Distal Sites Research Articles

Dental Implants with External Hex Inclined Shoulder in Full-Arch Immediate Loading Rehabilitations of the Maxilla.

Background: Coaxial implants with an inclined neck might overcome some problems related to angulation of the implant axis when using tilted implants. Therefore, the aim of the present work was to conduct a narrative review of the current literature and to present a case series comparing traditional and coaxial external hex implants in full-arch immediate loading rehabilitations of the maxilla. Methods: A total of 13 external hex tapered implants (Southern Implants) was inserted in the upper jaw of 3 patients. Each patient received two tilted implants in distal sites. In one randomly selected quadrant, the tilted implant was a standard implant, while a Co-Axis® implant with a 24° inclination of the implant shoulder was inserted on the other hemi-arch. Straight conical abutments were screwed on coaxial implants while multiunit abutments of appropriate inclination were screwed as needed on the other implants to correct their axes. Peri-implant bone level was recorded radiographically at T0 (delivery of the immediate loading prosthesis), and at 3, 6, 12, and 24 months of healing and then annually. Plaque index, probing depth, and bleeding on probing were also evaluated. Cumulative implant survival rate (CSR) was calculated, and biological or technical complications were recorded as well as the operator satisfaction towards the use of coaxial implants. Results: The preliminary data collected did not show significant differences in peri-implant tissues health and maintenance over time between the two implant types. No implants failed, and both implant types proved to be favorable for full-arch rehabilitation using tilted implants. Coaxial implants facilitated the prosthodontic procedures. However, a learning curve is required in order to optimize their insertion. Conclusions: Both implants proved to be reliable and suitable for achieving clinical success in full-arch immediate loading rehabilitations, but further research with longer follow-up and larger sample size is needed to confirm these preliminary outcomes.

Immediate Placement and Loading of Maxillary Single-Tooth with Scalloped Shape Implants and Digitalized Customized Restoration: A 5-Year Prospective Study of Marginal Bone Level.

To evaluate, within a period of 5 years, the bone level in mesial, distal, palatal, and buccal areas around scalloped shape implants immediately placed and loaded with temporary crowns fixed on final prefabricated abutments, and also to evaluate the thickness of buccal bone. 18 implants were inserted and loaded immediately using computer-assisted design/computer-assisted manufacturing technology on 18 patients to replace single tooth in the esthetic part of the maxilla. The marginal bone level across the scalloped implant neck was measured mesially and distally using intraoral standardized radiographs after crown fixations and 1, 3, and 5 years later. Cone beam computed tomography para-axial cuts images were used to measure bone level buccally and palatally from the implant neck to the implant-to bone contact after 5 years of loading and to evaluate the thickness of the buccal bone at the implant neck and 4 mm apically, immediately after implant placement and 5 years later. All implants were assessed clinically and radiologically after 5 years. No implant failure was recorded, and the average marginal bone variation on mesial and distal sites was 0.114 ± 0.135 mm at crown cementation, 0.239 ± 0.158 mm 1 year later, 0.233 ± 0.182 mm 3 years later, and 0.180 ± 0.182 mm 5 years later. Our findings indicate that at T0, the average thickness of the buccal bone was 2.27 mm at implant neck M0 (ranging from 1.9 to 2.4) and 2.33 mm at 4 mm apically to the implant neck M1 (ranging from 1.9 mm to 2.9 mm). By T4, the mean had decreased to 1.94 mm at M0 (with a range of 1.7 mm to 2.3 mm) and 2.14 mm at M1 (with a range of 1.8 mm to 2.4 mm). After 5 years the mean changes at buccal and palatal bone for all implants were +0.187 ± 0.52 mm and +0.06 ± 0.38 mm respectively. Minor prosthetic problems were observed over the five years: incisal ceramic chipping occurred in two crowns, and two crowns were replaced for esthetic reasons after one year. No loosening of crowns or abutments was reported. Scalloped neck implants demonstrated a comparable behavior to regular neck implants with similar designs in an immediate implantation and temporization protocol over a five-year period.

Fractional Lengthening of Forearm Flexor Tendons: A Cadaveric Biomechanical Analysis

Multiple procedures have been described for wrist and finger flexion contractures and spasticity. Fractional lengthening of forearm flexor tendons involves making parallel transverse tenotomies at the musculotendinous junction to elongate the muscle. Currently, there is limited literature to define the biomechanical consequences of this lengthening technique. Forty-eight flexor tendons were harvested from eight paired upper limbs including flexor carpi radialis, flexor carpi ulnaris, flexor pollicis longus, and flexor digitorum superficialis tendons. Each tendon that was lengthened was paired with the contralateral tendon as a control. A pair of transverse tenotomies were completed for the fractional lengthening. The first tenotomy was performed at the musculotendinous junction where the tendon narrowed to 75% of its maximal width. The second tenotomy was made 1 cm distal to the first. Tendon length was measured before and after fractional lengthening at a constant resting tension of 1 N. The maximum load at failure of each tendon and the mechanism of failure were each measured and compared with the contralateral side. After fractional lengthening, the mean increase in resting tendon length was 4 mm. When loaded to failure, the mean maximum load of fractionally lengthened tendons was 42% of the mean maximum load of intact tendons. All lengthened tendons failed at the distal tenotomy site. Fractional lengthening resulted in an increase of 3-6 mm (mean: 4 mm) in tendon length at resting tension. There was a significant loss in tensile strength and load to failure following fractional lengthening compared with an intact musculotendinous unit. The reduction in tensile strength following fractional lengthening results in loads at failure that are, in some cases, lower than the estimated forces required to perform basic tasks. Caution during the healing and rehabilitation period is warranted.

Determinants of right coronary artery occlusion in patients with inferior wall myocardial infarction: A cross-sectional study from Western India

ABSTRACT Introduction: In acute inferior wall myocardial infarction (IWMI) and occlusion of the right coronary artery (RCA), the most important element is to recognize the culprit artery and the location and size of the infarct, which is essential in the treatment. Adequate knowledge about clinical determinants can aid in prompt and effective management. Materials and Methods: A cross-sectional study was conducted to find determinants of RCA occlusion among 103 subjects with IWMI from rural western India. Results: About 86.4% of subjects were male. Females experienced IWMI at a later age than males (62.4 ± 10.3 vs. 58.6.1 ± 11.8 years). Bradycardia was more prevalent than tachycardia (44.66% vs. 15.53%). Males had a 5.17 times higher odds of having RCA involvement in IWMI. Subjects with deranged lipid profiles had 2.37 times higher odds of involvement of RCA. RCA involvement was significantly associated with bradycardia (P = 0.006, aOR = 15.31, 95% confidence interval [CI] = 2.21–105.7) but not with tachycardia (P = 0.903). Proximal site occlusion was significantly associated (P = 0.027; aOR = 0.103; 95% CI = 0.013–0.772) with bradycardia compared to distal sites. Triple-vessel disease had significantly (P < 0.05) 12.9 times higher odds than single-vessel disease. Total occlusion of RCA was 2.9 times higher than obstructive coronary artery disease. Conclusion: These findings suggest determinants associated with RCA involvement in IWMI, including gender, lipid profile, cardiac rhythm disturbances (particularly bradycardia), and the extent and site of coronary artery occlusion. Incorporating these factors into clinical practice, clinicians can better predict the underlying pathophysiology of IWMI, anticipate its hemodynamic and electrophysiologic consequences, and tailor management strategies accordingly.

Electromyographic Evaluation of Masseter and Temporalis Muscle Activity in Distal Extension Edentulous Patients After Replacing with Implants—A Prospective Clinical Trial

Aim: To assess the changes in muscle activity after replacing the missing teeth or teeth with implants in distal extension edentulous patients and to evaluate the patient’s satisfaction levels. Materials and Methods: This was a single-group experimental study, based on convenience sampling with a sample size of 40 patients. Only unilateral mandibular distal extension edentulous sites were included and endo-osseous dental implants were placed in delayed protocol. Electrical activities of the masseter and temporalis muscles were recorded at rest and clench before, immediately, and one month after the cementation using electromyography. Patient satisfaction was analyzed through a questionnaire. Descriptive statistics an inferential statistics with repeated measures of ANOVA followed by the Bonferroni post hoc test were done to analyze the study data. P value < .05 would be considered statistically significant for all the analyses. Results: Right temporalis showed significantly higher surface electromyography (sEMG) activity at one-month post cementation than baseline at rest and clench. Similarly, the left temporalis has shown significant sEMG activity at one-month post cementation than baseline at clench. However, there was no statistical difference with the right and left masseter. The implant-supported prosthesis was well received by the majority of patients. Conclusion: Definitive improvement in the electrical activity of the temporalis before and after the replacement of prosthesis was noticed with positive patient satisfaction levels with implant-supported prosthesis.

Rapid elucidation of agonist-driven regulation of the neurokinin 1 receptor using a GPCR phosphorylation immunoassay

Agonist-induced phosphorylation is a crucial step in the activation/deactivation cycle of G protein-coupled receptors (GPCRs), but direct determination of individual phosphorylation events has remained a major challenge. We have recently developed a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in 96-well plates, thus eliminating the need for western blot analysis. In the present study, we adapted this assay to three novel phosphosite-specific antibodies directed against the neurokinin 1 (NK1) receptor, namely pS338/pT339-NK1, pT344/pS347-NK1, and pT356/pT357-NK1. We found that substance P (SP) stimulated concentration-dependent phosphorylation of all three sites, which could be completely blocked in the presence of the NK1 receptor antagonist aprepitant. The other two endogenous ligands of the tachykinin family, neurokinin A (NKA) and neurokinin B (NKB), were also able to induce NK1 receptor phosphorylation, but to a much lesser extent than substance P. Interestingly, substance P promoted phosphorylation of the two distal sites more efficiently than that of the proximal site. The proximal site was identified as a substrate for phosphorylation by protein kinase C. Analysis of GPCR kinase (GRK)-knockout cells revealed that phosphorylation was mediated by all four GRK isoforms to similar extents at the T344/S347 and the T356/T357 cluster. Knockout of all GRKs resulted in abolition of all phosphorylation signals highlighting the importance of these kinases in agonist-mediated receptor phosphorylation. Thus, the 7TM phosphorylation assay technology allows for rapid and detailed analyses of GPCR phosphorylation.

Influence of proteolytic cleavage of ENaC's γ subunit upon Na+ and K+ handling.

The epithelial Na+ channel (ENaC) γ subunit is essential for homeostasis of Na+, K+, and body fluid. Dual γ subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146, in the mouse γ subunit). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (γQ4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short-circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of γQ4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γQ4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in γQ4/Q4 mice was not altered. γQ4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of γ subunit cleavage should not be used in isolation to evaluate ENaC activity. Furthermore, factors independent of γ subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo.NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) is activated in vitro by post-translational proteolysis. In vivo, low Na+ or high K+ diets enhance ENaC proteolysis, and proteolysis is hypothesized to contribute to channel activation in these settings. Using a mouse expressing ENaC with disruption of a key proteolytic cleavage site, this study demonstrates that impaired proteolytic activation of ENaC's γ subunit has little impact upon channel open probability or the ability of mice to adapt to low Na+ or high K+ diets.

Circulating tumor cells with metastasis-initiating competence survive fluid shear stress during hematogenous dissemination through CXCR4-PI3K/AKT signaling

To seed lethal secondary lesions, circulating tumor cells (CTCs) must survive all rate-limiting factors during hematogenous dissemination, including fluid shear stress (FSS) that poses a grand challenge to their survival. We thus hypothesized that CTCs with the ability to survive FSS in vasculature might hold metastasis-initiating competence. This study reported that FSS of physiologic magnitude selected a small subpopulation of suspended tumor cells in vitro with the traits of metastasis-initiating cells, including stemness, migration/invasion potential, cellular plasticity, and biophysical properties. These shear-selected cells generated local and metastatic tumors at the primary and distal sites efficiently, implicating their metastasis competence. Mechanistically, FSS activated the mechanosensitive protein CXCR4 and the downstream PI3K/AKT signaling, which were essential in shear-mediated selection of metastasis-competent CTCs. In summary, these findings conclude that CTCs with metastasis-initiating competence survive FSS during hematogenous dissemination through CXCR4-PI3K/AKT signaling, which may provide new therapeutic targets for the early prevention of tumor metastasis.

RUNX1 regulates promoter activity in the absence of cognate DNA binding motifs.

Runt-related transcription factor 1 (RUNX1) plays an important role in normal haematopoietic cell development and function, and its function is frequently disrupted in leukaemia. RUNX1 is widely recognised as a sequence-specific DNA binding factor that recognises the motif 5'-TG(T/C)GGT-3' in promoter and enhancer regions of its target genes. Moreover, RUNX1 fusion proteins, such as RUNX1-ETO formed by the t(8;21) translocation, retain the ability to recognise and bind to this sequence to elicit atypical gene regulatory effects on bona fide RUNX1 targets. However, our analysis of publicly available RUNX1 chromatin immunoprecipitation sequencing (ChIP-Seq) data has provided evidence challenging this dogma, revealing that this motif-specific model of RUNX1 recruitment and function is incomplete. Our analyses revealed that the majority of RUNX1 genomic localisation occurs outside of promoters, that 20% of RUNX1 binding sites lack consensus RUNX motifs, and that binding in the absence of a cognate binding site is more common in promoter regions compared to distal sites. Reporter assays demonstrate that RUNX1 can drive promoter activity in the absence of a recognised DNA binding motif, in contrast to RUNX1-ETO. RUNX1-ETO supresses activity when it is recruited to promoters containing a sequence specific motif, while interestingly, it binds but does not repress promoters devoid of a RUNX1 recognition site. These data suggest that RUNX1 regulation of target genes occurs through multiple mechanisms depending on genomic location, the type of regulatory element and mode of recruitment.

Leveraging Bidirectional Nature of Allostery To Inhibit Protein-Protein Interactions (PPIs): A Case Study of PCSK9-LDLR Interaction.

The protein PCSK9 (proprotein convertase subtilisin/Kexin type 9) negatively regulates the recycling of LDLR (low-density lipoprotein receptor), leading to an elevated plasma level of LDL. Inhibition of PCSK9-LDLR interaction has emerged as a promising therapeutic strategy to manage hypercholesterolemia. However, the large interaction surface area between PCSK9 and LDLR makes it challenging to identify a small molecule competitive inhibitor. An alternative strategy would be to identify distal cryptic sites as targets for allosteric inhibitors that can remotely modulate PCSK9-LDLR interaction. Using several microseconds long molecular dynamics (MD) simulations, we demonstrate that on binding with LDLR, there is a significant conformational change (population shift) in a distal loop (residues 211-222) region of PCSK9. Consistent with the bidirectional nature of allostery, we establish a clear correlation between the loop conformation and the binding affinity with LDLR. Using a thermodynamic argument, we establish that the loop conformations predominantly present in the apo state of PCSK9 would have lower LDLR binding affinity, and they would be potential targets for designing allosteric inhibitors. We elucidate the molecular origin of the allosteric coupling between this loop and the LDLR binding interface in terms of the population shift in a set of salt bridges and hydrogen bonds. Overall, our work provides a general strategy toward identifying allosteric hotspots: compare the conformational ensemble of the receptor between the apo and bound states of the protein and identify distal conformational changes, if any. The inhibitors should be designed to bind and stabilize the apo-specific conformations.

New insights into NO bonding in Tan sheep myoglobin for meat pigmentation: Spectroscopic and density functional theory investigations

Color of meat remains crucial quality attribute that attracts attention of researchers worldwide. Various strategies have been employed to manipulate meat color by generating nitric oxide (NO), which interacts with myoglobin (Mb) to yield the pinkish-red pigment, nitrosomyoglobin (MbFeⅡNO). However, the formation mechanism of MbFeⅡNO warrants further exploration at the molecular-structural level for meat pigmentation. Here, Tan sheep Mb was purified and characterized its primary structure via LC-MS/MS, which found it to be identical to sheep Mb. Circular dichroism spectroscopy revealed 70.05 % α-helix content in the secondary structure of Tan sheep Mb. UV-visible and resonance Raman spectroscopy monitored NO bonding in Tan sheep Mb, revealing that NO displaced H2O of MbFeⅢ, forming MbFeⅢNO, which subsequently transformed into low-spin six-coordination MbFeⅡNO. Additionally, three-dimensional structure of Tan sheep Mb was constructed utilizing Alphafold2, and theoretical simulations and calculations were performed through molecular docking and DFT. Results confirmed the synergistic effect of the strong metal-ligand and hydrogen bonding interactions among the NO, heme iron, and distal sites significantly contributed to the stable formation of MbFeⅡNO. Overall, this study offers a novel perspective on the mechanism of NO binding in Mb, potentially guiding the development of innovative strategies to mitigate meat color deterioration.

Snuffbox Versus Wrist Radiocephalic Arteriovenous Fistulas: 10 Years Experience.

Background The wrist radiocephalic arteriovenous fistula (RCAVF) is the initial suggested procedure for establishing hemodialysis vascular access (HVA) in the most distal site of the upper limb. The anatomical snuffbox arteriovenous fistula (SBAVF) is barely utilised, despite its remote location. In this study, we aimed to analyse and compare the results of SBAVF and RCAVF in terms of their maturity, patency, and failure rates. Methodology This descriptive, retrospective study compared outcomes between SBAVF and RCAVF in terms of maturation, patency, and failure. All patients with chronic kidney disease who attended and underwent either procedure at Betsi Cadwaladr University Health Board between 2013 and 2023 were studied. Results In a period of 10 years, 179 patients were included. Overall, 102 (57%) were male and 77 (43%) were female, with a male-to-female ratio of 1.3:1. Wrist radiocephalic fistula was the dominant type of surgery done in 76% (n = 136), while the snuffbox radiocephalic fistula was done in fewer than 24% (n = 43) of patients. Most patients underwent a successful arteriovenous (AV) fistula (n = 105, 58.7%), in contrast to 67 patients whose fistulas failed. There was a significant relationship between fistula failure and complications (p = 0.000). There was no significant effect of the fistula site, hypertension, diabetes, cardiac diseases, smoking, peripheral vascular disease, or central vein stenosis on the failure of the AV fistula (p = 0.127, 0.534, 0.510, 0.397, 0.017, 0.68, and 0.371, respectively). Conclusions The snuffbox AV fistula is a suitable and feasible first choice for patients on hemodynamic therapy.

Anatomical Structures at Risk in Percutaneous Distal Bunionette Correction

Introduction/Purpose: Bunionette deformity affects 23% of the population and has numerous surgical options, including a minimally invasive approach (MIA) to decrease complications. Percutaneous surgery is favored by patients as it decreases pain, inflammation, and noticeable incision sites. Three possible osteotomy sites have been identified: proximal, diaphyseal, and distal. The study investigates the anatomic structures at risk during distal osteotomy of bunionette deformity using a Shannon burr. The distal osteotomy site was on average greater than 8mm from the extensor digitorum longus and adductor digiti minimi while it was 1.64mm from the lateral dorsal cutaneous nerve. The study highlighted the challenges posed by minimally invasive approaches to treating bunionette deformity and the need for cautious consideration when utilizing percutaneous methods. Methods: Eleven fresh frozen below-knee amputated foot and ankle cadavers were used for this study. Cadavers were allowed to thaw for 12 hours at room temperature before performing any tissue handling. All cadavers were visually inspected for evidence of gross pathology. Fluoroscopic radiographs were used to inspect for evidence of preexisting pathology of the fifth metatarsal. All cadavers with gross pathology or radiographic fifth metatarsal pathology were excluded from the study. All procedures were performed by a fellowship trained orthopaedic foot and ankle surgeon. Results: All eleven cadavers met the inclusion criteria for this study. The average age of the patients was 66.6 +/- 20.1 years. There were five female feet and six male feet. There were seven right feet and four left feet. Distance from the percutaneous access site was measured to structures at potential risk. The closest structure at risk was the lateral dorsal cutaneous nerve (LDCN) (1.64 mm), which was contacted 2/11 times and transected both times. The extensor digitorum longus (EDL) and abductor digiti minimi (ADM) had no instances of contact during the procedure. Dissection allowed for the identification of structures at risk and the assessment of osteotomy. All procedures resulted in a complete osteotomy. Conclusion: Overall, distal percutaneous bunionette osteotomy is largely safe. Structures pertinent to the surgery were identified with ease and were intact after osteotomy with a Shannon burr. Anatomic variations of LDCN can present challenges to the procedure. Meticulous dissection and judicious use of fluoroscopy can help prevent further damage. Surgeons should be aware of anatomic variations in order to proceed safely.

(1184) - Left Ventricular Assist Device Implantation in Patient with Localized Stenosis at a Distal Anastomotic Site of the Ascending Aorta After Bentall's Surgery

(1184) - Left Ventricular Assist Device Implantation in Patient with Localized Stenosis at a Distal Anastomotic Site of the Ascending Aorta After Bentall's Surgery

Let's Get Rolling: Precise Control of Microfluidic Assay Conditions to Recapitulate Selectin-Mediated Rolling Interactions of the Leukocyte Adhesion Cascade.

The leukocyte adhesion cascade governs the recruitment of circulating immune cells from the vasculature to distal sites. The initial adhesive interactions between cell surface ligands displaying sialyl-LewisX (sLeX) and endothelial E- and P-selectins serve to slow the cells down enough to interact more closely with the surface, polarize, and exit into the tissues. Therefore, precise microfluidic assays are critical in modeling how well immune cells can interact and "roll" on selectins to slow down enough to complete further steps of the cascade. Here, we present a systematic protocol for selectin mediated rolling on recombinant surfaces and endothelial cell monolayers on polyacrylamide gels of varying stiffness. We also describe step-by-step the protocol for setting up and performing the experiment and how to analyze and present the data collected. This protocol serves to simplify and detail the procedure needed to investigate the initial selectin-mediated interactions of immune cells with the vasculature. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparing dishes for cell rolling experiments Basic Protocol 2: Fabrication of polyacrylamide gels for cell rolling experiments Alternate Protocol 1: Protein conjugation with N6 linker Alternate Protocol 2: HUVEC culturing for monolayers Basic Protocol 3: Conducting cell rolling experiments on polyacrylamide gels Basic Protocol 4: ImageJ analysis of cell rolling movies Basic Protocol 5: Quantification of Fc site density on a surface (e.g., for Fc chimeras).

Abstract 1449: Targeting squalene epoxidase for breast cancer brain metastasis: The evaluation of a new therapeutic target for brain extravasation and colonization using animal models

Abstract Breast cancer brain metastasis is one of the most common forms of breast cancer metastasis and a major cause of morbidity and mortality. Patients who develop brain metastases tend to be associated with progressive neurologic deficits and short overall survival, which represents an unmet medical need. Extensive research has been conducted to elucidate the mechanism of cancer metastasis with limited information gained as to how cancer cells extravasate and colonize to the brain. Recently, we identified a novel mechanism by which squalene epoxidase (SQLE), the second rate-limiting enzyme in cholesterol biosynthesis, plays a critical role in the process of breast cancer metastasis to the brain. Thus, toward a better understanding of the underlying molecular mechanisms and applications of molecular target(s) for brain metastasis therapy, murine models were employed for investigation of brain extravasation and colonization with intracardiac or direct intracranial injections of GFP/FLuc-labeled human MDA-MB-231 metastatic breast carcinoma cells. The MDA-MB-231 cells were found to metastasize to the brain and other organs following intracardiac implantation into nude mice and were further isolated from each tissue. Interestingly, the isolated brain-metastasized tumor cells (MDA-MB-231-BrM) exhibited a significant upregulation of SQLE expression when compared with those that metastasized to other distal sites. The essential role of SQLE in the specific steps of the breast cancer-to-brain metastatic process was evaluated by ex vivo immunofluorescence analysis and immunohistochemistry staining of brain slices from the study subjects. Our data demonstrate that high SQLE expression is essential for MDA-MB-231-BrM to extravasate into the parenchyma, as well as the formation of micro- and macro-metastases in the brain. Interestingly, we found that blood vessel co-option and the surrounding neuronal cells play key roles in supporting MDA-MB-231-BrM to develop brain macro-metastases. In vitro blood-brain barrier (BBB) models further demonstrated the critical role of SQLE in promoting tumor cell invasion and penetration through the BBB. To verify SQLE as an oncogenic factor that can be selected as a potential therapeutic target in suppressing breast cancer brain metastasis, we evaluated the inhibitory effects of terbinafine (a SQLE inhibitor) in both the MDA-MB-231-BrM orthotopic and intracardiac mouse models. Pharmacologic inhibition of SQLE by terbinafine suppressed MDA-MB-231-BrM tumor growth at the mammary fat pad and distal metastases to the brain, suggesting that targeting SQLE represents a therapeutic opportunity for breast cancer brain metastasis. Citation Format: Pony Yu-Ling Lee, Kun-Yuan Lin, Chao-Di Chang, Shan-Yun Cheng, Ya-Wen Hung, Chih-Chieh Yang, Yu-Hsien Chang, Chien-Chang Shen, Jia-Yun Yeh, Yung-Lung Yu, Shu-Ping Wang. Targeting squalene epoxidase for breast cancer brain metastasis: The evaluation of a new therapeutic target for brain extravasation and colonization using animal models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1449.

Abstract 1282: Gavage with Candida albicans leads to fungal colonization of colorectal tumors and decreased response to radiotherapy

Abstract The host microbiome in the gut and tumors have been shown to affect tumor growth and therapeutic response in cancer. Furthermore, the presence of specific fungal taxa are associated with tumor hypoxia, tumor progression, therapeutic response, and clinical outcomes in colorectal cancer (CRC). Preliminary data from our group found that the presence of Candida species increased tumor hypoxia and decreased overall survival in CRC. However, whether intratumoral Candida drive, support, or simply inhabit tumors with poor prognoses remains unclear. Here, we investigate a casual role for Candida albicans in tumor progression and therapy response using a syngeneic mouse model of microsatellite-instability high (MSI-H) colorectal cancer. We subcutaneously implanted MC38 colorectal cancer cells into C57BL/6 mice and then orally gavaged these mice with C. albicans, Saccharomyces cerevisiae, or PBS. The site of tumor formation was locally irradiated once (7.5 Gy), and growth of the tumor was measured over time. Differences in tumor volume were assessed by longitudinal mixed-effect models. Tumor sections were stained for fungi using calcofluor white (CFW) and underwent bulk RNA sequencing to detect differential gene expression, which were interpreted using gene set enrichment analysis (GSEA). Mice gavaged with C. albicans showed decreased response to radiation compared to mice gavaged with either S. cerevisiae (p<0.05) or PBS (p<0.001). Additionally, hyphae were observed in murine tumors gavaged with C. albicans, suggesting translocation of gavaged C. albicans from the gut to tumors. Further, tumors from mice gavaged with C. albicans displayed unique gene expression profiles, including decreased interferon alpha and IL-6 and STAT3 signaling, decreased oxidative phosphorylation, and decreased apoptosis-related gene expression. Here, we show that C. albicans may confer resistance to radiation therapy and affects immune and cancer cell activity in the MC38 (MSI-H) syngeneic in vivo model of colorectal cancer. Further, we show that hyphal fungi can be visualized in heterotopic murine tumors from our C. albicans gavage condition. These data establish that fungi can translocate from the gut to distal tumor sites, and that upon translocation, changes in gene expression and therapy response are observed. Future directions will explore the mechanism by which these effects occur and whether these findings can be leveraged to improve radiotherapy outcomes. Citation Format: Dennis J. Grencewicz, Alexander Loncar, Rebecca Hoyd, Aaditya Pallerla, Nyelia Williams, Martin Benej, McKenzie Kreamer, Yogita Mehra, Shiva Jahanbakhshi, Matthew Anderson, Nicholas Denko, Daniel Spakowicz. Gavage with Candida albicans leads to fungal colonization of colorectal tumors and decreased response to radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1282.

Abstract 2747: Chaperonin-containing TCP-1 promotes the release of exosomes from prostate and breast cancer cells

Abstract Coping with cancer recurrence and assessing metastatic risk are challenges for cancer patients that could be addressed with an effective diagnostic predictor. Exosomes, a subpopulation of extracellular vesicles released by tumor cells, can promote distal metastatic site development and could have promising diagnostic applications. However, the mechanisms controlling exosome secretion by cancer cells remain to be determined, which impedes clinical development. The actions of protein-folding chaperones could facilitate tumor exosome formation, payload loading, and extracellular release. Protein folding complexes link the expression of tumor-promoting oncogenes with the proteins driving oncogenic cancer behavior. Of these, the eukaryotic type II chaperonin, Chaperonin-Containing TCP-1 (CCT), folds many of the oncoproteins (e.g., KRAS, MYC, CDKs, STAT3, etc.) responsible for cancer growth and invasion. CCT is a complex machine composed of eight subunits, each encoded by a unique gene (cct1-8), which folds proteins in an ATP-dependent fashion. We and others showed that CCT is upregulated in breast, lung, and prostate cancer cells as well as neuroblastoma, leading to alterations in apoptosis, migration, cell morphology, and proliferation. Using T47D breast cancer and PC3 and 22rV1 prostate cancer cells, we exogenously expressed or depleted the second subunit of the CCT complex (CCT2). With these cells, we isolated exosomes from culture media by membrane-based affinity binding and examined exosomal RNA and protein content. In exosomes from prostate and breast cancer cells, we found abundant CCT2 subunit mRNA and protein in CD63+ exosomes ranging from 50 to 120 microns in size. When CCT2 was exogenously expressed in breast and prostate cancer cells, CCT2 mRNA and protein were higher in exosomes than other CCT subunits, and CCT2 was randomly distributed throughout the size range of exosomes. Of interest, an increase in CD63+ small exosomes (50-75 microns) was evident in cancer cells exogenously expressing CCT2 compared to mock-transfected and parental controls, suggesting a role for CCT2 in the exosomal secretory pathway. FACS-sorted CD63+ exosomes subjected to mass spectrometry yielded distinct protein content based on CCT2 expression. These findings are the first report of CCT2 driving exosome secretion in cancer cells and suggest that exosomes containing CCT2 RNA and protein are associated with metastatic potential. Exosomal CCT2 thus represents a promising diagnostic indicator for a liquid biopsy approach to monitor cancer recurrence and metastasis. Citation Format: Annette R. Khaled, Lam Truong, Colten Frank, Carolyn Dang, James Velazquez, Priya Gopalan, Sally Litherland. Chaperonin-containing TCP-1 promotes the release of exosomes from prostate and breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2747.

Integrated engineering at distal site and active center regulates stereoselectivity and activity of carbonyl reductase towards N-Boc-pyrrolidone

Green and efficient biosynthesis of two enantiomeric products of N-Boc-pyrrolidinol (NPBL) as key pharmaceutical blocks for treatment of cancer and HIV has received much attention. The precursor, N-Boc-pyrrolidone (NPBO), is considered as a difficult-to-reduce ketone by carbonyl reductases, because it has a bulky Boc group and sterically similar substitutions on either side of the carbonyl group resulting in low stereoselectivity and conversion. Moreover, activity enhancement concomitant with reversal of stereoselectivity is challenging. Carbonyl reductase (CpCR) from Candida parapsilosis shows 90 %ee for (S)-NBPL, but low catalytic efficiency. In this study, we considered both activity and selectivity, and proposed an integrated engineering strategy. The distal site was introduced from an activity perspective and the active pocket sites were introduced by virtual saturation mutagenesis from a stereoselectivity perspective. Mutants L34A/W116A (95 %ee (S)) and L34A/W116T/F285C/W286S (94 %ee (R)) were obtained with 26.8-fold and 2.9-fold higher catalytic efficiencies, respectively. Molecular dynamics simulations revealed the mechanism of stereoselective flip-flop and activity enhancement; mutation altered the substrate-binding mode and changed the shape and size of the cavities, thus contributing to the change in the ratio of active conformations in the pre-reactive state. This study, which differs from the traditional method of exchanging large and small pockets, provides a viable approach for the rational design of carbonyl reductases with high stereoselectivity for target substrates.

The gut-lung axis: the impact of the gut mycobiome on pulmonary diseases and infections.

The gastrointestinal tract contains a diverse microbiome consisting of bacteria, fungi, viruses and archaea. Although these microbes usually reside as commensal organisms, it is now well established that higher abundance of specific bacterial or fungal species, or loss of diversity in the microbiome can significantly affect development, progression and outcomes in disease. Studies have mainly focused on the effects of bacteria, however, the impact of other microbes, such as fungi, has received increased attention in the last few years. Fungi only represent around 0.1% of the total gut microbial population. However, key fungal taxa such as Candida, Aspergillus and Wallemia have been shown to significantly impact health and disease. The composition of the gut mycobiome has been shown to affect immunity at distal sites, such as the heart, lung, brain, pancreas, and liver. In the case of the lung this phenomenon is referred to as the 'gut-lung axis'. Recent studies have begun to explore and unveil the relationship between gut fungi and lung immunity in diseases such as asthma and lung cancer, and lung infections caused by viruses, bacteria and fungi. In this review we will summarize the current, rapidly growing, literature describing the impact of the gut mycobiome on respiratory disease and infection.