal sites of the lesion are not aligned. There has been no arthroscopic test described for such cases. Case Description We describe a simple and practical test that we have called the "bubble test" in a typical case to diagnose TFC central injuries, aiming to discriminate partial from complete lesions, which are eventually hard to differentiate through direct vision and arthroscopic probing, leading to a direct influence onto the treatment. The bubble test is performed with external compression of the distal radioulnar joint (DRUJ) aspect, with the thumb on the back of the DRUJ and the index and middle fingers onto the volar aspect. The abrupt compression of this region causes the passage of air mixed with synovial fluid from the DRUJ to the radiocarpal joint, generating bubble formation. Clinical Relevance The bubble test should correlate to physical examination and imaging (magnetic resonance imaging [MRI]) findings and is particularly useful in central and degenerative (Type II) TFC lesions in patients complaining of chronic pain on the ulnar region of the wrist, with no history of trauma and with inconclusive MRI findings. Once the diagnosis is confirmed, arthroscopic treatment consists of wide debridement of the lesion with resection of the injured tissue and the local inflammatory reaction. Thus, it is essential for the correct treatment to define the exact location of the lesion and to know whether the disc injury is complete.

Development of allosteric and selective CDK2 inhibitors for contraception with negative cooperativity to cyclin binding

Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception. However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs. In this paper, we describe the development and mechanism of action of type III inhibitors that bind CDK2 with nanomolar affinity. Notably, these anthranilic acid inhibitors exhibit a strong negative cooperative relationship with cyclin binding, which remains an underexplored mechanism for CDK2 inhibition. Furthermore, the binding profile of these compounds in both biophysical and cellular assays demonstrate the promise of this series for further development into a therapeutic selective for CDK2 over highly similar kinases like CDK1. The potential of these inhibitors as contraceptive agents is seen by incubation with spermatocyte chromosome spreads from mouse testicular explants, where they recapitulate Cdk2-/- and Spdya-/- phenotypes.

Metastases harbored in the maxillofacial region: Patient and cancer related characteristics and outcomes.

e18029 Background: Maxillofacial metastases from distal primary sites are rare, accounting for less than 1% of all newly diagnosed cancer in the head and neck region. Clinical manifestation is non-specific and lesions are often misdiagnosed as benign or inflammatory lesions, and mistreated. We sought to describe maxillofacial metastases and evaluate associations with treatment and patient related outcomes. Methods: Data from all patients diagnosed with cancer in the maxillofacial region between 2008-2020 were extracted retrospectively from the hospital registry, and those with radiological and histological evidence of metastatic lesions were included in the analysis. Patient demographics, clinical presentation, features of primary cancer and treatment were collected. Paired t-tests and univariate regression models were used to evaluate an association between variables and overall survival (OS). Results: Of 532 patients diagnosed with cancer in the maxillofacial region, 15 (2.8%) were found to have metastases from distal primary cancers and included in the study. The group comprised 8 males (53.3%) and 7 females (46.6%), mean age 69 years (range 43 -88) and most common primary malignancies were lung (27%), gastrointestinal (27%) and prostate (20%), with 60% adenocarcinoma histology and the rest SCC and undifferentiated carcinoma. Presenting symptoms included trismus, pain, and swelling in 40% of cases. Clinically, most intraoral manifestations were classic oral ulcerations while the most frequent extra oral features were diffuse pre-auricular and mandibular swelling. Mean time to diagnosis was 22 days (range 7-60), however, intraoral lesions were diagnosed numerically earlier than extra oral (18 vs 23 days, p = 0.5). The mandible was the primarily involved hard tissue location (40%), and the parotid, for soft tissue lesions (33%). Mean SUV for PET-CT scans was 4.4 (range 2.0-10.93) and CT demonstrated bone expansion and lysis (50% each) and periosteal reaction (33%). In total, 60% of patients in the study had a change to their treatment after diagnosing the metastasis, while 40% with direct radiation (30-70 Gy) to the metastatic site. Median OS was 5 months while 60% of patients in the study group survived less than 6 months. Despite being twice as likely to receive treatment (75% vs 37.5%), patients with intra oral metastases had significantly shorter survival than those with extra oral metastases (13.3 vs 2.9 months, 95% CI 3.025-17.689, p < 0.01). No other associations were observed between patient demographics, cancer type and treatment, with survival. Conclusions: The maxillofacial region appears to be a late site for cancer metastasis, with non-specific presentation. As patient survival outcomes improve across cancer types and the array of possible alternative diagnoses increases, clinicians should be aware of this entity to ensure rapid referral for diagnosis and palliative treatment.

Characteristics of local extension based on tumor distribution in nasopharyngeal carcinoma and proposed clinical target volume delineation.

e18068 Background: to summarize the characteristics of local extension of the eccentric and central nasopharyngeal carcinoma (NPC) by magnetic resonance imaging (MRI) and to improve clinical target volume (CTV) delineation. Methods: MRIs of 870 newly diagnosed NPC patients were reviewed. According to the tumor distribution feature, the NPCs were divided into eccentric and central lesions. Results: All local invasions presented as continuous invasion from gross lesions and structures adjacent to the nasopharynx are more likely to be invaded. There were 240 (27.6%) and 630 (72.4%) cases with central and eccentric lesions, respectively. The spread of eccentric lesions centered on the ipsilateral Rosenmüller’s fossa; and most anatomic sites had significantly higher invasion rates in ipsilateral side than the contralateral side ( P < 0.05); however, they were at low risk of concurrent bilateral tumor invasion ( < 10%) except the prevertebral muscle (15.4%) and nasal cavity (13.8%). The extension of central NPCs centered on the nasopharyngeal superior-posterior wall and was more common in superior-posterior direction; furthermore, the bilateral tumor invasion into the anatomical sites was common. Conclusions: Local invasion of NPC was characterized by continuous invasion from proximal to distal sites. The eccentric and central lesions showed different invasion features. Individual CTV delineation should be based on the distribution characteristics of tumors. The eccentric lesion had very low probability of invasion into the contralateral tissue, thus routine prophylactic radiation of contralateral parapharyngeal space and skull base foramina may not be necessary.

Flow recovery from distal pressure in linearized hemodynamics: an optimal control approach

The goal of this work is to derive a reliable stable and accurate inverse problem strategy for reconstructing cardiac output blood flow entering the ascending aorta from pressure measurements at a distal site of the arterial tree, assumed here to be the descending aorta. We assume that a reduced one-dimensional model of the aorta can be linearized around its steady state, resulting in a wave system with absorbing boundary condition at the outlet. Using this model, we attempt to reconstruct the inlet flow from a pressure measurement at the distal outlet. First, we investigate the observability of the problem and prove that the inversion of the input-output operator for the flow and pressure in the space of time-periodic solutions is ill-posed of degree one. We then develop a variational approach where we minimize the discrepancy between measurements and a simulated state and penalize the error with respect to a periodic state. It is shown that the penalty strategy is convergent and provides an efficient solution for the minimization. Numerical results illustrate the robustness of our approach to noise and the potential of our method to reconstruct inlet flow from real pressure recordings during anesthesia.

Novel reinforcement technique using pre-glued felt strip with Hydrofit® for a distal anastomotic site in acute type A aortic dissection.

In the surgical treatment of acute type A aortic dissection, performing the anastomosis in the dissected aorta is technically challenging due to the fragility of the dissected aortic wall. This study describes the reinforcement technique for the distal anastomotic site, using pre-glued felt strips with Hydrofit®. No intraoperative bleeding occurred at the anastomosis site of the distal anastomosis stump. Postoperative computed tomography revealed no new distal anastomotic entry. This technique is recommended for managing acute type A aortic dissection during distal aortic reinforcement.

Substituent Effects on the Carboxylation Step of Allyllic Substrates: A Density Functional Theory Study

AbstractNi(0/I) catalysed direct carboxylation of allyl alcohols represents an effective way to create β, γ‐unsaturated carboxylic acids, with the carboxylation to be a crucial elementary step. Using density functional theory (DFT) calculations, we investigated how substituents affected the carboxylation process. The energy barrier of the carboxylation process is relatively low in the case of Ni(0), which can be efficiently lowered down by electron‐donating group (at allyl‐α‐C). Other distal sites have less of an impact. The steric effect on this reaction is insignificant, although there is a certain threshold. Multiple linear regression was taken to fit the energy barrier of the carboxylation step using the NPA, Fukui function, and Wiberg bond index of the pre‐intermediate The results showed that the energy barrier of the carboxylation step can be easily predicted by the property parameter of the pre‐intermediate, eliminating the complex transition state calculation.

Redistribution of the chromatin remodeler Brg1 directs smooth muscle-derived adventitial progenitor-to-myofibroblast differentiation and vascular fibrosis.

Vascular smooth muscle-derived Sca1+ adventitial progenitor (AdvSca1-SM) cells are tissue-resident, multipotent stem cells that contribute to progression of vascular remodeling and fibrosis. Upon acute vascular injury, AdvSca1-SM cells differentiate into myofibroblasts and are embedded in perivascular collagen and the extracellular matrix. While the phenotypic properties of AdvSca1-SM-derived myofibroblasts have been defined, the underlying epigenetic regulators driving the AdvSca1-SM-to-myofibroblast transition are unclear. We show that the chromatin remodeler Smarca4/Brg1 facilitates AdvSca1-SM myofibroblast differentiation. Brg1 mRNA and protein were upregulated in AdvSca1-SM cells after acute vascular injury, and pharmacological inhibition of Brg1 by the small molecule PFI-3 attenuated perivascular fibrosis and adventitial expansion. TGF-β1 stimulation of AdvSca1-SM cells in vitro reduced expression of stemness genes while inducing expression of myofibroblast genes that was associated with enhanced contractility; PFI blocked TGF-β1-induced phenotypic transition. Similarly, genetic knockdown of Brg1 in vivo reduced adventitial remodeling and fibrosis and reversed AdvSca1-SM-to-myofibroblast transition in vitro. Mechanistically, TGF-β1 promoted redistribution of Brg1 from distal intergenic sites of stemness genes and recruitment to promoter regions of myofibroblast-related genes, which was blocked by PFI-3. These data provide insight into epigenetic regulation of resident vascular progenitor cell differentiation and support that manipulating the AdvSca1-SM phenotype will provide antifibrotic clinical benefits.

Interventions in Left Internal Mammary Artery

Left internal mammary artery (LIMA) grafts are the preferred arterial grafts as they are more durable. LIMA grafts can develop stenosis most commonly at the distal anastomotic site and less frequently involve the ostium or body of LIMA. They may occur in the immediate post-operative period due to technical faults or occur several years later. LIMA graft interventions are more challenging because of its long and tortuous course and frequently tend to develop spasm and dissection. It might be the only method of revascularization in some patients and needs to be undertaken on and off. LIMA grafts have been used for retrograde revascularization of chronic total occlusion of native coronary arteries.

Abstract IA19: Oncogenic KRAS and the regulation of the pancreatic cancer microenvironment.

Abstract Pancreatic ductal adenocarcinoma, the most common form of pancreatic cancer, is characterized by almost universal presence of oncogenic KRAS. A key characteristic of pancreatic cancer is the abundant accumulation of tumor microenvironment, rich in infiltrating immune cells and an extensive fibroblast population. Pancreatic cancer is preceded by precursor lesions known as Pancreatic Intraepithelial Neoplasia (PanIN); PanIN formation is accompanied by the formation of a pre-TME. Analysis of donor pancreas from healthy individual revealed that PanIN is common in human samples, and accompanied by pre-TME accumulation in human samples. We sought to determine whether oncogenic KRAS drives accumulation and maintenance of the pre-TME. For this purpose, we utilized the iKRAS mouse model of pancreatic cancer, which allows tissue-specific, inducible and reversible activation of KRASG12D, the most common mutant form in human pancreatic cancer. Activation of oncogenic KRAS in iKRAS mice leads to PanIN formation. We observed that fibroblast activation occurs upon KRAS activation and precedes the formation of overt lesions. Further, epithelial oncogenic KRAS is required to maintain fibroblast reprogramming in PanIN. To comprehensively understand changes in fibroblasts, we used single cell RNA sequencing, and observed that the fibroblast transcriptional program is extrinsically regulated by epithelial oncogenic KRAS. The reprogramming process can be modeled in vivo using conditioned media from pancreatic cancer cells expressing oncogenic KRAS, and is mediated by JAK/STAT3 signaling in fibroblasts. We then sought to determine whether the extrinsic effects of oncogenic KRAS induced reprogramming of fibroblasts at distal sites. We chose to study the lung, which is a common site of metastasis for pancreatic cancer. Our data show that lung fibroblasts are activated following expression of oncogenic KRAS in the pancreas; fibroblast activation is accompanied by phospho-Stat3 signaling, a readout of the JAK/Stat3 pathway, and precedes metastasis outgrowth in the pancreas. Thus, oncogenic KRAS extrinsically reprograms the local and systemic environments, possibly facilitating metastatic spread of pancreatic cancer. Citation Format: Marina Pasca di Magliano. Oncogenic KRAS and the regulation of the pancreatic cancer microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA19.

IL-1 signaling directs the prioritization of inflammatory sites in a model of post-operative pulmonary infection

Abstract Individuals recovering from surgery are at risk of nosocomial infections such as pneumonia. In this setting, the innate immune system faces competing inflammation in the injured skin and infected lung, raising the question of how the immune response prioritizes inflammatory sites. We hypothesized that the initiation of a lung infection would disrupt wound healing. Supporting this hypothesis, data from the ACS NSQIP database showed that the rate of laparotomy dehiscence in surgical patients with pneumonia was over double the rate of those without pneumonia. We next developed mouse models of post-operative pulmonary infection to assess cellular mechanisms. Using the tail skin excision and dorsal subcutaneous PVA sponge implantation wound healing models, we found that bacterial lung infection slowed the rate of wound closure and impaired wound neutrophil, monocyte, and cytokine/chemokine responses. Lung infection quickly suppressed wound fluid IL-1b and downstream chemokine concentrations within 6 hours of inoculation. IL-1 receptor antagonist (IL-1RA), an inhibitor of IL-1 signaling, was upregulated in the bronchoalveolar lavage fluid and plasma 6 hours after lung infection. Administration of IL-1RA to the wounds of uninfected mice phenocopied the effects of lung infection. Conversely, rIL-1b rescued wound inflammation when administered to the to the wounds of mice with lung infection, although this occurred at the expense of lung bacterial clearance. These results suggest that the infected lung induces IL-1RA systemically as a protective mechanism to tune inflammation at distal sites via IL-1b suppression, allowing the innate immune system to prioritize inflammatory insults. Supported by grants from Defense Advanced Research Projects Agency (DARPA), Brown University (Dean's Areas of Emerging New Science Award, Carney Institute Innovation Awards), and NIH (NIES T32-ES7272 Training in Environmental Pathology; NIGMS COBRE Award P20GM10935, NHLBI R01 126887),

Hemodynamic predictors for the onset of ischemic stroke due to carotid artery stenosis using a computational fluid dynamics analysis

Background: Carotid artery stenosis is one of the major diseases responsible for atherosclerotic ischemic stroke. It is difficult to predict the onset of cerebral infarction by means of their known risk factors, including age, sex, diabetes mellitus, hypertension, dyslipidemia, and smoking habit. The carotid artery shape itself is a risk factor for stenosis, in which hemodynamics may be involved. We conduct an observational clinical study using a computational fluid dynamics (CFD) technique to investigate hemodynamic risk factors for carotid stenosis progression by enrolling and prospectively examining patients with carotid artery stenosis who underwent three-dimensional imaging of the carotid artery and carotid echocardiography. A total of 546 patients were registered in this study, which is designated the NHO Carotid CFD Study. To investigate hemodynamic predictors for the onset of ischemic stroke, we performed a multivariate analysis of 90 cases with carotid stenosis with an area stenosis rate of 50% to 85% among those enrolled in this clinical study, after adjustment for known risks. Methods: Patient-specific geometries of the carotid arteries were segmented and reconstructed from the volume data set of three-dimensional computed tomography angiography images in Digital Imaging and Communications in Medicine (DICOM) format, using Mimics and 3-matic (Materialise, Leuven, Belgium). Pulsatile blood flow was simulated using a CFD software package, ANSYS CFX (ANSYS, PA, USA), based on the Navier-Stokes equations for incompressible fluid. Individual vessel diameters and peak-systolic and end-diastolic velocities were measured with the Doppler ultrasound technique on common carotid arteries and internal carotid arteries of each patient to compute the corresponding flow rates. The stenotic carotid artery defined three regions: the “stenotic site” where the stenosis is present, the “proximal site” from the lower end of the stenosis to 10 mm proximal, and the “distal site” from the upper end of the stenosis to 10 mm distal. Results: Metrics related to proximal wall shear stress (WSS) disturbances, such as proximal transverse WSS (transWSS) (odds ratio (OR) 2.42, 95% confidence interval (95%CI) 1.32 to 5.00), proximal normalized transWSS (NtransWSS) (OR 1.71, 95%CI 1.02 to 3.03), and the stenotic/proximal ratio of transWSS (1/OR 1.59, 95%CI 0.39 to 0.88), had significant correlations with a history of cerebral infarction on the ipsilateral side of the stenotic artery. Conclusions: Our present analysis suggested that carotid arteries with the same degree of stenosis may be more likely to have a cerebral infarction in cases with higher transWSS in the proximal site than the stenotic one. In patients with these hemodynamic risks, underlying diseases should be controlled more strictly with imaging examinations at shorter intervals. This study was supported by a Grant-in-Aid for Japanese National Hospital Organization Multi-Center Clinical Research, and AMED under Grant Number JP15gm0810006h0301. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A heme pocket aromatic quadrupole modulates gas binding to cytochrome c′-β: Implications for NO sensors

The structural basis by which gas-binding heme proteins control their interactions with NO, CO, and O2 is fundamental to enzymology, biotechnology, and human health. Cytochromes c' (cyts c') are a group of putative NO-binding heme proteins that fall into two families: the well-characterized four alpha helix bundle fold (cyts c'-α) and an unrelated family with a large beta-sheet fold (cyts c'-β) resembling that of cytochromes P460. A recent structure of cyt c'-β from Methylococcus capsulatus Bath revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas-binding site. This feature, dubbed the "Phe cap," is highly conserved within the sequences of other cyts c'-β but is absent in their close homologs, the hydroxylamine-oxidizing cytochromes P460, although some do contain a single Phe residue. Here, we report an integrated structural, spectroscopic, and kinetic characterization of cyt c'-β from Methylococcus capsulatus Bath complexes with diatomic gases, focusing on the interaction of the Phe cap with NO and CO. Significantly, crystallographic and resonance Raman data show that orientation of the electron-rich aromatic ring face of Phe 32 toward distally bound NO or CO is associated with weakened backbonding and higher off rates. Moreover, we propose that an aromatic quadrupole also contributes to the unusually weak backbonding reported for some heme-based gas sensors, including the mammalian NO sensor, soluble guanylate cyclase. Collectively, this study sheds light on the influence of highly conserved distal Phe residues on heme-gas complexes of cytochrome c'-β, including the potential for aromatic quadrupoles to modulate NO and CO binding in other heme proteins.

Distinct role of AtCuAOβ- and RBOHD-driven H2O2 production in wound-induced local and systemic leaf-to-leaf and root-to-leaf stomatal closure.

Polyamines (PAs) are ubiquitous low-molecular-weight aliphatic compounds present in all living organisms and essential for cell growth and differentiation. The developmentally regulated and stress-induced copper amine oxidases (CuAOs) oxidize PAs to aminoaldehydes producing hydrogen peroxide (H2O2) and ammonia. The Arabidopsis thaliana CuAOβ (AtCuAOβ) was previously reported to be involved in stomatal closure and early root protoxylem differentiation induced by the wound-signal MeJA via apoplastic H2O2 production, suggesting a role of this enzyme in water balance, by modulating xylem-dependent water supply and stomata-dependent water loss under stress conditions. Furthermore, AtCuAOβ has been shown to mediate early differentiation of root protoxylem induced by leaf wounding, which suggests a whole-plant systemic coordination of water supply and loss through stress-induced stomatal responses and root protoxylem phenotypic plasticity. Among apoplastic ROS generators, the D isoform of the respiratory burst oxidase homolog (RBOH) has been shown to be involved in stress-mediated modulation of stomatal closure as well. In the present study, the specific role of AtCuAOβ and RBOHD in local and systemic perception of leaf and root wounding that triggers stomatal closure was investigated at both injury and distal sites exploiting Atcuaoβ and rbohd insertional mutants. Data evidenced that AtCuAOβ-driven H2O2 production mediates both local and systemic leaf-to-leaf and root-to-leaf responses in relation to stomatal movement, Atcuaoβ mutants being completely unresponsive to leaf or root wounding. Instead, RBOHD-driven ROS production contributes only to systemic leaf-to-leaf and root-to-leaf stomatal closure, with rbohd mutants showing partial unresponsiveness in distal, but not local, responses. Overall, data herein reported allow us to hypothesize that RBOHD may act downstream of and cooperate with AtCuAOβ in inducing the oxidative burst that leads to systemic wound-triggered stomatal closure.

Engineered skin bacteria induce antitumor T cell responses against melanoma

Certain bacterial colonists induce a highly specific T cell response. A hallmark of this encounter is that adaptive immunity develops preemptively, in the absence of an infection. However, the functional properties of colonist-induced T cells are not well defined, limiting our ability to understand anticommensal immunity and harness it therapeutically. We addressed both challenges by engineering the skin bacterium Staphylococcus epidermidis to express tumor antigens anchored to secreted or cell-surface proteins. Upon colonization, engineered S. epidermidis elicits tumor-specific T cells that circulate, infiltrate local and metastatic lesions, and exert cytotoxic activity. Thus, the immune response to a skin colonist can promote cellular immunity at a distal site and can be redirected against a target of therapeutic interest by expressing a target-derived antigen in a commensal.

Cone Beam CT Assessment of Bone Width of Upper and Lower Jaws for Dental Implant Placement: An Iraqi Study.

Implantology focuses on the measurement of bone thickness in both the lower and upper jaws. This study aimed to measure and compare alveolar bone thickness of the upper and lower jaws at single edentate sites and cortical bone thickness of their mesial and distal dentate sites. Thickness of alveolar bone thickness was measured in 80 upper and 80 lower implant edentate sites and that of buccal and lingual cortical plates of their mesial and distal dentate sites using Cone beam CT. The bone thickness of the edentulous sites was recorded at 3 points (crestal bone, five mm from the crest, and ten mm from the crest), while the bone thickness of the dentate sites was determined at four points (crestal bone, midroot bone, mid of the alveolar bone housing, and apical portion). An increased amount of bone was measured from the crest to the apical portion of the dentate sites on the buccal and lingual sides of both jaws with a highly significant difference detected among all the tested points (P < 0.0001). No statistical difference was detected between the means of buccal bone width at the first 3 points, except at point 4 (the apical portion), where the mean of the lower jaw (3.35 ± 0.54) was significantly larger than that of the upper jaw (3.17 ± 0.55) (P = 0.04). Bone width measured in the edentulous sites showed a gradual increase from the crest to the apical portions in both jaws. Bone thickness at the coronal levels is low and susceptible to resorption compared to the apical portions regardless of the dentate state.

Commensal microbiota regulate renal gene expression in a sex-specific manner.

The gut microbiome impacts host gene expression not only in the colon but also at distal sites including the liver, white adipose tissue, and spleen. The gut microbiome also influences the kidney and is associated with renal diseases and pathologies; however, a role for the gut microbiome to modulate renal gene expression has not been examined. To determine if microbes modulate renal gene expression, we used whole organ RNA sequencing to compare gene expression in C57Bl/6 mice that were germ free (lacking gut microbiota) versus conventionalized (gut microbiota reintroduced using an oral gavage of a fecal slurry composed of mixed stool). 16S sequencing showed that male and female mice were similarly conventionalized, although Verrucomicrobia was higher in male mice. We found that renal gene expression was differentially regulated in the presence vs. absence of microbiota and that these changes were largely sex specific. Although microbes also influenced gene expression in the liver and large intestine, most differentially expressed genes (DEGs) in the kidney were not similarly regulated in the liver or large intestine. This demonstrates that the influence of the gut microbiota on gene expression is tissue specific. However, a minority of genes (n = 4 in males and n = 6 in females) were similarly regulated in all three tissues examined, including genes associated with circadian rhythm (period 1 in males and period 2 in females) and metal binding (metallothionein 1 and metallothionein 2 in both males and females). Finally, using a previously published single-cell RNA-sequencing dataset, we assigned a subset of DEGs to specific kidney cell types, revealing clustering of DEGs by cell type and/or sex.NEW & NOTEWORTHY It is unknown whether the microbiome influences host gene expression in the kidney. Here, we utilized an unbiased, bulk RNA-sequencing approach to compare gene expression in the kidneys of male and female mice with or without gut microbiota. This report demonstrates that renal gene expression is modulated by the microbiome in a sex- and tissue-specific manner.