Personalized nutritional management in the transition from non-dialysis dependent chronic kidney disease to dialysis.

Dialysis has been the dominant treatment regimen in end-stage kidney disease as a means to remove uremic waste products and to maintain electrolyte, acid base, and fluid balance. However, given that dialysis may not always provide a survival benefit nor improved quality of life in certain subpopulations, there is growing recognition of the need for conservative and preservative management as an alternative treatment strategy for advanced chronic kidney disease (CKD). Personalized nutritional management tailored to patient's sociodemographics, social needs, psychological status, health literacy level, and preferences is a key component of conservative and preservative care, as well as in the management of patients transitioning from non-dialysis dependent CKD to dialysis. In this review, we discuss the nutritional and metabolic alterations that ensue in CKD; the rationale for low-protein diets in the conservative and preservative management of advanced CKD; the role of plant-based diets in kidney health; emerging data on dietary potassium and sodium intake on CKD outcomes; and the practical implementation of dietary interventions in advanced kidney disease.

A Compound that Inhibits Glycolysis in Prostate Cancer Controls Growth of Advanced Prostate Cancer.

Metastatic castration-resistant prostate cancer remains incurable regardless of recent therapeutic advances. Prostate cancer tumors display highly glycolytic phenotypes as the cancer progresses. Non-specific inhibitors of glycolysis have not been utilized successfully for chemotherapy, because of their penchant to cause systemic toxicity. This study reports the preclinical activity, safety, and pharmacokinetics of a novel small molecule preclinical candidate, BKIDC-1553, with antiglycolytic activity. We tested a large battery of prostate cancer cell lines for inhibition of cell proliferation, in vitro. Cell cycle, metabolic and enzymatic assays were used to demonstrate their mechanism of action. A human PDX model implanted in mice and a human organoid were studied for sensitivity to our BKIDC preclinical candidate. A battery of pharmacokinetic experiments, absorption, distribution, metabolism, and excretion experiments, and in vitro and in vivo toxicology experiments were carried out to assess readiness for clinical trials. We demonstrate a new class of small molecule inhibitors where antiglycolytic activity in prostate cancer cell lines is mediated through inhibition of hexokinase 2. These compounds display selective growth inhibition across multiple prostate cancer models. We describe a lead BKIDC-1553 that demonstrates promising activity in a preclinical xenograft model of advanced prostate cancer, equivalent to that of enzalutamide. BKIDC-1553 demonstrates safety and pharmacologic properties consistent with a compound that can be taken into human studies with expectations of a good safety margin and predicted dosing for efficacy. This work supports testing BKIDC-1553 and its derivatives in clinical trials for patients with advanced prostate cancer.

A Compound that Inhibits Glycolysis in Prostate Cancer Controls Growth of Advanced Prostate Cancer.

Purpose: Metastatic castration-resistant prostate cancer remains incurable regardless of recent therapeutic advances. Prostate cancer tumors display highly glycolytic phenotypes as the cancer progresses. Non-specific inhibitors of glycolysis have not been utilized successfully for chemotherapy, because of their penchant to cause systemic toxicity. This study reports the preclinical activity, safety, and pharmacokinetics of a novel small molecule preclinical candidate, BKIDC-1553, with antiglycolytic activity. Experimental design: We tested a large battery of prostate cancer cell lines for inhibition of cell proliferation, in vitro. Cell cycle, metabolic and enzymatic assays were used to demonstrate their mechanism of action. A human PDX model implanted in mice and a human organoid were studied for sensitivity to our BKIDC preclinical candidate. A battery of pharmacokinetic experiments, absorption, distribution, metabolism, and excretion experiments, and in vitro and in vivo toxicology experiments were carried out to assess readiness for clinical trials. Results: We demonstrate a new class of small molecule inhibitors where antiglycolytic activity in prostate cancer cell lines is mediated through inhibition of hexokinase 2. These compounds display selective growth inhibition across multiple prostate cancer models. We describe a lead BKIDC-1553 that demonstrates promising activity in a preclinical xenograft model of advanced prostate cancer, equivalent to that of enzalutamide. BKIDC-1553 demonstrates safety and pharmacologic properties consistent with a compound that can be taken into human studies with expectations of a good safety margin and predicted dosing for efficacy. Conclusion: This work supports testing BKIDC-1553 and its derivatives in clinical trials for patients with advanced prostate cancer.

Impact of Human Recombinant Irisin on Tissue-Engineered Skeletal Muscle Structure and Function.

Tissue engineering of exogenous skeletal muscle units (SMUs) through isolation of muscle satellite cells from muscle biopsies is a potential treatment method for acute volumetric muscle loss (VML). A current issue with this treatment process is the limited capacity for muscle stem cell (satellite cell) expansion in cell culture, resulting in a decreased ability to obtain enough cells to fabricate SMUs of appropriate size and structural quality and that produce native levels of contractile force. This study determined the impact of human recombinant irisin on the growth and development of three-dimensional (3D) engineered skeletal muscle. Muscle satellite cells were cultured without irisin (control) or with 50, 100, or 250 ng/mL of irisin supplementation. Light microscopy was used to analyze myotube formation with particular focus placed on the diameter and density of the monotubes during growth of the 3D SMU. Following the formation of 3D constructs, SMUs underwent measurement of maximum tetanic force to analyze contractile function, as well as immunohistochemical staining, to characterize muscle structure. The results indicate that irisin supplementation with 250 ng/mL significantly increased the average diameter of myotubes and increased the proliferation and differentiation of myoblasts in culture but did not have a consistent significant impact on force production. In conclusion, supplementation with 250 ng/mL of human recombinant irisin promotes the proliferation and differentiation of myotubes and has the potential for impacting contractile force production in scaffold-free tissue-engineered skeletal muscle.

FAF1 Gene Involvement in Pituitary Corticotroph Tumors.

Cushing's disease (CD) is caused by rare pituitary corticotroph tumors that lead to corticotropin (ACTH) excess. Variants in FAF1, a pro-apoptotic protein involved in FAS-induced cell death, have been implicated in malignant disorders but the involvement of FAF1 in pituitary tumors has not been studied. Genetic data from patients with CD were reviewed for variants in FAF1 gene. Knockout mice (KO) were followed to assess the development of any pituitary disorder or cortisol excess. AtT-20 cells were used to study the effects of the variants of interest on ACTH secretion and cell proliferation. Three variants of interest were identified in 5 unique patients, two of which had rare allele frequency in genomic databases and were predicted to be likely pathogenic. KO mice were followed over time and no difference in their length/weight was noted. Additionally, KO mice did not develop any pituitary lesions and retained similar corticosterone secretion with wild type. AtT-20 cells transfected with FAF1 variants of interest or WT expression plasmids showed no significant difference in cell death or Pomc gene expression. However, in silico prediction models suggested significant differences in secondary structures of the produced proteins. In conclusion, we identified two FAF1 variants in patients diagnosed with CD with a potential pathogenic effect on the protein function and structure. Our in vitro and in vivo studies did not reveal an association of FAF1 defects with pituitary tumorigenesis and further studies may be needed to understand any association.

Acute Carpal Tunnel Syndrome from Horseshoe Abscess: A Case Report.

CASE: A 53-year-old man presented with a puncture wound to his right thumb, resulting in a horseshoe abscess, its infectious clinical picture muddled by dense paresthesias from the ensuing acute carpal tunnel syndrome. He was treated with irrigation and debridement of the thumb and small finger flexor tendon sheaths, carpal tunnel release, and bootlacing and dermal substitute application to the proximal forearm with eventual split-thickness skin grafting. CONCLUSION: Although a horseshoe abscess resulting in acute carpal tunnel syndrome is a rare entity unquantified in the literature, the treating orthopaedic surgeon should be aware of its association and initiate appropriate treatment accordingly.

E3 ubiquitin ligase ZBTB25 suppresses beta coronavirus infection through ubiquitination of the main viral protease MPro.

The main protease of severe acute respiratory syndrome coronavirus 2, Mpro, is a key viral protein essential for viral infection and replication. Mpro has been the target of many pharmacological efforts; however, the host-specific regulation of Mpro protein remains unclear. Here, we report the ubiquitin-proteasome-dependent degradation of Mpro protein in human cells, facilitated by the human E3 ubiquitin ligase ZBTB25. We demonstrate that Mpro has a short half-life that is prolonged via proteasomal inhibition, with its Lys-100 residue serving as a potential ubiquitin acceptor. Using in vitro binding assays, we observed ZBTB25 and Mpro bind to each other in vitro, and using progressive deletional mapping, we further uncovered the required domains for this interaction. Finally, we used an orthologous beta-coronavirus infection model and observed that genetic ablation of ZBTB25 resulted in a more highly infective virus, an effect lost upon reconstitution of ZBTB25 to deleted cells. In conclusion, these data suggest a new mechanism of Mpro protein regulation as well as identify ZBTB25 as an anticoronaviral E3 ubiquitin ligase.

Retinal vascular remodeling in photoreceptor degenerative disease.

Abnormal vasculature in the retina, specifically tortuous vessels and capillary degeneration, is common in many of the most prevalent retinal degenerative diseases, currently affecting millions of people across the world. However, the formation and development of abnormal vasculature in the context of retinal degenerative diseases are still poorly understood. The FVB/N (rd1) and rd10 mice are well-studied animal models of retinal degenerative diseases, but how photoreceptor degeneration leads to vascular abnormality in the diseases remains to be elucidated. Here, we used advancements in confocal microscopy, immunohistochemistry, and image analysis software to systematically characterize the pathological vasculature in the FVB/N (rd1) and rd10 mice, known as a chronic, rapid and slower retinal degenerative model, respectively. We demonstrated that there was plexus-specific vascular degeneration in the retinal trilaminar vascular network paralleled to photoreceptor degeneration in the diseased retinas. We also quantitatively analyzed the vascular structural architecture in the wild-type and diseased retinas to provide valuable information on vascular remodeling in retinal degenerative disease.

Long-term outcome analysis of Y90 radioembolization in hepatocellular carcinoma.

Background: Yttrium-90 (Y90) radioembolization is a catheter-based therapy for hepatocellular carcinoma (HCC). Multiple trials have evaluated the efficacy of Y90 in HCC; however, few have assessed long-term hepatic function. This study aimed to evaluate a clinical real-world experience of Y90 effectiveness and long-term impact on hepatic function. Methods: A single-center retrospective chart review was performed for patients with Child-Pugh (CP) class A or B who received Y90 for primary HCC between 2008 and 2016. Model for end-stage liver disease (MELD) and CP scores were calculated on the day of treatment and 1, 3, 6, 12, and 24 months post-procedure. Results: Of the 134 patients included, the mean age was 60 years old and median overall survival (OS) from date of diagnosis was 28 months [95% confidence interval (CI): 22.21-38.05]. Patients with CP class A (85%) had a median progression-free survival (PFS) of 3 months (95% CI: 2.99-5.55) and median OS of 17 months (95% CI: 9.59-23.10) from date of Y90 treatment compared to a median PFS of 4 months (95% CI: 2.07-8.28) and OS of 8 months (95% CI: 4.60-15.64) for patients with CP class B. MELD scores were significantly higher post-treatment than pre-treatment, with significant recovery at 24 months. No significant differences were seen between cancer stage and OS, while PFS and cancer stage did show difference between cancer stage 1 and 3 with longer median PFS seen in stage 1. Conclusions: While our study supports the literature for OS in Y90-treated patients, we found a shorter PFS in this population. This may reflect the differences between the utilization of RECIST in clinical trials and clinical radiology practice in determining progression. Significant factors associated with OS were age, MELD, CP scores and portal vein thrombosis (PVT). For PFS, CP score and stage at diagnosis were significant. Increasing MELD scores over time likely reflected a combination of radioembolization-induced liver disease, liver decompensation or progression of HCC. The downtrend at 24 months is likely due to long term survivors with significant benefit from therapy with no long-term complications from Y90.

OntoTrek: 3D visualization of application ontology class hierarchies.

An application ontology often reuses terms from other related, compatible ontologies. The extent of this interconnectedness is not readily apparent when browsing through larger textual presentations of term class hierarchies, be it Manchester text format OWL files or within an ontology editor like Protege. Users must either note ontology sources in term identifiers, or look at ontology import file term origins. Diagrammatically, this same information may be easier to perceive in 2 dimensional network or hierarchical graphs that visually code ontology term origins. However, humans, having stereoscopic vision and navigational acuity around colored and textured shapes, should benefit even more from a coherent 3-dimensional interactive visualization of ontology that takes advantage of perspective to offer both foreground focus on content and a stable background context. We present OntoTrek, a 3D ontology visualizer that enables ontology stakeholders-students, software developers, curation teams, and funders-to recognize the presence of imported terms and their domains, ultimately illustrating how projects can capture knowledge through a vocabulary of interwoven community-supported ontology resources.

Tolvaptan-induced isolated elevation of bilirubin in a patient with Gilbert syndrome.

Tolvaptan is the current standard of treatment for autosomal dominant polycystic kidney disease. It operates by acting on V2 receptors and blocks vasopressin interactions, causing a reduction in the rate of renal cyst growth and preserving kidney function. The current known risks of tolvaptan involve a serious liver injury characterized by an elevation in total bilirubin and alanine transaminase and aspartate transaminase levels. In this report, we document a unique liver injury characterized by an elevated bilirubin with normal alanine transaminase and aspartate transaminase levels in a patient who is homozygous for the UGT1A1 consistent with Gilbert syndrome.

Exploratory Study of the Association of Genetic Factors With Recovery of Adrenal Function in Cushing Disease.

Successful treatment of endogenous Cushing disease (CD) is often followed by a period of adrenal insufficiency (AI). We performed an exploratory study on genetic factors potentially involved in the hypothalamic-pituitary-adrenal (HPA) axis recovery in patients with CD after remission. We identified 90 patients who achieved remission after surgery and had a minimum of 3 months follow-up. Variants in a selected panel of genes that were rare in the general population and predicted as damaging in silico were retrieved from whole exome sequencing analysis. We did not identify any variant with significant correlation with recovery time after adjusting for multiple comparisons. On gene-specific analysis the BAG1 gene showed a correlation with shorter duration of postsurgical AI, but both patients with BAG1 variants later experienced a recurrence. After excluding patients with recurrence, no statistical association was recorded. To conclude, we did not identify a strong genetic modifier of HPA recovery in this exploratory study.

Influenza-associated hospitalisation and mortality rates among global Indigenous populations; a systematic review and meta-analysis.

BACKGROUND: More than 50 million influenza infections and over 100,000 deaths from influenza occur annually. While Indigenous populations experience an inequitable influenza burden, the magnitude of this inequity has not previously been estimated on a global scale. This study compared rates of influenza-associated hospitalisation and mortality between Indigenous and non-Indigenous populations globally. METHODS: A systematic review and meta-analysis was conducted including literature published prior to 13 July 2021. Eligible articles either reported a rate ratio (RR) comparing laboratory-confirmed influenza-associated hospitalisation and/or mortality between an Indigenous population and a corresponding benchmark population, or reported sufficient information for this to be calculated using publicly available data. Findings were reported by country/region and pooled by country and period (pandemic/seasonal) when multiple studies were available using a random-effects model. The I2 statistic assessed variability between studies. RESULTS: Thirty-six studies (moderate/high quality) were included; all from high or high-middle income countries. The pooled influenza-associated hospitalisation RR (HRR) for indigenous compared to benchmark populations was 5·7 (95% CI: 2·7-12·0) for Canada, 5·2 (2.9-9.3) for New Zealand, and 5.2 (4.2-6.4) for Australia. Of the Australian studies, the pooled HRR for seasonal influenza was 3.1 (2·7-3·5) and for pandemic influenza was 6·2 (5·1-7·5). Heterogeneity was slightly higher among studies of pandemic influenza than seasonal influenza. The pooled mortality RR was 4.1 (3·0-5.7) in Australia and 3·3 (2.7-4.1) in the United States. CONCLUSIONS: Ethnic inequities in severe influenza persist and must be addressed by reducing disparities in the underlying determinants of health. Influenza surveillance systems worldwide should include Indigenous status to determine the extent of the disease burden among Indigenous populations. Ethnic inequities in pandemic influenza illustrate the need to prioritise Indigenous populations in pandemic response plans.

Feasibility of using an app-based coaching intervention to improve provider communication about HPV vaccination.

Provider communication training is effective for increasing HPV vaccination rates among U.S. adolescents. However, such trainings often rely on in-person meetings, which can be burdensome for providers and costly to implement. To evaluate the feasibility of Checkup Coach, an app-based coaching intervention, to improve provider communication about HPV vaccination. In 2021, we offered Checkup Coach to providers in 7 primary care clinics in a large integrated delivery system. Participating providers (n = 19) attended a 1-h interactive virtual workshop that taught 5 high-quality practices for recommending HPV vaccination. Providers then had 3 months of access to our mobile app, which offered ongoing communication assessments, tailored tips for addressing parents' concerns, and a dashboard of their clinic's HPV vaccination coverage. Online surveys assessed pre-/post-intervention changes in providers' perceptions and communication behaviors. Compared to baseline, more providers reported high-quality HPV vaccine recommendation practices at 3-month follow-up (47% vs. 74%, p < .05). Providers' knowledge, self-efficacy, and shared commitment to improving HPV vaccination also improved (all p < .05). Although we found improvements in several other cognitions after the workshop, these changes did not retain statistical significance at 3 months. About three-quarters (78%) of providers used the mobile app, logging 2.3 sessions on average. Most providers agreed the app was easy to use (mean = 4.7/5.0), a convenient way to get vaccination data (mean = 4.6/5.0), and a tool they would recommend (mean = 4.3/5.0). Our app-based coaching intervention demonstrated feasibility and warrants additional evaluation as a novel mode for training providers to improve their HPV vaccine communication.

The aim of this study was to evaluate the feasibility of Checkup Coach, an app-based coaching intervention to improve provider communication about HPV vaccination, by offering the app to providers in 7 primary care clinics in a large integrated delivery system. Participating providers attended a 1-h interactive virtual workshop that taught high-quality HPV vaccine recommendation practices. For the following 3 months, providers used the app for ongoing communication assessments, tailored tips for addressing parents' concerns, and a dashboard of their clinic's HPV vaccination rates. Online surveys assessed pre- and post-intervention changes in providers' perceptions and communication practices. The percentage of providers reporting high-quality HPV vaccine recommendation practices increased from baseline to follow-up. Providers also reported higher HPV vaccine-related knowledge, self-efficacy, and shared commitment at 3 months. Providers agreed that the app was easy to use, a convenient way to get vaccination data, and a tool they would recommend. Our app-based coaching intervention demonstrated feasibility and warrants additional evaluation as a novel mode for training providers to improve their HPV vaccine communication.

Functional and clinical studies reveal pathophysiological complexity of CLCN4-related neurodevelopmental condition.

Missense and truncating variants in the X-chromosome-linked CLCN4 gene, resulting in reduced or complete loss-of-function (LOF) of the encoded chloride/proton exchanger ClC-4, were recently demonstrated to cause a neurocognitive phenotype in both males and females. Through international clinical matchmaking and interrogation of public variant databases we assembled a database of 90 rare CLCN4 missense variants in 90 families: 41 unique and 18 recurrent variants in 49 families. For 43 families, including 22 males and 33 females, we collated detailed clinical and segregation data. To confirm causality of variants and to obtain insight into disease mechanisms, we investigated the effect on electrophysiological properties of 59 of the variants in Xenopus oocytes using extended voltage and pH ranges. Detailed analyses revealed new pathophysiological mechanisms: 25% (15/59) of variants demonstrated LOF, characterized by a "shift" of the voltage-dependent activation to more positive voltages, and nine variants resulted in a toxic gain-of-function, associated with a disrupted gate allowing inward transport at negative voltages. Functional results were not always in line with in silico pathogenicity scores, highlighting the complexity of pathogenicity assessment for accurate genetic counselling. The complex neurocognitive and psychiatric manifestations of this condition, and hitherto under-recognized impacts on growth, gastrointestinal function, and motor control are discussed. Including published cases, we summarize features in 122 individuals from 67 families with CLCN4-related neurodevelopmental condition and suggest future research directions with the aim of improving the integrated care for individuals with this diagnosis.

Correlation between Streptococcus mutans levels in dental plaque and saliva of children.

PURPOSE: This study was designed to compare the levels of Streptococcus mutans (S. mutans) in saliva with those in occlusal plaque on posterior teeth at different stages of dentition, and to explore the correlation with caries experience to determine the most suitable source of S. mutans for research. METHODS: Samples of saliva and occlusal plaque were collected from 83 patients (aged 3-17 years) over three months. S. mutans levels were determined by culture-based selective plating, morphological identification, and S.mutans-specific monoclonal antibody labeling. RESULTS: The mean age of the participants was 8.8 (±3.7) years, and 74.7% of them were Hispanic. Mean caries experience for children with primary, mixed, and permanent dentition was 5.2 (±4.7), 4.0 (±3.3), and 0.8 (±1.3), respectively. Children with primary and mixed dentition had a higher caries experience than children with permanent dentition (P < 0.01), despite having similar S. mutans levels and total bacteria. A positive correlation was observed between S. mutans levels in plaque and those in saliva, but not between S. mutans levels and caries experience. It was noteworthy that plaque samples harbored higher S. mutans levels (>105 CFU/mL) than saliva samples. CONCLUSION: Both plaque and saliva samples are useful sources for S. mutans isolation. S. mutans levels from both sources were not significantly correlated with caries experience, but occlusal plaque had greater sensitivity for quantification of high S. mutans levels.

Measured weight loss as a precursor to cancer diagnosis: retrospective cohort analysis of 43 302 primary care patients.

BACKGROUND: Unexpected weight loss is a presenting feature of cancer in primary care. Data from primary care are lacking to quantify how much weight loss over what period should trigger further investigation for cancer. This research aimed to quantify cancer diagnosis rates associated with measured weight change in people attending primary care. METHODS: Retrospective cohort study of primary care electronic health records data linked to the Surveillance, Epidemiology, and End Results cancer registry (Integrated healthcare delivery system in Washington State, United States). Multivariable Cox regression incorporating time varying covariates using splines to model non-linear associations (age, percentage weight change, and weight change interval). Fifty thousand randomly selected patients aged 40 years and over followed for up to 9 years (1 January 2006 to 31 December 2014). Outcome measures are hazard ratios (95% confidence intervals) to quantify the association between percentage weight change and cancer diagnosis for all cancers combined, individual cancer sites and stages; percentage risk of cancer diagnosis within 6 months of the end of each weight change episode; and the positive predictive value for cancer diagnosis. RESULTS: There were 43 302 included in the analysis after exclusions. Over 287 858 patient-years of follow-up, including 24 272 (56.1%) females, 23 980 (55.4%) aged 40 to 59 years, 15 113 (34.9%) 60 to 79 years, and 4209 (9.7%) aged 80 years and over. Adjusted hazard ratios (95% confidence interval) for cancer diagnosis in a 60 years old ranged from 1.04 (1.02 to 1.05, P < 0.001) for 1% weight loss to 1.44 (1.23 to 1.68, P < 0.001) for 10%. An independent linear association was observed between percentage weight loss and increasing cancer risk. The absolute risk of cancer diagnosis increased with increasing age (up to 85 years) and as the weight change measurement interval decreased (<1 year). The positive predictive value for a cancer diagnosis within 1 year of ≥5% measured weight loss in a 60 to 69 years old was 3.41% (1.57% to 6.37%) in men and 3.47% (1.68% to 6.29%) in women. The risk of cancer diagnosis was significantly increased for pancreatic, myeloma, gastro-oesophageal, colorectal, breast, stage II and IV cancers. CONCLUSIONS: Weight loss is a sign of undiagnosed cancer regardless of the interval over which it occurs. Guidelines should resist giving an arbitrary cut-off for the interval of weight loss and focus on the percentage of weight loss and the patient's age. Future studies should focus on the association between diagnostic evaluation of weight change and risk of cancer mortality.

Combinatorial and Machine Learning Approaches for Improved Somatic Variant Calling From Formalin-Fixed Paraffin-Embedded Genome Sequence Data.

Formalin fixation of paraffin-embedded tissue samples is a well-established method for preserving tissue and is routinely used in clinical settings. Although formalin-fixed, paraffin-embedded (FFPE) tissues are deemed crucial for research and clinical applications, the fixation process results in molecular damage to nucleic acids, thus confounding their use in genome sequence analysis. Methods to improve genomic data quality from FFPE tissues have emerged, but there remains significant room for improvement. Here, we use whole-genome sequencing (WGS) data from matched Fresh Frozen (FF) and FFPE tissue samples to optimize a sensitive and precise FFPE single nucleotide variant (SNV) calling approach. We present methods to reduce the prevalence of false-positive SNVs by applying combinatorial techniques to five publicly available variant callers. We also introduce FFPolish, a novel variant classification method that efficiently classifies FFPE-specific false-positive variants. Our combinatorial and statistical techniques improve precision and F1 scores compared to the results of publicly available tools when tested individually.