Racial Differences in Choroidal Vascularity Index in Healthy Patients: Novel Insights.

BACKGROUND AND OBJECTIVE: Choroidal vascularity index (CVI) measures the ratio of blood vessels in the choroid to the total choroidal area. We aimed to compare CVI between young Black and White patients without a history of ocular or systemic disease. PATIENTS AND METHODS: We used a previously validated algorithm for shadow compensation and choroidal vessel binarization to measure CVI across the Early Treatment of Diabetic Retinopathy Study grid. RESULTS: Black patients had a lower CVI (ß = -0.05, P < 0.001) compared to White patients. Choroidal volume or luminal volume did not significantly differ with respect to race, whereas there was a trend for Black patients to have a greater stromal volume (ß = 3.08, P = 0.01). CONCLUSIONS: Black patients have a lower CVI than do White patients, likely due to a greater proportion of stromal volume. Further study of this parameter is warranted to validate the findings of this exploratory study. [Ophthalmic Surg Lasers Imaging Retina 2024;55:30-38.].

Metformin Use and Age-Related Macular Degeneration in Patients Without Diabetes.

Importance: Metformin use may protect against the development of age-related macular degeneration (AMD) based on results from observational studies. However, its potential effectiveness among patients without diabetes remains unclear. Objective: To assess the association between metformin use and the development of AMD in patients without diabetes. Design, Setting, and Participants: This case-control study used data from 2006 to 2017 in the Merative MarketScan Research Database, a nationwide insurance claims database that includes between 27 and 57 million patients in the US with primary or Medicare supplemental health insurance. Cases with AMD and controls without AMD aged 55 years or older were matched 1:1 by year, age, anemia, hypertension, region, and Charlson Comorbidity Index score. Then, cases and matched controls without a diagnosis of diabetes were selected. In subgroup analyses, cases with dry AMD and their matched controls were identified to explore the association between metformin use and AMD staging in patients without diabetes. Data were analyzed between March and September 2023. Exposures: Exposure to metformin in the 2 years prior to the index date (ie, date of AMD diagnosis in cases and date of a randomly selected eye examination for controls) was assessed from the claims database and categorized into quartiles based on cumulative dose (1-270, 271-600, 601-1080, and >1080 g/2 y). Exposure to other antidiabetic medications was also noted. Main Outcomes and Measures: Odds of new-onset AMD development as assessed by multivariable conditional logistic regression after adjusting for known risk factors for AMD, including female sex, hyperlipidemia, smoking, and exposures to other antidiabetic medications. Asymptotic Cochran-Armitage tests for trend were also performed. Results: We identified 231 142 patients with any AMD (mean [SD] age, 75.1 [10.4] years; 140 172 females [60.6%]) and 232 879 matched controls without AMD (mean [SD] age, 74.9 [10.5] years; 133 670 females [57.4%]), none of whom had a diagnosis of diabetes. The sample included 144 147 cases with dry AMD that were matched to 144 530 controls. In all, 2268 (1.0%) cases and 3087 controls (1.3%) were exposed to metformin in the 2 years before their index visit. After data adjustment, exposure to any metformin was associated with reduced odds of any AMD development (adjusted odds ratio [AOR], 0.83; 95% CI, 0.74-0.87), specifically in the dosing quartiles of 1 to 270, 271 to 600, and 601 to 1080 g/2 y. Any metformin use was also associated with a reduced odds of developing dry AMD (AOR, 0.85; 95% CI, 0.79-0.92), specifically in the dosing quartiles of 1 to 270 and 271 to 600 g/2 y. Adjusted odds ratios for any AMD and dry AMD development did not differ across the dosing quartiles. Asymptotic Cochran-Armitage tests for trend revealed 2-sided P = .51 and P = .66 for the any and dry AMD samples, respectively. Conclusions and Relevance: In this case-control study of a population without a diagnosis of diabetes, metformin use was associated with reduced odds of developing AMD. This association does not appear to be dose dependent. These findings provide further impetus to study metformin's usefulness in protecting against AMD in prospective clinical trials.

Association of Metformin and Other Diabetes Medication Use and the Development of New-Onset Dry Age-Related Macular Degeneration: A Case-Control Study.

Purpose: To investigate if metformin use is associated with decreased odds of developing new non-neovascular ("dry") age-related macular degeneration (AMD). Methods: Case-control study examining 194,135 cases with diagnoses of new-onset AMD between 2008 and 2017 and 193,990 matched controls in the Merative MarketScan Research Databases. The diabetic subgroup included 49,988 cases and 49,460 controls. Multivariable conditional logistic regressions identified the risks of exposures on the development of dry AMD. Main outcome measures were odds ratios (ORs) of developing dry AMD with metformin use. Results: In multivariable conditional logistic regression, any metformin use was associated with decreased odds of developing dry AMD (OR = 0.97; 95% confidence interval [CI], 0.95-0.99). This protective effect was noted for cumulative 2-year doses of metformin of 1 to 270 g (OR = 0.93; 95% CI, 0.90-0.97) and 271 to 600 g (OR = 0.92; 95% CI, 0.89-0.96). In a diabetic subgroup, metformin use below 601 g per 2 years decreased the odds of developing dry AMD (1-270 g: OR = 0.95; 95% CI, 0.91-0.99; 271-600 g: OR = 0.92; 95% CI, 0.89-0.96). Unlike in diabetic patients with diabetic retinopathy, diabetic patients without diabetic retinopathy had decreased odds of developing dry AMD with any metformin use (OR = 0.97; 95% CI, 0.94-0.998) and cumulative two-year doses of 1 to 270 g (OR 0.96; 95% CI, 0.91-0.998) and 271 to 600 g (OR = 0.92; 95% CI, 0.88-0.96). Conclusions: Metformin use was associated with decreased odds of developing dry AMD. The protective effect was observed for cumulative 2-year doses below 601 g. In diabetics, this association persisted, specifically in those without diabetic retinopathy. Therefore, metformin may be a strategy to prevent development of dry AMD.

Autologous humanized PDX modeling for immuno-oncology recapitulates features of the human tumor microenvironment.

BACKGROUND: Interactions between immune and tumor cells are critical to determining cancer progression and response. In addition, preclinical prediction of immune-related drug efficacy is limited by interspecies differences between human and mouse, as well as inter-person germline and somatic variation. To address these gaps, we developed an autologous system that models the tumor microenvironment (TME) from individual patients with solid tumors. METHOD: With patient-derived bone marrow hematopoietic stem and progenitor cells (HSPCs), we engrafted a patient's hematopoietic system in MISTRG6 mice, followed by transfer of patient-derived xenograft (PDX) tissue, providing a fully genetically matched model to recapitulate the individual's TME. We used this system to prospectively study tumor-immune interactions in patients with solid tumor. RESULTS: Autologous PDX mice generated innate and adaptive immune populations; these cells populated the TME; and tumors from autologously engrafted mice grew larger than tumors from non-engrafted littermate controls. Single-cell transcriptomics revealed a prominent vascular endothelial growth factor A (VEGFA) signature in TME myeloid cells, and inhibition of human VEGF-A abrogated enhanced growth. CONCLUSIONS: Humanization of the interleukin 6 locus in MISTRG6 mice enhances HSPC engraftment, making it feasible to model tumor-immune interactions in an autologous manner from a bedside bone marrow aspirate. The TME from these autologous tumors display hallmarks of the human TME including innate and adaptive immune activation and provide a platform for preclinical drug testing.

Trends in Secondary Intraocular Lens Surgery among Vitreoretinal Surgeons.

OBJECTIVE: To identify changes in secondary lens techniques over time and to determine common complications of each technique. DESIGN: Retrospective cohort study. PARTICIPANTS: All patients in the database from January 2015 to December 2021 who underwent secondary lens placement (anterior chamber intraocular lens [IOL, ACIOL], scleral-fixated IOL [SFIOL], or scleral-sutured IOL [SSIOL]). METHODS: Rates of secondary IOL surgery techniques were analyzed in 3597 participants in a nationwide aggregated electronic health care database using 2-sample independent t tests. Rates of postoperative rhegmatogenous retinal detachment (RRD) after secondary IOL surgery were assessed using chi-square test of proportion. Postoperative visual acuity (VA) was assessed using 2-sample independent t tests. MAIN OUTCOME MEASURES: The primary outcome was change in IOL technique over time. Secondary data points examined were the development of postoperative RRD after secondary IOL surgery, VA changes, the development of endophthalmitis, suture erosion, haptic erosion, or corneal edema after IOL surgery. RESULTS: Anterior chamber IOL use decreased over the 7-year period from 93% of cases to 36% of cases (P < 0.0001), while SFIOL use increased from 3% to 34% (P < 0.0001) and SSIOL use increased from 4% to 30% (P < 0.0001). Visual acuity increased for each surgical technique (ACIOL: 44.1 vs. 49.2 ETDRS letters, P < 0.001; SFIOL: 48.7 vs. 57.6 letters, P < 0.001; SSIOL: 51.5 vs. 61.2 letters, P < 0.001), with larger VA gains seen in SFIOL and SSIOL use (ACIOL vs. SFIOL, P = 0.004; ACIOL vs. SSIOL, P = 0.002; SFIOL vs. SSIOL, P = 0.64). Average RRD rates did not significantly differ between techniques. Rates of endophthalmitis, haptic erosion, and suture erosion were low and did not significantly differ between techniques. Rates of corneal edema were significantly higher in ACIOL cases (vs. SFIOL, P < 0.0001; vs. SSIOL, P < 0.0001). CONCLUSIONS: Rates of ACIOL implantation performed by vitreoretinal surgeons have decreased over time with more vitreoretinal surgeons electing to place either an SFIOL or SSIOL toward the end of the study period; complication profiles among the 3 techniques may be similar. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Androstadienone sensitivity is associated with attention to emotions, social interactions, and sexual behavior in older U.S. adults.

Δ 4,16-androstadien-3-one (androstadienone) is a putative human pheromone often linked to sexual attraction in young adults, although specific associations with sexual behavior are not yet established. Androstadienone also serves a broader social-emotional function beyond the sexual domain, specifically tuning the brain to efficiently process emotional information. Whether these effects persist throughout the lifespan into post-reproductive life is unknown. In a laboratory study of older adults, those with greater androstadienone odor sensitivity paid greater attention to subliminal emotional information, specifically, angry faces (p = 0.05), with a similar relationship to happy faces. In contrast, the physical odor n-butanol (a control) did not affect emotional attention (p = 0.49). We then extended this laboratory research and determined whether sensitivity to androstadienone affects the everyday lives of older adults by measuring their social and sexual behavior. In this second study, we surveyed in a nationally representative sample of US older adults living in their homes (National Social Life and Aging Project, 62-90 years; n = 2,086), along with their sensitivity to androstadienone, general olfactory function, health and demographics. Greater sensitivity to androstadienone was associated with richer social lives: having more friends, increased communication with close friends and family, and more participation in organized social events and volunteer activities (all p's ≤ 0.05, generalized linear models, adjusted for age and gender). It was also associated with more recent sexual activity, more frequent sexual thoughts, and viewing sex as an important part of life (all p's ≤ 0.05). General olfactory function did not explain these associations, supporting a specialized function for this pheromone during everyday life, and expanding its role to social life as well as sexual behavior, likely mediated by enhanced attention to emotional information.

Computerized Texture Analysis of Optical Coherence Tomography Angiography of Choriocapillaris in Normal Eyes of Young and Healthy Subjects.

Computerized texture analysis uses higher-order mathematics to identify patterns beyond what the naked eye can recognize. We tested its feasibility in optical coherence tomography angiography imaging of choriocapillaris. Our objective was to determine sets of parameters that provide coherent and consistent output when applied to a homogeneous, healthy group of patients. This observational cross-sectional study involved 19 eyes of 10 young and healthy Caucasian subjects. En-face macular optical coherence tomography angiography of superficial choriocapillaris was obtained by the RTVue-XR Avanti system. Various algorithms were used to extract texture features. The mean and standard deviation were used to assess the distribution and dispersion of data points in each metric among eyes, which included: average gray level, gray level yielding 70% threshold and 30% threshold, balance, skewness, energy, entropy, contrast, edge mean gradient, root-mean-square variation, and first moment of power spectrum, which was compared between images, showing a highly concordant homology between all eyes of participants. We conclude that computerized texture analysis for en-face optical coherence tomography angiography images of choriocapillaris is feasible and provides values that are coherent and tightly distributed around the mean in a homogenous, healthy group of patients. Homology of blob size among subjects may represent a "repeat pattern" in signal density and thus a perfusion in the superficial choriocapillaris of healthy young individuals of the same ethnic background.

NKG2A-checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA-E.

NKG2A has emerged as a new immunotherapy target and its blockade with the novel immune checkpoint inhibitor (ICI) monalizumab can boost both NK cell and CD8+ T cell responses. NKG2A forms heterodimers with CD94 and binds to the human non-classical MHC class I molecule HLA-E. HLA-E forms complexes with a limited set of peptides mainly derived from the leader sequences of the classical MHC class I molecules (HLA-A, HLA-B and HLA-C) and the non-classical class I paralogue HLA-G, and it is well established that the interaction between CD94/NKG2x receptors and its ligand HLA-E is peptide-sensitive. Here, we have evaluated peptide dependence of NKG2A-mediated inhibition and the efficiency of interference by monalizumab in a transcriptional T cell reporter system. NKG2A inhibition was mediated by cell-expressed HLA-E molecules stably presenting disulfate-trapped peptide ligands. We show that different HLA-class I leader peptides mediate varying levels of inhibition. We have used NKG2A/NKG2C chimeric receptors to map the binding site of NKG2A and NKG2C blocking antibodies. Furthermore, we determined the functional EC50 values of blocking NKG2A antibodies and show that they greatly depend on the HLA-leader peptide presented by HLA-E. Monalizumab was less effective in augmenting NK cell-mediated killing of target cells displaying HLA-G peptide on HLA-E, than cells expressing HLA-E complexed with HLA-A, HLA-B and HLA-C peptides. Our results indicate that peptides displayed by HLA-E molecules on tumour cells might influence the effectivity of NKG2A-ICI therapy and potentially suggest novel approaches for patient stratification, for example, based on tumoral HLA-G levels.

Transposon Mutagenesis-Guided CRISPR/Cas9 Screening Strongly Implicates Dysregulation of Hippo/YAP Signaling in Malignant Peripheral Nerve Sheath Tumor Development.

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive, genomically complex, have soft tissue sarcomas, and are derived from the Schwann cell lineage. Patients with neurofibromatosis type 1 syndrome (NF1), an autosomal dominant tumor predisposition syndrome, are at a high risk for MPNSTs, which usually develop from pre-existing benign Schwann cell tumors called plexiform neurofibromas. NF1 is characterized by loss-of-function mutations in the NF1 gene, which encode neurofibromin, a Ras GTPase activating protein (GAP) and negative regulator of RasGTP-dependent signaling. In addition to bi-allelic loss of NF1, other known tumor suppressor genes include TP53, CDKN2A, SUZ12, and EED, all of which are often inactivated in the process of MPNST growth. A sleeping beauty (SB) transposon-based genetic screen for high-grade Schwann cell tumors in mice, and comparative genomics, implicated Wnt/ß-catenin, PI3K-AKT-mTOR, and other pathways in MPNST development and progression. We endeavored to more systematically test genes and pathways implicated by our SB screen in mice, i.e., in a human immortalized Schwann cell-based model and a human MPNST cell line, using CRISPR/Cas9 technology. We individually induced loss-of-function mutations in 103 tumor suppressor genes (TSG) and oncogene candidates. We assessed anchorage-independent growth, transwell migration, and for a subset of genes, tumor formation in vivo. When tested in a loss-of-function fashion, about 60% of all TSG candidates resulted in the transformation of immortalized human Schwann cells, whereas 30% of oncogene candidates resulted in growth arrest in a MPNST cell line. Individual loss-of-function mutations in the TAOK1, GDI2, NF1, and APC genes resulted in transformation of immortalized human Schwann cells and tumor formation in a xenograft model. Moreover, the loss of all four of these genes resulted in activation of Hippo/Yes Activated Protein (YAP) signaling. By combining SB transposon mutagenesis and CRISPR/Cas9 screening, we established a useful pipeline for the validation of MPNST pathways and genes. Our results suggest that the functional genetic landscape of human MPNST is complex and implicate the Hippo/YAP pathway in the transformation of neurofibromas. It is thus imperative to functionally validate individual cancer genes and pathways using human cell-based models, to determinate their role in different stages of MPNST development, growth, and/or metastasis.

R-spondin 2 Drives Liver Tumor Development in a Yes-Associated Protein-Dependent Manner.

Each year, more than 25,000 people succumb to liver cancer in the United States, and this neoplasm represents the second cause of cancer-related death globally. R-spondins (RSPOs) are secreted regulators of Wnt signaling that function in development and promote tissue stem cell renewal. In cancer, RSPOs 2 and 3 are oncogenes first identified by insertional mutagenesis screens in tumors induced by mouse mammary tumor virus and by transposon mutagenesis in the colonic epithelium of rodents. RSPO2 has been reported to be activated by chromosomal rearrangements in colorectal cancer and overexpressed in a subset of hepatocellular carcinoma. Using human liver tumor gene expression data, we first discovered that a subset of liver cancers were characterized by high levels of RSPO2 in contrast to low levels in adjacent nontumor tissue. To determine if RSPOs are capable of inducing liver tumors, we used an in vivo model from which we found that overexpression of RSPO2 in the liver promoted Wnt signaling, hepatomegaly, and enhanced liver tumor formation when combined with loss of transformation-related protein 53 (Trp53). Moreover, the Hippo/yes-associated protein (Yap) pathway has been implicated in many human cancers, influencing cell survival. Histologic and gene expression studies showed activation of Wnt/ß-catenin and Hippo/Yap pathways following RSPO2 overexpression. We demonstrate that knockdown of Yap1 leads to reduced tumor penetrance following RSPO2 overexpression in the context of loss of Trp53. Conclusion: RSPO2 overexpression leads to tumor formation in the mouse liver in a Hippo/Yap-dependent manner. Overall, our results suggest a role for Yap in the initiation and progression of liver tumors and uncover a novel pathway activated in RSPO2-induced malignancies. We show that RSPO2 promotes liver tumor formation in vivo and in vitro and that RSPO2's oncogenic activity requires Hippo/Yap activation in hepatocytes. Both RSPO2 and YAP1 are suggested to represent novel druggable targets in Wnt-driven tumors of the liver.

Recurrent Brain Arteriovenous Malformations (AVMs): A Systematic Review.

OBJECTIVE: Risk factors for the recurrence of surgically excised brain arteriovenous malformations (AVMs) are poorly understood. In addition, ideal follow-up imaging paradigms to catch AVM recurrences are not well defined. We present a systematic review on risk factors for the recurrence of surgically resected AVMs and identify potential theories of recurrence. METHODS: A literature search was performed by a reference librarian, and after screening, 14 case reports and 16 case series were left for inclusion in the review. All possible data were abstracted by 2 authors, and the results were tabulated and descriptive statistics (mean, range; and proportions) were reported. No formal statistical analysis was performed as part of this study. RESULTS: Systematic review of the literature revealed 73 patients with a surgically resected AVM that recurred. The average age of first AVM presentation was 13.8 years, and most patients presented with hemorrhage (90%). After angiographically confirmed complete surgical resection, average time to AVM recurrence was 4.2 years. Rate of recurrence was 2.7% in adult series or case reports (n = 8). When we analyzed only pediatric case reports or series (n = 12), the average rate of recurrence was 9.5% but was as high as almost 14% in a series with compulsory short-term follow-up serial imaging. Four (5.5%) patients experienced re-recurrence of AVM after complete surgical excision of first AVM recurrence. CONCLUSIONS: AVM recurrence after complete surgical resection is a recognized risk that occurs primarily in children. Follow-up imaging within 1 year of surgery is strongly indicated for pediatric patients with surgically resected AVMs, even with postoperative angiographically confirmed complete excision.

Dual nucleotide specificity determinants of an infection aborting anticodon nuclease.

The anticodon nuclease (ACNase) PrrC is silenced by a DNA restriction-modification (RM) protein and activated by a phage T4-encoded restriction inhibitor. The activation is driven by GTP hydrolysis while dTTP, which accumulates during the infection, stabilizes the active form. We show here, first, that the ABC-ATPase N-domains of PrrC can accommodate the two nucleotides simultaneously. Second, mutating a sequence motif that distinguishes the N-domain of PrrC from typical ABC-ATPases implicates three residues in the specificity for dTTP. Third, failure to bind dTTP or its deprivation hastened the centrifugal sedimentation of PrrC, possibly due to exposed sticky PrrC surfaces. Fourth, dTTP inhibited the GTPase activity of PrrC, probably by preventing GDP from leaving. These observations, correlated with relevant traits of a related ACNase, further suggest that PrrC utilizes GTP at canonical ABC-ATPase sites and binds dTTP to distinct sites exposed upon disruption of the ACNase-silencing interaction with the RM partner.

Phage T4-induced DNA breaks activate a tRNA repair-defying anticodon nuclease.

The natural role of the conserved bacterial anticodon nuclease (ACNase) RloC is not known, but traits that set it apart from the homologous phage T4-excluding ACNase PrrC could provide relevant clues. PrrC is silenced by a genetically linked DNA restriction-modification (RM) protein and turned on by a phage-encoded DNA restriction inhibitor. In contrast, RloC is rarely linked to an RM protein, and its ACNase is regulated by an internal switch responsive to double-stranded DNA breaks. Moreover, PrrC nicks the tRNA substrate, whereas RloC excises the wobble nucleotide. These distinctions suggested that (i) T4 and related phage that degrade their host DNA will activate RloC and (ii) the tRNA species consequently disrupted will not be restored by phage tRNA repair enzymes that counteract PrrC. Consistent with these predictions we show that Acinetobacter baylyi RloC expressed in Escherichia coli is activated by wild-type phage T4 but not by a mutant impaired in host DNA degradation. Moreover, host and T4 tRNA species disrupted by the activated ACNase were not restored by T4's tRNA repair system. Nonetheless, T4's plating efficiency was inefficiently impaired by AbaRloC, presumably due to a decoy function of the phage encoded tRNA target, the absence of which exacerbated the restriction.

A DNA break inducer activates the anticodon nuclease RloC and the adaptive immunity in Acinetobacter baylyi ADP1.

Double-stranded DNA breaks (DSB) cause bacteria to augment expression of DNA repair and various stress response proteins. A puzzling exception educes the anticodon nuclease (ACNase) RloC, which resembles the DSB responder Rad50 and the antiviral, translation-disabling ACNase PrrC. While PrrC's ACNase is regulated by a DNA restriction-modification (R-M) protein and a phage anti-DNA restriction peptide, RloC has an internal ACNase switch comprising a putative DSB sensor and coupled ATPase. Further exploration of RloC's controls revealed, first, that its ACNase is stabilized by the activating DNA and hydrolysed nucleotide. Second, DSB inducers activated RloC's ACNase in heterologous contexts as well as in a natural host, even when R-M deficient. Third, the DSB-induced activation of the indigenous RloC led to partial and temporary disruption of tRNA(Glu) and tRNA(Gln). Lastly, accumulation of CRISPR-derived RNA that occurred in parallel raises the possibility that the adaptive immunity and RloC provide the genotoxicated host with complementary protection from impending infections.

The wobble nucleotide-excising anticodon nuclease RloC is governed by the zinc-hook and DNA-dependent ATPase of its Rad50-like region.

The conserved bacterial anticodon nuclease (ACNase) RloC and its phage-excluding homolog PrrC comprise respective ABC-adenosine triphosphatase (ATPase) and ACNase N- and C-domains but differ in three key attributes. First, prrC is always linked to an ACNase silencing, DNA restriction-modification (R-M) locus while rloC rarely features such linkage. Second, RloC excises its substrate's wobble nucleotide, a lesion expected to impede damage reversal by phage transfer RNA (tRNA) repair enzymes that counteract the nick inflicted by PrrC. Third, a distinct coiled-coil/zinc-hook (CC/ZH) insert likens RloC's N-region to the universal DNA damage checkpoint/repair protein Rad50. Previous work revealed that ZH mutations activate RloC's ACNase. Data shown here suggest that RloC has an internal ACNase silencing/activating switch comprising its ZH and DNA-break-responsive ATPase. The existence of this control may explain the lateral transfer of rloC without an external silencer and supports the proposed role of RloC as an antiviral contingency acting when DNA restriction is alleviated under genotoxic stress. We also discuss RloC's possible evolution from a PrrC-like ancestor.

Phage T4-induced dTTP accretion bolsters a tRNase-based host defense.

The anticodon nuclease (ACNase) PrrC is silenced in Escherichia coli by an associated DNA restriction-modification protein, activated by the phage T4-encoded anti-DNA restriction factor Stp and counteracted by T4's tRNA repair enzymes polynucleotide kinase and RNA ligase 1. Hence, only tRNA repair-deficient phages succumb to PrrC's restriction. PrrC's ABC-ATPase motor domains are implicated in driving its activation by hydrolyzing GTP and in stabilizing the activated ACNase by avidly binding dTTP. The latter effect has been associated with dTTP's accumulation early in T4 infection when PrrC is activated. In agreement, delayed dTTP accumulation caused by dCMP deaminase deficiency coincided with impaired manifestation of PrrC's ACNase activity. This impairment did not suffice to suppress the PrrC-mediated restriction of tRNA repair deficient phage but was synthetically suppressive with a leaky stp mutation that only partly impairs PrrC's activation. Presumably, ability to gauge dTTP's changing level helps confine PrrC's toxicity to its viral target.

RloC: a wobble nucleotide-excising and zinc-responsive bacterial tRNase.

SUMMARY: The conserved bacterial protein RloC, a distant homologue of the tRNA(Lys) anticodon nuclease (ACNase) PrrC, is shown here to act as a wobble nucleotide-excising and Zn(++)-responsive tRNase. The more familiar PrrC is silenced by a genetically linked type I DNA restriction-modification (R-M) enzyme, activated by a phage anti-DNA restriction factor and counteracted by phage tRNA repair enzymes. RloC shares PrrC's ABC ATPase motifs and catalytic ACNase triad but features a distinct zinc-hook/coiled-coil insert that renders its ATPase domain similar to Rad50 and related DNA repair proteins. Geobacillus kaustophilus RloC expressed in Escherichia coli exhibited ACNase activity that differed from PrrC's in substrate preference and ability to excise the wobble nucleotide. The latter specificity could impede reversal by phage tRNA repair enzymes and account perhaps for RloC's more frequent occurrence. Mutagenesis and functional assays confirmed RloC's catalytic triad assignment and implicated its zinc hook in regulating the ACNase function. Unlike PrrC, RloC is rarely linked to a type I R-M system but other genomic attributes suggest their possible interaction in trans. As DNA damage alleviates type I DNA restriction, we further propose that these related perturbations prompt RloC to disable translation and thus ward off phage escaping DNA restriction during the recovery from DNA damage.

Combined axillary reverse mapping (ARM) technique for breast cancer patients requiring axillary dissection.

BACKGROUND: The objective of axillary reverse mapping (ARM) is to preserve the main lymphatic chain-with both the nodes and the ducts-in relation to lymphatic arm drainage (LAD) during an axillary dissection (AD). METHODS: From July 2006 to March 2008, 23 patients with stage II or III breast carcinoma requiring an AD underwent an ARM procedure. Identification of the ARM nodes relied on an isotope injection into the web space of the ipsilateral hand. During AD, the radioactive ARM node was localized above the second intercostal brachial nerve, and blue dye was directly injected inside the node to visualize the efferent ducts, constituting the lymphatic ARM chain. The blue and radioactive nodes constituted the ARM sampling, while other nodes were considered part of the AD. RESULTS: Metastatic lymph node involvement was identified in the AD in 20 of 23 patients, with an average of 4.4 (1-11) nodes involved and an average of 10.7 (7-20) lymph nodes removed. The ARM sampling was performed in 21 of 23 patients (91%), with an average of 1.6 ARM nodes removed. In 18 of these 21 patients (86%), the nodes relating to ARM sampling had no metastatic involvement. There were 3 patients (14%) who demonstrated metastatic involvement of the ARM sampling, and all had pN3a (N+ > 9) involvement of the axilla. CONCLUSION: This technique of combined isotopic and blue dye ARM and findings must now be validated. A multicentric study is planned to confirm this data.

Parallel dimerization of a PrrC-anticodon nuclease region implicated in tRNALys recognition.

The optional Escherichia coli restriction tRNase PrrC represents a family of potential antiviral devices widespread among bacteria. PrrC comprises a functional C-domain of unknown structure and regulatory ABC/ATPase-like N-domain. The possible involvement of a C-domain sequence in tRNA(Lys) recognition was investigated using a matching end-protected 11-meric peptide. This mimic, termed here LARP (Lys-anticodon recognizing peptide) UV-cross-linked tRNA(Lys) anticodon stem-loop (ASL) analogs and inhibited their PrrC-catalyzed cleavage. Trimming LARP or introducing in it inactivating PrrC missense mutations impaired these activities. LARP appeared to mimic its matching protein sequence in ability to dimerize in parallel, as inferred from the following results. First, tethering Cys to the amino- or carboxy-end of LARP dramatically enhanced the ASL-cross-linking and PrrC-inhibiting activities under suitable redox conditions. Second, Cys-substitutions in a C-domain region containing the sequence corresponding to LARP elicited specific intersubunit cross-links. The parallel dimerization of PrrC's C-domains and expected head-to-tail dimerization of its N-domains further suggest that the NTPase and tRNA(Lys)-binding sites of PrrC arise during distinct assembly stages of its dimer of dimers form.