Perimacular Atrophy Following Voretigene Neparvovec-Rzyl Treatment in the Setting of Previous Contralateral Eye Treatment With a Different Viral Vector.

Purpose: To report on cases of unilateral perimacular atrophy after treatment with voretigene neparvovec-rzyl, in the setting of previous contralateral eye treatment with a different viral vector. Design: Single-center, retrospective chart review. Methods: In this case series, four patients between the ages of six and 11 years old with RPE65-related retinopathy were treated unilaterally with rAAV2-CB-hRPE65 as part of a gene augmentation clinical trial (NCT00749957). Six to 10 years later the contralateral eyes were treated with the Food and Drug Administration-approved drug, voretigene neparvovec-rzyl. Best-corrected visual acuity (BCVA), fundus photos, ocular coherence tomography, two-color dark-adapted perimetry, full field stimulus threshold testing (FST), and location of subretinal bleb and chorioretinal atrophy were evaluated. Results: Three out of four patients showed unilateral perimacular atrophy after treatment with voretigene, ranging from five to 22 months after treatment. Areas of robust visual field improvement were followed by areas of chorioretinal atrophy. Despite perimacular changes, BCVA, FST, and subjective improvements in vision and nyctalopia were maintained. Perimacular atrophy was not observed in the first eye treated with the previous viral vector. Conclusions: We observed areas of robust visual field improvement followed by perimacular atrophy in voretigene treated eyes, as compared to the initially treated contralateral eyes. Translational Relevance: Caution is advised when using two different viral vectors between eyes in gene therapy. This may become an important issue in the future with increasing gene therapy clinical trials for inherited retinal dystrophies.

Update on Hypermetabolism in Pediatric Burn Patients.

Despite advancements in pediatric burn care, the profound hypermetabolic response associated with severe burns remains a multifaceted challenge throughout the continuum of care. Understanding the various physiologic disturbances that constitute hypermetabolism is crucial for a thorough evaluation and for implementing appropriate surgical and nonsurgical interventions. In this article, we describe the pathophysiology and treatment of hypermetabolism in pediatric burn patients with a focus on reducing resting energy requirements, minimizing infection, and optimizing nutrition for patients undergoing frequent surgical intervention.

Lost in Translation: Multilingual Analysis of Online Breast Cancer Information.

INTRODUCTION: Patients use the internet to learn more about health conditions. Non-English-speaking patients may face additional challenges. The quality of online breast cancer information, the most common cancer in women, is uncertain. This study aims to examine the quality of online breast cancer information for English and non-English-speaking patients. METHODS: Three search engines were queried using the terms: "how to do a breast examination," "when do I need a mammogram," and "what are the treatment options for breast cancer" in English, Spanish, and Chinese. For each language, 60 unique websites were included and classified by type and information source. Two language-fluent reviewers evaluated website quality using the Journal of American Medical Association benchmark criteria (0-4) and the DISCERN tool (1-5), with higher scores representing higher quality. Scores were averaged for each language. Health On the Net code presence was noted. Inter-rater reliability between reviewers was assessed. RESULTS: English and Spanish websites most commonly originated from US sources (92% and 80%, respectively) compared to Chinese websites (33%, P < 0.001). The most common website type was hospital-affiliated for English (43%) and foundation/advocacy for Spanish and Chinese (43% and 45%, respectively). English websites had the highest and Chinese websites the lowest mean the Journal of American Medical Association (2.2 ± 1.4 versus 1.0 ± 0.8, P = 0.002) and DISCERN scores (3.5 ± 0.9 versus 2.3 ± 0.6, P < 0.001). Health On the Net code was present on 16 (8.9%) websites. Inter-rater reliability ranged from moderate to substantial agreement. CONCLUSIONS: The quality of online information on breast cancer across all three languages is poor. Information quality was poorest for Chinese websites. Improvements to enhance the reliability of breast cancer information across languages are needed.

Trimethylsilyldiazomethane Disassembly at a Three-Fold Symmetric Iron Site.

The reaction of equimolar trimethylsilyldiazomethyllithium (LiTMSD) with high spin (S = 2) PhB(AdIm)3FeCl (PhB(AdIm)3- = tris(3-adamantylimidazol-2-ylidene)phenylborate) affords the corresponding N-nitrilimido complex PhB(AdIm)3Fe-N═N═C(SiMe3). This complex can be converted to the thermodynamically more favorable C-isocyanoamido isomer PhB(AdIm)3Fe-C═N═N(SiMe3) by reaction with an additional equivalent of LiTMSD. While the iron(II) complexes are four-coordinate, the diazomethane is bound side-on in the iron(I) congener PhB(AdIm)3Fe(N,N'-κ2-N2C(H)Si(CH3)3). The latter complex adopts high spin (S = 3/2) ground state and features an unusually weak C-H bond. Photolysis of the iron(II) complexes induces N═N bond cleavage, with the iron(II) cyanide PhB(AdIm)3Fe-C≡N and iron(IV) nitride PhB(AdIm)3Fe≡N complexes being the major products of the reaction. The same products are obtained when the iron(I) complex is photolyzed or treated with a fluoride source. The trimethylsilyldiazomethane-derived ligand disassembly reactions are contrasted with those observed for related tris(carbene)amine complexes.

Comparison of Cyclic and Linear PEG Conjugates.

Bioconjugation of polymers to proteins is a method to impart improved stability and pharmacokinetic properties to biologic systems. However, the precise effects of polymer architecture on the resulting bioconjugates are not well understood. Particularly, cyclic polymers are known to possess unique features such as a decreased hydrodynamic radius when compared to their linear counterparts of the same molecular weight, but have not yet been studied. Here, we report the first bioconjugation of a cyclic polymer, poly(ethylene glycol) (PEG), to a model protein, T4 lysozyme, containing a single engineered cysteine residue (V131C). We compare the stability and activity of this conjugate with those of a linear PEG-T4 lysozyme analogue of similar molecular weight. Furthermore, we used molecular dynamics (MD) simulations to determine the behavior of the polymer-protein conjugates in solution. We introduce cyclic polymer-protein conjugates as potential candidates for the improvement of biologic therapeutics.

Glycosaminoglycans' for brain health: Harnessing glycosaminoglycan based biomaterials for treating central nervous system diseases and in-vitro modeling.

Dysfunction of the central nervous system (CNS) following traumatic brain injuries (TBI), spinal cord injuries (SCI), or strokes remains challenging to address using existing medications and cell-based therapies. Although therapeutic cell administration, such as stem cells and neuronal progenitor cells (NPCs), have shown promise in regenerative properties, they have failed to provide substantial benefits. However, the development of living cortical tissue engineered grafts, created by encapsulating these cells within an extracellular matrix (ECM) mimetic hydrogel scaffold, presents a promising functional replacement for damaged cortex in cases of stroke, SCI, and TBI. These grafts facilitate neural network repair and regeneration following CNS injuries. Given that natural glycosaminoglycans (GAGs) are a major constituent of the CNS, GAG-based hydrogels hold potential for the next generation of CNS healing therapies and in vitro modeling of CNS diseases. Brain-specific GAGs not only offer structural and biochemical signaling support to encapsulated neural cells but also modulate the inflammatory response in lesioned brain tissue, facilitating host integration and regeneration. This review briefly discusses different roles of GAGs and their related proteoglycan counterparts in healthy and diseases brain and explores current trends and advancements in GAG-based biomaterials for treating CNS injuries and modeling diseases. Additionally, it examines injectable, 3D bioprintable, and conductive GAG-based scaffolds, highlighting their clinical potential for in vitro modeling of patient-specific neural dysfunction and their ability to enhance CNS regeneration and repair following CNS injury in vivo.

What Proportion of BRCA-Associated Breast Cancer Is Human Epidermal Growth Factor 2-Low and Eligible for Additional Targeted Therapy?

INTRODUCTION: DESTINY B04 provided clinical meaning to a new classification of human epidermal growth factor 2 (HER2) expression in breast cancer: HER2-low. Patients with germline breast cancer type 1 gene pathogenic variants (gBRCA1) often develop triple negative breast cancer (TNBC), but the proportion who could be classified as HER2-low and qualify for an additional targeted therapy option is unknown. This study aims to characterize the proportion of gBRCA1 or germline breast cancer type 2 gene pathogenic variants patients for whom these novel targeted therapies may be an option. METHODS: We performed a retrospective chart review of patients with gBRCA1/2 treated at our institution for invasive breast cancer from 2000 to 2021. Synchronous or metachronous contralateral breast cancers were recorded separately. HER2 status was determined by immunohistochemistry and fluorescence in situ hybridization. We excluded patients without complete HER2 data. RESULTS: Among the 95 breast cancers identified in our cohort of 85 gBRCA1/2 patients, 41 (43%) were TNBC, 38 (40%) were hormone receptor positive (HR+)/HER2-negative, and 16 (17%) were HER2-positive based on standard conventions. We found that 82% of the HR+/HER2-cancers and 66% of TNBCs would be reclassified as HER2-low. After stratifying by BRCA gene status, 64% of cancers in patients with gBRCA1 and 58% of cancers in patients with germline breast cancer type 2 gene pathogenic variants were HER2-low. CONCLUSIONS: A significant portion of gBRCA1/2 patients who were previously diagnosed with TNBC or HR+/HER2- breast cancer would now be classified as HER2-low and could be considered for the use of trastuzumab deruxtecan in the metastatic setting. Outcome differences from therapy changes in this cohort should now be assessed.

Variation in DCD Liver Transplant Protocols Among Transplant Centers in the United States.

Background: Variation in donation after circulatory death (DCD) organ recovery and liver transplant practices exist among transplant centers. This study aimed to evaluate these practices among centers in the United States. Methods: Scientific Registry of Transplant Recipients data were accessed to identify centers that performed liver transplantation in 2021 and 2022. Surveys were sent to transplant centers that consistently performed ≥5 DCD liver transplants per year. Results: DCD liver transplants were performed by 95 centers (65.1%) of the 146 liver transplant centers in the United States. Survey results were recorded from 42 centers that consistently performed ≥5 DCD liver transplants per year, with a 59.5% response rate. Withdrawal-to-asystole and agonal time were used to define donor warm ischemia time (WIT) in 16% and 84% centers, respectively. Fifty-six percent of the centers did not use oxygen saturation to define donor WIT. Systolic blood pressure cutoffs used to define agonal time varied between 50 and 80 mm Hg, donor age cutoffs ranged between 55 and 75 y, and cold ischemia times varied between 4 and 10 h. Seventy-six percent of centers used normothermic machine perfusion for DCD liver transplantation. Conclusions: This study highlights the wide variation in use, recovery, and definition of donor WIT. Using national data to rigorously define best practices will encourage greater utilization of this important donor resource.

Redox-Active Boron Clusters.

ConspectusIn this Account, we discuss our group's research over the past decade on a class of functionalized boron clusters with tunable chemical and physical properties, with an emphasis on accessing and controlling their redox behavior. These clusters can be thought of as three-dimensional aromatic systems that have distinct redox behavior and photophysical properties compared to their two-dimensional organic counterparts. Specifically, our lab has studied the highly tunable, multielectron redox behavior of B12(OR)12 clusters and applied these molecules in various settings. We first discuss the spectroscopic and electrochemical characterization of B12(OR)12 clusters in various oxidation states, followed by their use as catholytes and/or anolytes in redox flow batteries and chemical dopants in conjugated polymers. Additionally, the high oxidizing potential and visible light-absorbing nature of fluoroaryl-functionalized B12(OR)12 clusters have been leveraged by our group to generate weakly coordinating, photoexcitable species that can promote photooxidation chemistry.We have further translated these solution-phase studies of B12(OR)12 clusters to the solid state by using the precursor [B12(OH)12]2- cluster as a robust building block for hybrid metal oxide materials. Specifically, we have shown that the boron cluster can act as a thermally stable cross-linking material, which enhances electron transport between metal oxide nanoparticles. We applied this structural motif to create TiO2- and WO3-containing materials that showed promising properties as photocatalysts and electroactive materials for supercapacitors. Building on this concept, we later discovered that B12(OCH3)12, the smallest of the B12(OR)12 family, could retain its redox behavior in the solid state, a previously unseen phenomenon. We successfully harnessed this unique behavior for solid-state energy storage by implementing this boron cluster as a cathode-active material in a Li-ion prototype cell device. Recently, our group has also explored how to tune the redox properties of clusters other than B12(OR)12 species by synthesizing a library of vertex-differentiated clusters containing both B-OR and B-halogen groups. Due to the additive qualities of different functional groups on the cluster, these species allow access to a region of electrochemical potentials previously inaccessible by fully substituted closo-dodecaborate alkoxy-based derivatives.Lastly, we discuss our research into smaller-sized redox-active polyhedral boranes (B6- and B10-based cluster cores). Interestingly, these clusters show significantly less redox stability and reversibility than their dodecaborate-based counterparts. While exploring the functionalization of closo-hexaborate to create fully substituted derivates (i.e., [B6R6Hfac]-), we observed unique oxidative decomposition pathways for this cluster system. Consequently, we leveraged this oxidative instability to generate useful alkyl boronate esters via selective chemical oxidation. We further explored a closo-decaborate cluster as a platform to access electrophilic [B10H13]+ species capable of directly borylating arene compounds with unique regioselectivity. Upon chemical oxidation of the arylated decaborate clusters, we successfully synthesized various aryl boronate esters, establishing the generality of the oxidative cluster deconstruction concept.Overall, our work shows that boron clusters are an appealing class of redox-active molecules, and this fundamental and understudied property can be leveraged for constructing novel materials with tunable physical and electrochemical properties, as well as producing unique chemical reagents for small molecule synthesis.

Social Determinants of Cardiovascular Risk Factors Among Asian American Subgroups.

BACKGROUND: Social determinants of health (SDOH) play a significant role in the development of cardiovascular risk factors. We investigated SDOH associations with cardiovascular risk factors among Asian American subgroups. METHODS AND RESULTS: We utilized the National Health Interview Survey, a nationally representative survey of US adults, years 2013 to 2018. SDOH variables were categorized into economic stability, neighborhood and social cohesion, food security, education, and health care utilization. SDOH score was created by categorizing 27 SDOH variables as 0 (favorable) or 1 (unfavorable). Self-reported cardiovascular risk factors included diabetes, high cholesterol, high blood pressure, obesity, insufficient physical activity, suboptimal sleep, and nicotine exposure. Among 6395 Asian adults aged ≥18 years, 22.1% self-identified as Filipino, 21.6% as Asian Indian, 21.0% as Chinese, and 35.3% as other Asian. From multivariable-adjusted logistic regression models, each SD increment of SDOH score was associated with higher odds of diabetes among Chinese (odds ratio [OR], 1.45; 95% CI, 1.04-2.03) and Filipino (OR, 1.24; 95% CI, 1.02-1.51) adults; high blood pressure among Filipino adults (OR, 1.28; 95% CI, 1.03-1.60); insufficient physical activity among Asian Indian (OR, 1.42; 95% CI, 1.22-1.65), Chinese (OR, 1.58; 95% CI, 1.33-1.88), and Filipino (OR, 1.24; 95% CI, 1.06-1.46) adults; suboptimal sleep among Asian Indian adults (OR, 1.20; 95% CI, 1.01-1.42); and nicotine exposure among Chinese (OR, 1.56; 95% CI, 1.15-2.11) and Filipino (OR, 1.50; 95% CI, 1.14-1.97) adults. CONCLUSIONS: Unfavorable SDOH are associated with higher odds of cardiovascular risk factors in Asian American subgroups. Culturally specific interventions addressing SDOH may help improve cardiovascular health among Asian Americans.

Engineering water exchange is a safe and effective method for magnetic resonance imaging in diverse cell types.

Aquaporin-1 (Aqp1), a water channel, has garnered significant interest for cell-based medicine and in vivo synthetic biology due to its ability to be genetically encoded to produce magnetic resonance signals by increasing the rate of water diffusion in cells. However, concerns regarding the effects of Aqp1 overexpression and increased membrane diffusivity on cell physiology have limited its widespread use as a deep-tissue reporter. In this study, we present evidence that Aqp1 generates strong diffusion-based magnetic resonance signals without adversely affecting cell viability or morphology in diverse cell lines derived from mice and humans. Our findings indicate that Aqp1 overexpression does not induce ER stress, which is frequently associated with heterologous expression of membrane proteins. Furthermore, we observed that Aqp1 expression had no detrimental effects on native biological activities, such as phagocytosis, immune response, insulin secretion, and tumor cell migration in the analyzed cell lines. These findings should serve to alleviate any lingering safety concerns regarding the utilization of Aqp1 as a genetic reporter and should foster its broader application as a noninvasive reporter for in vivo studies.

A protocol for distilling animal body water from biological samples and measuring oxygen and hydrogen stable isotopes via cavity ring-down spectroscopy.

The application of stable isotope analysis (SIA) to the fields of ecology and animal biology has rapidly expanded over the past three decades, particularly with regards to water analysis. SIA now provides the opportunity to monitor migration patterns, examine food webs, and assess habitat changes in current and past study systems. While carbon and nitrogen SIA of biological samples have become common, analyses of oxygen or hydrogen are used more sparingly despite their promising utility for tracing water sources and animal metabolism. Common ecological applications of oxygen or hydrogen SIA require injecting enriched isotope tracers. As such, methods for processing and analyzing biological samples are tailored for enriched tracer techniques, which require lower precision than other techniques given the large signal-to-noise ratio of the data. However, instrumentation advancements are creating new opportunities to expand the applications of high-throughput oxygen and hydrogen SIA. To support these applications, we update methods to distill and measure water derived from biological samples with consistent precision equal to, or better than, ± 0.1 ‰ for δ17O, ± 0.3 ‰ for δ18O, ± 1 ‰ for δ2H, ± 2 ‰ for d-excess, and ± 15 per meg for Δ17O.

A single-cell atlas enables mapping of homeostatic cellular shifts in the adult human breast.

Here we use single-cell RNA sequencing to compile a human breast cell atlas assembled from 55 donors that had undergone reduction mammoplasties or risk reduction mastectomies. From more than 800,000 cells we identified 41 cell subclusters across the epithelial, immune and stromal compartments. The contribution of these different clusters varied according to the natural history of the tissue. Age, parity and germline mutations, known to modulate the risk of developing breast cancer, affected the homeostatic cellular state of the breast in different ways. We found that immune cells from BRCA1 or BRCA2 carriers had a distinct gene expression signature indicative of potential immune exhaustion, which was validated by immunohistochemistry. This suggests that immune-escape mechanisms could manifest in non-cancerous tissues very early during tumor initiation. This atlas is a rich resource that can be used to inform novel approaches for early detection and prevention of breast cancer.

5-HT1F receptor agonism induces mitochondrial biogenesis and increases cellular function in brain microvascular endothelial cells.

Introduction: Vascular and mitochondrial dysfunction are well-established consequences of multiple central nervous system (CNS) disorders, including neurodegenerative diseases and traumatic injuries. We previously reported that 5-hydroxytryptamine 1F receptor (5-HT1FR) agonism induces mitochondrial biogenesis (MB) in multiple organ systems, including the CNS. Methods: Lasmiditan is a selective 5-HT1FR agonist that is FDA-approved for the treatment of migraines. We have recently shown that lasmiditan treatment induces MB, promotes vascular recovery and improves locomotor function in a mouse model of spinal cord injury (SCI). To investigate the mechanism of this effect, primary cerebral microvascular endothelial cells from C57bl/6 mice (mBMEC) were used. Results: Lasmiditan treatment increased the maximal oxygen consumption rate, mitochondrial proteins and mitochondrial density in mBMEC, indicative of MB induction. Lasmiditan also enhanced endothelial cell migration and tube formation, key components of angiogenesis. Trans-endothelial electrical resistance (TEER) and tight junction protein expression, including claudin-5, were also increased with lasmiditan, suggesting improved barrier function. Finally, lasmiditan treatment decreased phosphorylated VE-Cadherin and induced activation of the Akt-FoxO1 pathway, which decreases FoxO1-mediated inhibition of claudin-5 transcription. Discussion: These data demonstrate that lasmiditan induces MB and enhances endothelial cell function, likely via the VE-Cadherin-Akt-FoxO1-claudin-5 signaling axis. Given the importance of mitochondrial and vascular dysfunction in neuropathologies, 5-HT1FR agonism may have broad therapeutic potential to address multiple facets of disease progression by promoting MB and vascular recovery.

The impact of patient-reported frailty on cardiovascular outcomes in elderly patients after non-ST-acute coronary syndrome.

BACKGROUND: As life expectancy increases, the population of older individuals with coronary artery disease and frailty is growing. We aimed to assess the impact of patient-reported frailty on the treatment and prognosis of elderly early survivors of non-ST-elevation acute coronary syndrome (NSTE-ACS). METHODS: Frailty data were obtained from two prospective trials, POPular Age and the POPular Age Registry, which both assessed elderly NSTE-ACS patients. Frailty was assessed one month after admission with the Groningen Frailty Indicator (GFI) and was defined as a GFI-score of 4 or higher. In these early survivors of NSTE-ACS, we assessed differences in treatment and 1-year outcomes between frail and non-frail patients, considering major adverse cardiovascular events (MACE, including cardiovascular mortality, myocardial infarction, and stroke) and major bleeding. RESULTS: The total study population consisted of 2192 NSTE-ACS patients, aged ≥70 years. The GFI-score was available in 1320 patients (79 ± 5 years, 37% women), of whom 712 (54%) were considered frail. Frail patients were at higher risk for MACE than non-frail patients (9.7% vs. 5.1%, adjusted hazard ratio [HR] 1.57, 95% confidence interval [CI] 1.01-2.43, p = 0.04), but not for major bleeding (3.7% vs. 2.8%, adjusted HR 1.23, 95% CI 0.65-2.32, p = 0.53). Cubic spline analysis showed a gradual increase of the risk for clinical outcomes with higher GFI-scores. CONCLUSIONS: In elderly NSTE-ACS patients who survived 1-month follow-up, patient-reported frailty was independently associated with a higher risk for 1-year MACE, but not with major bleeding. These findings emphasize the importance of frailty screening for risk stratification in elderly NSTE-ACS patients.

Isolation and monoculture of functional primary astrocytes from the adult mouse spinal cord.

Astrocytes are a widely heterogenic cell population that play major roles in central nervous system (CNS) homeostasis and neurotransmission, as well as in various neuropathologies, including spinal cord injury (SCI), traumatic brain injury, and neurodegenerative diseases, such as amyotrophic lateral sclerosis. Spinal cord astrocytes have distinct differences from those in the brain and accurate modeling of disease states is necessary for understanding disease progression and developing therapeutic interventions. Several limitations to modeling spinal cord astrocytes in vitro exist, including lack of commercially available adult-derived cells, lack of purchasable astrocytes with different genotypes, as well as time-consuming and costly in-house primary cell isolations that often result in low yield due to small tissue volume. To address these issues, we developed an efficient adult mouse spinal cord astrocyte isolation method that utilizes enzymatic digestion, debris filtration, and multiple ACSA-2 magnetic microbead purification cycles to achieve an astrocyte monoculture purity of ≅93-98%, based on all markers assessed. Importantly, the isolated cells contain active mitochondria and express key astrocyte markers including ACSA-1, ACSA-2, EAAT2, and GFAP. Furthermore, this isolation method can be applied to the spinal cord of male and female mice, mice subjected to SCI, and genetically modified mice. We present a primary adult mouse spinal cord astrocyte isolation protocol focused on purity, viability, and length of isolation that can be applied to a multitude of models and aid in targeted research on spinal-cord related CNS processes and pathologies.

Exploring the potential of water channels for developing MRI reporters and sensors without the need for exogenous contrast agents.

Genetically encoded reporters for magnetic resonance imaging (MRI) offer a valuable technology for making molecular-scale measurements of biological processes within living organisms with high anatomical resolution and whole-organ coverage without relying on ionizing radiation. However, most MRI reporters rely on contrast agents, typically paramagnetic metals and metal complexes, which often need to be supplemented exogenously to create optimal contrast. To eliminate the need for contrast agents, we previously introduced aquaporin-1, a mammalian water channel, as a new reporter gene for the fully autonomous detection of genetically labeled cells using diffusion-weighted MRI. In this study, we aimed to expand the toolbox of diffusion-based genetic reporters by modulating aquaporin membrane trafficking and harnessing the evolutionary diversity of water channels across species. We identified a number of new water channels that functioned as diffusion-weighted reporter genes. In addition, we show that loss-of-function variants of yeast and human aquaporins can be leveraged to design first-in-class diffusion-based sensors for detecting the activity of a model protease within living cells.

Robustness of cancer microbiome signals over a broad range of methodological variation.

In 2020, we identified cancer-specific microbial signals in The Cancer Genome Atlas (TCGA) [1]. Multiple peer-reviewed papers independently verified or extended our findings [2-12]. Given this impact, we carefully considered concerns by Gihawi et al. [13] that batch correction and database contamination with host sequences artificially created the appearance of cancer type-specific microbiomes. (1) We tested batch correction by comparing raw and Voom-SNM-corrected data per-batch, finding predictive equivalence and significantly similar features. We found consistent results with a modern microbiome-specific method (ConQuR [14]), and when restricting to taxa found in an independent, highly-decontaminated cohort. (2) Using Conterminator [15], we found low levels of human contamination in our original databases (~1% of genomes). We demonstrated that the increased detection of human reads in Gihawi et al. [13] was due to using a newer human genome reference. (3) We developed Exhaustive, a method twice as sensitive as Conterminator, to clean RefSeq. We comprehensively host-deplete TCGA with many human (pan)genome references. We repeated all analyses with this and the Gihawi et al. [13] pipeline, and found cancer type-specific microbiomes. These extensive re-analyses and updated methods validate our original conclusion that cancer type-specific microbial signatures exist in TCGA, and show they are robust to methodology.